ANG4043, a novel brain-penetrant peptide-mAb conjugate, is efficacious against HER2-positive intracranial tumors in mice.

Anti-HER2 monoclonal antibodies (mAb) have been shown to reduce tumor size and increase survival in patients with breast cancer, but they are ineffective against brain metastases due to poor brain penetration. In previous studies, we identified a peptide, known as Angiopep-2 (An2), which crosses the blood-brain barrier (BBB) efficiently via receptor-mediated transcytosis, and, when conjugated, endows small molecules and peptides with this property. Extending this strategy to higher molecular weight biologics, we now demonstrate that a conjugate between An2 and an anti-HER2 mAb results in a new chemical entity, ANG4043, which retains in vitro binding affinity for the HER2 receptor and antiproliferative potency against HER2-positive BT-474 breast ductal carcinoma cells. Unlike the native mAb, ANG4043 binds LRP1 clusters and is taken up by LRP1-expressing cells. Measuring brain exposure after intracarotid delivery, we demonstrate that the new An2-mAb conjugate penetrates the BBB with a rate of brain entry (Kin) of 1.6 × 10(-3) mL/g/s. Finally, in mice with intracranially implanted BT-474 xenografts, systemically administered ANG4043 increases survival. Overall, this study demonstrates that the incorporation of An2 to the anti-HER2 mAb confers properties of increased uptake in brain endothelial cells as well as BBB permeability. These characteristics of ANG4043 result in higher exposure levels in BT-474 brain tumors and prolonged survival following systemic treatment. Moreover, the data further validate the An2-drug conjugation strategy as a way to create brain-penetrant biologics for neuro-oncology and other CNS indications.

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.

Phase I study of GRN1005 in recurrent malignant glioma.

PURPOSE: GRN1005 is a peptide-drug conjugate with the ability to penetrate the blood-brain barrier (BBB) and tumor cells by targeting the low-density lipoprotein receptor-related protein-1. We conducted a first-in-human phase I trial of GRN1005 in patients with recurrent glioma. METHODS: Patients received GRN1005 by intravenous infusion every 3 weeks. Doses were escalated using a modified Fibonacci scheme. Study objectives included safety, tolerability, identification of the maximum tolerated dose (MTD), pharmacokinetics, and preliminary evidence of efficacy. Tumor extracted from patients undergoing surgery following administration of GRN1005 was analyzed to determine whether therapeutic concentrations of GRN1005 were achieved. RESULTS: Sixty-three patients received GRN1005 at doses of 30 to 700 mg/m(2) every 3 weeks. Therapy was well tolerated with neutropenia, leucopenia, and fatigue as the most frequent drug-associated grade 3/4 or higher toxicities. The MTD was 650 mg/m(2) every 3 weeks. Dose-limiting toxicities were grade 3 mucositis and grade 4 neutropenia. There was no evidence of central nervous system toxicity or antibody production. Pharmacokinetic analysis showed that exposure to GRN1005 was dose proportional. We observed one complete and two partial responses. Eight of 27 patients dosed ≥ 420 mg/m(2) had stable disease, which lasted a median of 51 days. Therapeutic concentrations of GRN1005 and free paclitaxel were shown in tumor tissue of surgical patients dosed with ≥ 200 mg/m(2). CONCLUSION: GRN1005 delivers paclitaxel across the BBB and achieves therapeutic concentrations in tumor tissue. It has similar toxicity to paclitaxel and appears to have activity in recurrent glioma. The recommended phase II dose is 650 mg/m(2) every 3 weeks.

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.

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 alpha(2)-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.

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.

Once-daily administration of CJC-1295, a long-acting growth hormone-releasing hormone (GHRH) analog, normalizes growth in the GHRH knockout mouse.

Although the majority of children with isolated growth hormone (GH) deficiency have a good growth response to GH-releasing hormone (GHRH), the use of this therapeutic agent is limited by its very short half-life. Indeed, we have shown that, in mice with GHRH gene ablation (GHRH knockout; GHRHKO), even twice-daily injections of a GHRH analog are unable to normalize growth. CJC-1295 is a synthetic GHRH analog that selectively and covalently binds to endogenous albumin after injection, thereby extending its half-life and duration of action. We report the effects of CJC-1295 administration in GHRHKO animals. Three groups of 1-wk-old GHRHKO mice were treated for 5 wk with 2 microg of CJC-1295 at intervals of 24, 48, and 72 h. Placebo-treated GHRHKO mice and mice heterozygous for the GHRHKO allele served as controls. GHRHKO animals receiving daily doses of CJC-1295 exhibited normal body weight and length. Mice treated every 48 and 72 h reached higher body weight and length than placebo-treated animals, without full growth normalization. Femur and tibia length remained normal in animals treated every 24 and 48 h. Relative lean mass and subcutaneous fat mass were normal in all treated groups. CJC-1295 caused an increase in total pituitary RNA and GH mRNA, suggesting that proliferation of somatotroph cells had occurred, as confirmed by immunohistochemistry images. These findings demonstrate that treatment with once-daily administration of CJC-1295 is able to maintain normal body composition and growth in GHRHKO mice. The same dose is less effective when administered every 48 or 72 h.

Prolonged stimulation of growth hormone (GH) and insulin-like growth factor I secretion by CJC-1295, a long-acting analog of GH-releasing hormone, in healthy adults.

CONTEXT: Therapeutic use of GHRH to enhance GH secretion is limited by its short duration of action. OBJECTIVE: The objective of this study was to examine the pharmacokinetic profile, pharmacodynamic effects, and safety of CJC-1295, a long-acting GHRH analog. DESIGN: The study design was two randomized, placebo-controlled, double-blind, ascending dose trials with durations of 28 and 49 d. SETTING: The study was performed at two investigational sites. PARTICIPANTS: Healthy subjects, ages 21-61 yr, were studied. INTERVENTIONS: CJC-1295 or placebo was administered sc in one of four ascending single doses in the first study and in two or three weekly or biweekly doses in the second study. MAIN OUTCOME MEASURES: The main outcome measures were peak concentrations and area under the curve of GH and IGF-I; standard pharmacokinetic parameters were used for CJC-1295. RESULTS: After a single injection of CJC-1295, there were dose-dependent increases in mean plasma GH concentrations by 2- to 10-fold for 6 d or more and in mean plasma IGF-I concentrations by 1.5- to 3-fold for 9-11 d. The estimated half-life of CJC-1295 was 5.8-8.1 d. After multiple CJC-1295 doses, mean IGF-I levels remained above baseline for up to 28 d. No serious adverse reactions were reported. CONCLUSIONS: Subcutaneous administration of CJC-1295 resulted in sustained, dose-dependent increases in GH and IGF-I levels in healthy adults and was safe and relatively well tolerated, particularly at doses of 30 or 60 microg/kg. There was evidence of a cumulative effect after multiple doses. These data support the potential utility of CJC-1295 as a therapeutic agent.

A Phase II, multicenter, randomized, double-blind, placebo-controlled crossover study of CJC-1008--a long-acting, parenteral opioid analgesic--in the treatment of postherpetic neuralgia.

INTRODUCTION: CJC-1008 is a chemical modification of the opioid peptide dynorphin A (1-13) (Dyn A) that promotes dynorphin's covalent attachment to human serum albumin in vivo after administration, thus prolonging its duration of action. The primary objective of this study was to evaluate the preliminary efficacy and safety of CJC-1008 as compared with placebo in patients with postherpetic neuralgia (PHN). METHODS: Patients with PHN were assigned 1:1 to receive active study medication or placebo. After dosing, measurements were made every 15 minutes for the first hour; at two, three, four, six, and eight hours postdose; and during return visits to the study site after two, seven, and 28 days (as necessary), as well as during precrossover and exit visits. These measurements examined: 1) overall pain intensity, 2) pain intensity for each individual PHN type, 3) categorical overall pain intensity, 4) categorical pain relief and 5) adverse events (AEs). When PHN pain intensity returned to baseline and/or at patients' first request for rescue analgesia other than acetaminophen (typically around 28 days after dosing but sometimes as soon as two days postdose), patients were to cross over to the alternative treatment and be monitored on the same schedule. RESULTS: A substantial placebo response was observed, but the analgesic effect observed in the active group was greater than that in the placebo group for the first eight hours. By 24 hours, the difference was not significant. A total of 29 out of 30 patients (96 percent) experienced at least one treatment-emergent AE during active drug treatment, while 14 of 27 patients (52 percent) reported such AEs during placebo treatment. Of the AEs occurring within the first eight hours after dosing, 97 percent were reported during treatment with active drug and 3 percent were reported during treatment with placebo. The majority of these AEs were mild in intensity. DISCUSSION: This study provides evidence of a greater analgesic effect when using CJC-1008 compared to placebo in patients with PHN. However, the effect only lasted through eight hours postdose and diminished by 24 hours. This study provides evidence of a peripheral action of dynorphin, since CJC-1008 does not cross the blood-brain barrier.

Synthesis and evaluation of insulin-human serum albumin conjugates.

A series of human insulin maleimido derivatives with short and long linkers was synthesized by exploiting the variations in the pK(a) values and environment of the three amino groups present in the protein. The syntheses were accomplished in organic solvent because of maleimide's instability in basic aqueous media. The derivatives thus obtained were conjugated to the free thiol on Cys34 of human serum albumin (HSA) and purified. A structure-activity relationship based on in vitro receptor binding and activation results for this series of insulin-HSA conjugates showed that the best compounds were attached at the B1 position of insulin with either short or long linkers. Two conjugates were administered subcutaneously to streptozotocin-induced diabetic rats and found to possess blood glucose normalizing activity up to 8 h post-administration. The return to diabetic plasma glucose levels was not observed within the time frame of the experiment (48 h). In comparison, the insulin-treated group's normalization activity lasted 2 h and returned to a diabetic level at 8 h. The onset of the conjugate activities were delayed by 1 h when compared to the activity of human insulin. The study results led to the identification of CJC-1575 as a potent and long lasting human insulin analogue.

Identification of CJC-1131-albumin bioconjugate as a stable and bioactive GLP-1(7-36) analog.

A series of analogs of GLP-1(7-36) amide containing a Nepsilon-(2-[2-[2-(3-maleimidopropylamido)ethoxy]ethoxy]acetyl)lysine has been synthesized and the resulting derivatives were bioconjugated to Cys34 of human serum albumin (HSA). The GLP-1-HSA bioconjugates were analyzed in vitro to assess the stabilizing effect of bioconjugation in the presence of DPP-IV as well as GLP-1 receptor binding and activation. Compound 9 (CJC-1131) having the point of attachment to albumin at the C-terminal of GLP-1 and a D-alanine substitution at position 8 was identified as having the best combination of stability and bioactivity.

Synthesis and in vitro analysis of atrial natriuretic peptide-albumin conjugates.

Atrial natriuretic peptide (ANP) is a clinically useful anti-hypertensive hormone. Maleimide derivatives of ANP have been synthesized and conjugated to cysteine-34 of human serum albumin. The conjugates were analyzed to assess their stability, receptor binding affinity and ability to stimulate guanylyl-cyclase activity in rat lung fibroblasts.

Development and characterization of a glucagon-like peptide 1-albumin conjugate: the ability to activate the glucagon-like peptide 1 receptor in vivo.

The rapid degradation of native glucagon-like peptide 1 (GLP-1) by dipeptidyl peptidase-IV (DPP-IV) has fostered new approaches for generation of degradation-resistant GLP-1 analogues. We examined the biological activity of CJC-1131, a DPP-IV-resistant drug affinity complex (DAC) GLP-1 compound that conjugates to albumin in vivo. The CJC-1131 albumin conjugate bound to the GLP-1 receptor (GLP-1R) and activated cAMP formation in heterologous fibroblasts expressing a GLP-1R. CJC-1131 lowered glucose in wild-type mice, but not in GLP-1R-/- mice. Basal glucose and glycemic excursion following glucose challenge remained significantly reduced 10-12 h following a single injection of CJC-1131. Twice daily administration of CJC-1131 to db/db mice significantly reduced glycemic excursion following oral and IP glucose challenge (P < 0.01 to 0.05) but did not significantly lower body weight during the 4-week study period. Levels of random fed glucose were significantly lower in CJC-1131-treated +/+ and db/db mice and remained significantly lower even 1 week following discontinuation of CJC-1131 administration. CJC-1131 increased levels of pancreatic proinsulin mRNA transcripts, percent islet area, and the number of bromodeoxyuridine-positive islet cells. These findings demonstrate that an albumin-conjugated DAC:GLP-1 mimics the action of native GLP-1 and represents a new approach for prolonged activation of GLP-1R signaling.