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.

[1]  Maxime Culot,et al.  Modelling of the blood–brain barrier in drug discovery and development , 2007, Nature Reviews Drug Discovery.

[2]  T. Terasaki,et al.  Functional characterization of the brain-to-blood efflux clearance of human amyloid-β peptide (1–40) across the rat blood–brain barrier , 2006, Neuroscience Research.

[3]  R. Gabathuler,et al.  Approaches to transport therapeutic drugs across the blood–brain barrier to treat brain diseases , 2010, Neurobiology of Disease.

[4]  A. Brenner,et al.  Safety, Pharmacokinetics, and Activity of GRN1005, a Novel Conjugate of Angiopep-2, a Peptide Facilitating Brain Penetration, and Paclitaxel, in Patients with Advanced Solid Tumors , 2011, Molecular Cancer Therapeutics.

[5]  I. Kissin The Development of New Analgesics Over the Past 50 Years: A Lack of Real Breakthrough Drugs , 2010, Anesthesia and analgesia.

[6]  F. St-Gelais,et al.  The role of neurotensin in central nervous system pathophysiology: what is the evidence? , 2006, Journal of psychiatry & neuroscience : JPN.

[7]  S. Pizzo,et al.  Clearance and binding of two electrophoretic "fast" forms of human alpha 2-macroglobulin. , 1981, The Journal of biological chemistry.

[8]  P. Sarret,et al.  Intrathecal administration of NTS1 agonists reverses nociceptive behaviors in a rat model of neuropathic pain , 2012, European journal of pain.

[9]  R. Melzack,et al.  The formalin test: a validation of the weighted-scores method of behavioural pain rating , 1993, Pain.

[10]  A. V. van Zonneveld,et al.  The Second and Fourth Cluster of Class A Cysteine-rich Repeats of the Low Density Lipoprotein Receptor-related Protein Share Ligand-binding Properties* , 1999, The Journal of Biological Chemistry.

[11]  I. Mikhailenko,et al.  Recognition of α2-Macroglobulin by the Low Density Lipoprotein Receptor-related Protein Requires the Cooperation of Two Ligand Binding Cluster Regions* , 2001, The Journal of Biological Chemistry.

[12]  M. Lafrance,et al.  Altered morphine-induced analgesia in neurotensin type 1 receptor null mice , 2010, Neuroscience.

[13]  M. De Hert,et al.  Cost of disorders of the brain in Europe. , 2006, European journal of neurology.

[14]  Christophe Rousselle,et al.  Development of an In Situ Mouse Brain Perfusion Model and its Application to mdr1a P-Glycoprotein-Deficient Mice , 2000, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[15]  S. Buga,et al.  The management of pain in metastatic bone disease. , 2012, Cancer control : journal of the Moffitt Cancer Center.

[16]  W. Pardridge,et al.  Blood-brain barrier delivery. , 2007, Drug discovery today.

[17]  A. Gaultier,et al.  Identification of the Low Density Lipoprotein (LDL) Receptor-related Protein-1 Interactome in Central Nervous System Myelin Suggests a Role in the Clearance of Necrotic Cell Debris* , 2012, The Journal of Biological Chemistry.

[18]  T. Yaksh,et al.  Structure-antinociceptive activity of neurotensin and some novel analogues in the periaqueductal gray region of the brainstem , 1991, Brain Research.

[19]  T. Heffner,et al.  Reduced amide bond neurotensin 8-13 mimetics with potent in vivo activity , 1995 .

[20]  R. Béliveau,et al.  New Angiopep-modified doxorubicin (ANG1007) and etoposide (ANG1009) chemotherapeutics with increased brain penetration. , 2010, Journal of medicinal chemistry.

[21]  S. Ishibashi [LDL-receptor-related protein]. , 1994, Nihon rinsho. Japanese journal of clinical medicine.

[22]  P. Steeg,et al.  Uptake of ANG1005, A Novel Paclitaxel Derivative, Through the Blood-Brain Barrier into Brain and Experimental Brain Metastases of Breast Cancer , 2009, Pharmaceutical Research.

[23]  E. Richelson,et al.  Diverse Roles of Neurotensin Agonists in the Central Nervous System , 2013, Front. Endocrinol..

[24]  R. Gabathuler Development of new peptide vectors for the transport of therapeutic across the blood-brain barrier. , 2010, Therapeutic delivery.

[25]  A. Beaudet,et al.  Regional and cellular distribution of low affinity neurotensin receptor mRNA in adult and developing mouse brain , 1998, The Journal of comparative neurology.

[26]  S. Pizzo,et al.  Physical properties of human α2-macroglobulin following reaction with methylamine and trypsin , 1982 .

[27]  P. Sexton,et al.  Allosteric Ligands of the Glucagon-Like Peptide 1 Receptor (GLP-1R) Differentially Modulate Endogenous and Exogenous Peptide Responses in a Pathway-Selective Manner: Implications for Drug Screening , 2010, Molecular Pharmacology.

[28]  M. Zhuo,et al.  Translational investigation and treatment of neuropathic pain , 2012, Molecular pain.

[29]  S. Hunskaar,et al.  The formalin test: an evaluation of the method , 1992, Pain.

[30]  J. Os,et al.  Cost of disorders of the brain in Europe 2010 , 2011, European Neuropsychopharmacology.

[31]  D. Strickland,et al.  LDL receptor-related protein 1: unique tissue-specific functions revealed by selective gene knockout studies. , 2008, Physiological reviews.

[32]  C. Woolf,et al.  Overcoming obstacles to developing new analgesics , 2010, Nature Medicine.

[33]  Y. Qi,et al.  NT79: A novel neurotensin analog with selective behavioral effects , 2010, Brain Research.

[34]  Michel Demeule,et al.  Identification and Design of Peptides as a New Drug Delivery System for the Brain , 2008, Journal of Pharmacology and Experimental Therapeutics.

[35]  R. Leduc,et al.  Elucidation of the Structure–Activity Relationships of Apelin: Influence of Unnatural Amino Acids on Binding, Signaling, and Plasma Stability , 2012, ChemMedChem.

[36]  A. Beaudet,et al.  Cellular distribution of neurotensin receptors in rat brain: Immunohistochemical study using an antipeptide antibody against the cloned high affinity receptor , 1996, The Journal of comparative neurology.

[37]  J. Sawynok,et al.  The Formalin Test: Characteristics and Usefulness of the Model , 2003 .

[38]  R. Lecomte,et al.  Behavioral, Medical Imaging and Histopathological Features of a New Rat Model of Bone Cancer Pain , 2010, PloS one.

[39]  R. Gabathuler Blood-brain barrier transport of drugs for the treatment of brain diseases. , 2009, CNS & neurological disorders drug targets.

[40]  T. Tanaka,et al.  Stability of a novel hexapeptide, (Me)Arg-Lys-Pro-Trp-tert-Leu-Leu-OEt, with neurotensin activity, in aqueous solution and in the solid state. , 1990, Chemical & pharmaceutical bulletin.

[41]  P. Vogel,et al.  Time course of the hypothermic response to continuously administered neurotensin , 2007, Neuropeptides.

[42]  T. Mikkelsen,et al.  Phase I Study of GRN1005 in Recurrent Malignant Glioma , 2013, Clinical Cancer Research.

[43]  P. Mcgonigle Peptide therapeutics for CNS indications. , 2012, Biochemical pharmacology.

[44]  A. Beaudet,et al.  Distribution of neurotensin binding sites in rat brain: A light microscopic radioautographic study using monoiodo [125I]Tyr3-neurotensin , 1987, Neuroscience.

[45]  Gary J. Bennett,et al.  A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man , 1988, Pain.

[46]  E. Richelson,et al.  Highly potent neurotensin analog that causes hypothermia and antinociception. , 2000, European Journal of Pharmacology.

[47]  K. Orth,et al.  Molecular dissection of ligand binding sites on the low density lipoprotein receptor-related protein. , 1994, The Journal of biological chemistry.

[48]  P. Turner The Blood-Brain Barrier in Health and Disease , 1987 .

[49]  H. Duvernoy,et al.  The vascularization of the human cerebellar cortex , 1983, Brain Research Bulletin.

[50]  P. Sarret,et al.  Spinal NTS1 receptors regulate nociceptive signaling in a rat formalin tonic pain model , 2008, Journal of neurochemistry.

[51]  M. Danhof,et al.  Drug transport across the blood-brain barrier , 1992, Pharmaceutisch Weekblad.

[52]  R. Mahley,et al.  Atherogenic remnant lipoproteins: role for proteoglycans in trapping, transferring, and internalizing. , 2007, The Journal of clinical investigation.

[53]  Ashley M. Jacobi,et al.  Spinal NTS2 receptor activation reverses signs of neuropathic pain , 2013, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[54]  Jean Martínez,et al.  Synthesis and biological effects of c(Lys-Lys-Pro-Tyr-Ile-Leu-Lys-Lys-Pro-Tyr-Ile-Leu) (JMV2012), a new analogue of neurotensin that crosses the blood-brain barrier. , 2008, Journal of medicinal chemistry.

[55]  J. Kennedy Neuropathic pain: molecular complexity underlies continuing unmet medical need. , 2007, Journal of medicinal chemistry.

[56]  J. Wettstein,et al.  Comparative Antipsychotic Profiles of Neurotensin and a Related Systemically Active Peptide Agonist , 1997, Peptides.

[57]  D. Strickland,et al.  Sequence identity between the alpha 2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor. , 1990, The Journal of biological chemistry.

[58]  B. Clineschmidt,et al.  Neurotensin: antinocisponsive action in rodents. , 1979, European journal of pharmacology.

[59]  P. Soubrié,et al.  Biochemical and pharmacological activities of SR 142948A, a new potent neurotensin receptor antagonist. , 1997, The Journal of pharmacology and experimental therapeutics.

[60]  G. Gebhart,et al.  Neurotensin activation of the NTR1 on spinally-projecting serotonergic neurons in the rostral ventromedial medulla is antinociceptive , 2005, Pain.

[61]  R. Béliveau,et al.  Involvement of the low‐density lipoprotein receptor‐related protein in the transcytosis of the brain delivery vector Angiopep‐2 , 2008, Journal of neurochemistry.

[62]  E. Richelson,et al.  Analgesic synergy of neurotensin receptor subtype 2 agonist NT79 and morphine , 2011, Behavioural pharmacology.

[63]  E. Richelson,et al.  NT69L, a novel analgesic, shows synergy with morphine , 2009, Brain Research.

[64]  S. L. Gonias,et al.  Embryonic Fibroblasts That Are Genetically Deficient in Low Density Lipoprotein Receptor-related Protein Demonstrate Increased Activity of the Urokinase Receptor System and Accelerated Migration on Vitronectin* , 1997, The Journal of Biological Chemistry.

[65]  A. Lipkowski,et al.  Neurotensin and neurotensin receptors: characteristic, structure-activity relationship and pain modulation--a review. , 2013, European Journal of Pharmacology.

[66]  C. Nemeroff,et al.  Neurotensin-induced antinociception in mice: antagonism by thyrotropin-releasing hormone. , 1981, The Journal of pharmacology and experimental therapeutics.

[67]  P. Kosson,et al.  PK20, a new opioid-neurotensin hybrid peptide that exhibits central and peripheral antinociceptive effects , 2010, Molecular pain.

[68]  D. Dubuisson,et al.  The formalin test: A quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats , 1977, Pain.

[69]  M. K. Hadden,et al.  Comparison of N-terminal modifications on neurotensin(8-13) analogues correlates peptide stability but not binding affinity with in vivo efficacy. , 2009, Journal of medicinal chemistry.

[70]  K. Abe,et al.  Pharmacokinetics of novel hexapeptides with neurotensin activity in rats. , 1993, Biological and Pharmaceutical Bulletin.

[71]  P. Quesenberry,et al.  Regulation of macrophage alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein by lipopolysaccharide and interferon-gamma. , 1993, The Journal of clinical investigation.

[72]  W. Pardridge The blood-brain barrier: Bottleneck in brain drug development , 2005, NeuroRx : the journal of the American Society for Experimental NeuroTherapeutics.

[73]  A micro-imaging study linking bone cancer pain with tumor growth and bone resorption in a rat model , 2013, Clinical & Experimental Metastasis.

[74]  A. Beaudet,et al.  Potent Spinal Analgesia Elicited through Stimulation of NTS2 Neurotensin Receptors , 2005, The Journal of Neuroscience.

[75]  S. Pizzo,et al.  Physical properties of human alpha 2-macroglobulin following reaction with methylamine and trypsin. , 1982, Biochimica et biophysica acta.

[76]  A. Beaudet,et al.  Chapter VI Neurotensin receptors in the central nervous system , 2002 .

[77]  C. D. Stern,et al.  Handbook of Chemical Neuroanatomy Methods in Chemical Neuroanatomy. Edited by A. Bjorklund and T. Hokfelt. Elsevier, Amsterdam, 1983. Cloth bound, 548 pp. UK £140. (Volume 1 in the series). , 1986, Neurochemistry International.

[78]  R. Béliveau,et al.  Transport characteristics of a novel peptide platform for CNS therapeutics , 2009, Journal of cellular and molecular medicine.

[79]  F. Hughes,et al.  Identification and functional characterization of a stable, centrally active derivative of the neurotensin (8-13) fragment as a potential first-in-class analgesic. , 2010, Journal of medicinal chemistry.

[80]  W. Pardridge,et al.  Biopharmaceutical drug targeting to the brain , 2010, Journal of drug targeting.

[81]  R. Béliveau,et al.  Antitumour activity of ANG1005, a conjugate between paclitaxel and the new brain delivery vector Angiopep‐2 , 2008, British journal of pharmacology.

[82]  T. Willnow,et al.  Genetic deficiency in low density lipoprotein receptor-related protein confers cellular resistance to Pseudomonas exotoxin A. Evidence that this protein is required for uptake and degradation of multiple ligands. , 1994, Journal of cell science.

[83]  G. Balboni,et al.  Conformational Dynamics of Kir3.1/Kir3.2 Channel Activation Via δ-Opioid Receptors , 2013, Molecular Pharmacology.

[84]  Michel Bouvier,et al.  Multiplexing of multicolor bioluminescence resonance energy transfer. , 2010, Biophysical journal.

[85]  C. Nemeroff,et al.  Alterations in nociception and body temperature after intracisternal administration of neurotensin, beta-endorphin, other endogenous peptides, and morphine. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[86]  G. Pasternak,et al.  Systemically and Topically Active Antinociceptive Neurotensin Compounds , 2010, Journal of Pharmacology and Experimental Therapeutics.

[87]  R. Béliveau,et al.  Influence of glioma tumour microenvironment on the transport of ANG1005 via low-density lipoprotein receptor-related protein 1 , 2011, British Journal of Cancer.

[88]  F. Zintl,et al.  Varicella vaccination in children after bone marrow transplantation , 1997, Bone Marrow Transplantation.

[89]  M. Lafrance,et al.  Involvement of NTS2 receptors in stress-induced analgesia , 2010, Neuroscience.

[90]  D. Holtzman,et al.  Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier. , 2000, The Journal of clinical investigation.

[91]  M. Aapro,et al.  Bone health management in patients with breast cancer: current standards and emerging strategies. , 2012, Breast.

[92]  A. Sinha,et al.  Evidence-Based Review of the Pharmacoeconomics Related to the Management of Chronic Nonmalignant Pain , 2010, Journal of pain & palliative care pharmacotherapy.

[93]  P. Dobner Neurotensin and pain modulation , 2006, Peptides.

[94]  Y. Pang,et al.  In vitro binding and CNS effects of novel neurotensin agonists that cross the blood–brain barrier , 1999, Neuropharmacology.

[95]  S. Leeman,et al.  Widespread expression in adult rat forebrain of mRNA encoding high‐affinity neurotensin receptor , 1998, The Journal of comparative neurology.

[96]  A. Beaudet,et al.  Immunohistochemical distribution of NTS2 neurotensin receptors in the rat central nervous system , 2003, The Journal of comparative neurology.