Abstract:
Neurotensin analogs selective for neurotensin receptor subtype 2 are described. These include hexapeptides (NT(8-13)) and pentapeptides (NT(9-13)) having a D-3,1-naphthyl-alanine, D-3,2-naphthyl-alanine, an alanine derivative such as cyclohexylalanine, or 1,2,3,4-tetrahydroisoquinoline at position 11. Methods of treating pain by administering these neurotensin analogs are also described.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    Polypeptides as well as many other types of compounds such as neurotransmitters and drugs can exert profound effects on the body. For example, neurotensin (NT) induces antinociception and hypothermia upon direct administration to brain. Systemic administration of NT does not induce these effects since NT is rapidly degraded by proteases and has poor blood brain barrier permeability. 
         [0002]    Neurotensin is a tridecapeptide with the amino acid sequence pyroGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH. Most, if not all, of the activity mediated by NT(1-13) is mediated by the 6 amino acid fragment, NT(8-13), which does not exist naturally in vivo. In order to observe pharmacological effects of either NT or NT(8-13) in the nervous system, each has to be administered directly into the brain or the spinal cord. Intravenous injection of NT and its fragments, however, causes hypotension, as well as other pharmacological effects. (See Carraway, R. et al. J B IOL  C HEM  248:6854-61 (1973) and Carraway, R. E. et al. “Structural requirements for the biological activity of neurotensin, a new vasoactive peptide.” In Fourth American Peptide Symposium. Edited by R Walter and J Meienhofer, Ann Arbor Science Publishers Inc., p. 679-85 (1975)) 
         [0003]    Neurotensin acts as a neurotransmitter or neuromodulator in the central nervous system (CNS), interacting largely with dopaminergic systems. (See Tyler-McMahon, B. M. et al. R EGUL  P EPT  93:125-36 (2000) and Binder, E. B. et al. P HARMACOL  R EV  53:453-86 (2001)) In addition, it has been known for a long time that neurotensin, when injected into brain, is a potent antinociceptive agent, operating by a μ-opioid independent mechanism. (See Clineschmidt, B. V. et al. E UR  J P HARMACOL  46:395-6 (1977) and Clineschmidt, B. V. et al. E UR  J P HARMACOL  54:129-39 (1979)) In fact, on a molar basis, NT is more potent than morphine as an antinociceptive agent. (See Nemeroff, C. B. et al. P ROC  N ATL  A CAD  S CI  USA 76:5368-71 (1979) and Al-Rodhan, N. R. et al. B RAIN  R ESEARCH  557:227-35 (1991)) 
         [0004]    Neurotensin and its analogs are also potent analgesics in animals. NT is produced in the brain, spinal cord dorsal horn, hypothalamus, and gut. NT receptors involved in the treatment of central pain may be different from those involved in the treatment of peripheral pain. Additionally, NT administration is associated with not just analgesia but also hypotension (unrelated to histamine release), fall in basal temperature, and decreased food intake leading to weight loss. NT has also been known to induce tolerance, increase gastrointestinal transit, induce diarrhea, and exhibit antipsychotic and antiparkinsonian effects (Boules, M. et al., Peptides 27:2523-33 (2006)). 
         [0005]    Neurotensin mediates its effects through at least 3 different receptors. (See Boules, M. et al. “NTS1 neurotensin receptor” In xPharm. Edited by S J Enna and D B Bylund. New York City, Elsevier, Inc. (2004); Boules, M. et al. “NTS2 neurotensin receptor” In xPharm. Edited by S J Enna and D B Bylund. New York City, Elsevier, Inc. (2004); and Boules, M. et al. “NTS3 neurotensin receptor” In xPharm. Edited by S J Enna and D B Bylund. New York City, Elsevier, Inc. (2004)) The first neurotensin receptor (NTS1) was molecularly cloned from rat brain (see Tanaka, K. et al. N EURON  4:847-54 (1990)) and human brain (see Watson, M. et al. M AYO  C LINIC  P ROCEEDINGS  68:1043-8 (1993)). The second neurotensin receptor (NTS2), which in binding assays is sensitive to the antihistamine levocabastine, has been cloned from mouse (see Mazella, J. et al. J N EUROSCI  16:5613-20 (1996), rat (see Chalon, P. et al. FEBS L ETTERS  386:91-4 (1996), and human (see Vita, N. et al. S OCIETY FOR  N EUROSCIENCE  23:394 [abstract] (1997)). Both NTS1 and NTS2 are 7-transmembrane spanning, G-protein coupled receptors. A third neurotensin receptor (NTS3) is a transmembrane protein, but spans the membrane only once and is identical to the protein called “sortilin.” (See Mazella, J. et al. J B IOL  C HEM  273:26273-6 (1998)). Recent data suggest that NTS3 has a function in inflammatory processes in the central nervous system. (See Martin, S. et al. J N EUROSCI  23:1198-205 (2003)) NT and NT(8-13) have highest affinity for NTS1, followed by NTS2 and NTS3. These peptides have over 1000-fold lower affinity for NTS3, as compared to that for NTS1. (See Kokko, K. P. et al. J M ED  C HEM  46:4141-8 (2003)). It is likely that both NTS1 and NTS2 mediate the antinociceptive effects of NT (see Dobner, P. R. P EPTIDES  27:2405-14 (2006)), while NTS1 mediates the hypotensive effects, among others. 
         [0006]    In addition to the antihistamine levocabastine, which has selectivity for NTS2, there are two other non-peptide neurotensin receptor antagonists. One antagonist, SR48692 (see Gully, D. et al. P ROC  N ATL  A CAD  S CI  USA 90:65-9 (1993)), has relatively high affinity for both NTS1 and NTS2, with selectivity for NTS1. (See Chalon, P. et al. FEBS L ETTERS  386:91-4 (1996)). SR48692 has very low affinity for NTS3. (See Mazella, J. et al. J B IOL  C HEM  273:26273-6 (1998)). Consistent with its relative selectivity for NTS1, in vivo SR48692 does not block all the effects of neurotensin. Another antagonist, SR142948A (see Gully, D. et al. J P HARMACOL  E XP  T HER  280:802-12 (1997), has a broader spectrum of activity in vivo against NT and is considered non-selective in binding to NTS1 and NTS2. Its affinity for NTS3 is unknown. Levocabastine may be a partial agonist/antagonist at NTS2. (See Dubuc, I. et al. E UR  J P HARMACOL  381:9-12 (1999)) 
         [0007]    There are many known neurotensin receptor agonists that are non-selective for NTS1 or NTS2 and that are active in the central nervous system (CNS) after peripheral administration (e.g., subcutaneously or intraperitoneally). (See Tyler, B. M. et al. N EUROPHARMACOLOGY  38:1027-34 (1999); Cusack, B. et al. B RAIN  R ES  856:48-54 (2000); Boules, M. et al. B RAIN  R ES  919:1-11 (2001); Kokko, K. P. et al. N EUROPHARMACOLOGY  48:417-25 (2005); and Hadden, M. K. et al. N EUROPHARMACOLOGY  (2005)). Such results indicate that these non-selective compounds pass the blood-brain barrier (BBB). There are also a few compounds that are relatively selective and potent at rodent NTS2 (e.g., JMV 431) (See Dubuc, I. et al. J N EUROSCI  19:503-10 (1999)) For the published NTS2-selective compounds, however, all studies employed their direct injection into brain (see Dubuc, I. et al. J N EUROSCI  19:503-10 (1999)) or into spinal cord (see Sarret, P. et al. J N EUROSCI  25:8188-96 (2005)) to elicit pharmacological effects. Therefore, it is assumed that these compounds do not penetrate the BBB. 
         [0008]    Over the years, Doctor Richelson and his team have designed, synthesized, and tested in vitro and in vivo over 60 peptides that are largely analogs of NT(8-13) and NT(9-13). From these studies, a large amount of structure-activity data were gathered, which led to defining the binding site for NT(8-13) at rat and human NTS1. (See Pang, Y. P. et al. J B IOL  C HEM  271:15060-8 (1996)) In addition, brain-penetrating analogs that bind with improved affinity to human NTS1 have been developed, largely as a result of the incorporation into these peptides of a novel amino acid, neo-Trp. (See Fauq, A. H. et al. T ETRAHEDRON:  A SYMMETRY  9:4127-34 (1998)) This amino acid is a regioisomer of tryptophan. U.S. Patents have been issued for this new amino acid and peptides that contain it, specifically many of the NT agonists developed in the laboratory of Dr. Richelson. (See U.S. Pat. Nos. 6,214,790; 6,765,099; and 7,098,307) 
         [0009]    In their series of peptides studied at hNTS1 and hNTS2, about one-half of the compounds had essentially the same affinities for both hNTS1 and hNTS2. Furthermore, there was a strong correlation between the log K d  (equilibrium dissociation constant) at hNTS1 and the log K d  at hNTS2 for the peptides, indicating that the binding site for these peptides at the hNTS2 is in a region with high homology to the binding site in the hNTS1. 
         [0010]    The key binding segment of the NTS1 receptor was previously shown to be the third outer loop of this putative seven-helix transmembrane spanning receptor. (See Pang, Y. P. et al. J B IOL  C HEM  271:15060-8 (1996); Cusack, B. et al. J B IOL  C HEM  271:15054-9 (1996); and Cusack, B. et al. B IOCHEM  P HARMACOL  60:793-801 (2000)) From their computer modeling studies, the binding site for NT(8-13) was determined to be primarily composed of eight residues—Phe 326 , Ile 329 , Trp 334 , Phe 337 , Tyr 339 , Phe 341 , Tyr 342 , and Tyr 344 —in the human NTS1. (See Pang, Y. P. et al. J B IOL  C HEM  271:15060-8 (1996)) Seven of the eight hydrophobic residues form an aromatic core of the NT(8-13) binding site or “pocket” in human NTS1. 
         [0011]    The human NTS1 (hNTS1) contains 418 amino acids, while hNTS2 is 8 amino acids shorter. Alignment of these receptors shows only about 33% identity of amino acids. The putative third extracellular loop for hNTS1 encompasses amino acids 326-345: FCYISDEQWTPFLYDFYHYF; while the corresponding region for hNTS2 spans amino acids 320-339: YCYVPDDAWTDPLYNFYHYF. In this region, the amino acid identity between the two receptors is still only 60%, but nearly twice as great as the overall figure for these receptors. Of the eight residues of the proposed binding site in hNTS1 (see Pang, Y. P. et al. J B IOL  C HEM  271:15060-8 (1996)), five (63%) are identical to those in hNTS2. All the aromatic residues in the third extracellular loop of the two receptors are conserved. In addition, those three residues that are different in the third extracellular loop have almost the same preference for adopting a loop conformation, based upon Chou and Fasman probabilities (see Chou, P. Y. et al. B IOCHEMISTRY  13:211-22 (1974)). From this sequence analogy and from the binding data, the binding site at the hNTS2 is likely composed of eight residues, namely, Tyr 320  Val 323  Trp 328  Pro 331  Tyr 333  Phe 335  Tyr 336  Tyr 338 . The binding pocket of the hNTS2 is just a bit smaller than that of the hNTS1. At the hNTS1, the low affinity of NT50, which is the most selective compound for the hNTS2, is probably due to the steric hindrance introduced most likely by Gln 333 , which is next to the key residue Trp 334  in the hNTS1 and mutated to Ala in hNTS2. 
         [0012]    From antisense studies, it appears that the hypothermic effects of neurotensin are mediated by NTS1 in rats and in mice, while antinociceptive effects of NT are mediated by activation of NTS1 in rats and NTS2 in mice. (See Tyler, B. M. et al. P ROC  N ATL  A CAD  S CI  USA 96: 7053-58 (1999) and Dubuc, I. et al. J N EUROSCI  19: 503-10 (1999)). 
         [0013]    Curiously, in vitro, antagonists and agonists at the NTS1 have opposite effects at the NTS2. Thus, from studies with the molecularly cloned NTS2, the expected antagonists, SR 48692 and SR 142948A behave as agonists, while NT and other agonists behave as antagonists or partial agonists. (See Vita, N. et al. E UR  J P HARMACOL  360: 265-72 (1998) and Yamada, M. et al. L IFE  S CI  62: L375-PL380 (1998)). These results are also made more interesting in light of the in vivo studies suggesting that the antagonists SR 48692 and SR 142948A have no intrinsic activities. (See Gully, D. et al. J P HARMACOL  E XP  T HER  280: 802-12 (1997)). Thus, there is a need for selective NTS1 and NTS2 agonists for in vivo experimentation. 
         [0014]    Furthermore, NTS2 has been shown to regulate pain. Therefore, we have discovered that compounds selective for NTS2 are effective and selective to treat pain while unexpectedly reducing or eliminating hypotensive effects. Thus, it would be advantageous to discover and develop drugs that selectively regulate NTS2. 
       SUMMARY OF THE INVENTION 
       [0015]    In one embodiment of the invention, neurotensin analogs that are hexapeptides designated NT(8-13) having a D-3,1-naphthyl-alanine at position 11 are described. Additionally, the neurotensin analog may include an N-methyl-arginine at position 8. Additionally, or in the alternative, the neurotensin analog may include a tert-leucine at position 12. Additionally, or in the alternative, the neurotensin analog may include a diaminobutyric acid at position 9. Additionally, or in the alternative, the neurotensin analog may include a Lysine (D or L) at position 8 or 9. Additionally, or in the alternative, the neurotensin analog may include an Ornithine (D or L) at position 9. 
         [0016]    In an alternative embodiment, neurotensin analogs that are pentapeptides designated NT(9-13) having a D-3,1-naphthyl-alanine (D or L) at position 11 are described. Additionally, the neurotensin analog may include a diaminobutyric acid at position 9. In the alternative, the neurotensin analog may additionally include a Lysine (D or L) at position 9. Additionally, or in the alternative, the neurotensin analog may include a tert-leucine at position 12. 
         [0017]    In one embodiment of the invention, neurotensin analogs that are hexapeptides designated NT(8-13) having a D-3,2-naphthyl-alanine at position 11 are described, with the proviso that positions 8 and 9 are not Lysine. Additionally, the neurotensin analog may include an N-methyl-arginine at position 8. Additionally, or in the alternative, the neurotensin analog may include a tert-leucine at position 12. Additionally, or in the alternative, the neurotensin analog may include a diaminobutyric acid at position 9. Additionally, or in the alternative, the neurotensin analog may include an Ornithine (D or L) at position 9. 
         [0018]    In one embodiment of the invention, neurotensin analogs that are hexapeptides designated NT(8-13) having a D-3,2-naphthyl-alanine at position 11 and an Arginine or an Arginine derivative at position 8 and/or position 9, i.e., at at least one of positions 8 or 9, are described. The Arginine may have an L or D configuration. The Arginine derivative may be N-methyl-arginine. Additionally, or in the alternative, the neurotensin analog may include a diaminobutyric acid at position 9. Additionally, or in the alternative, the neurotensin analog may include a Lysine at position 9. Additionally, or in the alternative, the neurotensin analog may include a tert-leucine at position 12. In one embodiment, the neurotensin analog may have an Arginine at both positions 8 and 9. In another embodiment, the neurotensin analog may have an N-methyl-arginine at position 8. In another embodiment, the hexapeptide has the Arginine or the Arginine derivative at position 8 and an Ornithine at position 9. In another alternative embodiment, the hexapeptide has a Lysine at position 8 and an Arginine at position 9. 
         [0019]    In another embodiment, neurotensin analogs that are pentapeptides designated NT(9-13) having a D-3,2-naphthyl-alanine at position 11 are described. The D-3,2-naphthyl-alanine may have a D or L configuration. Additionally, the neurotensin analog may include a tert-leucine at position 12. Additionally, or in the alternative, the neurotensin analog may include a Lysine at position 9. Additionally, or in the alternative, the neurotensin analog may include a diaminobutyric acid at position 9. 
         [0020]    In an alternative embodiment, neurotensin analogs that are hexapeptides designated NT(8-13) having an Alanine derivative at position 11 are described. In one embodiment, the Alanine derivative may be cyclohexylalanine. 
         [0021]    In an alternative embodiment, neurotensin analogs that are hexapeptides designated NT(8-13) having a 1,2,3,4-tetrahydroisoquinoline at position 11 are described. Additionally, the neurotensin analog may include an N-methyl-arginine at position 8. Additionally, or in the alternative, the neurotensin analog may include a Lysine (D or L) at position 8 and/or position 9, i.e., at at least one of positions 8 or 9. Additionally, or in the alternative, the neurotensin analog may include a tert-leucine at position 12. Additionally, or in the alternative, the neurotensin analog may include an Ornithine (D or L) at position 9. Additionally, or in the alternative, the neurotensin analog may include a diaminobutyric acid at position 9. 
         [0022]    In another embodiment, neurotensin analogs that are pentapeptides designated NT(9-13) having a 1,2,3,4-tetrahydroisoquinoline at position 11 are described. Additionally, or in the alternative, the neurotensin analog may include a diaminobutyric acid at position 9. Additionally, or in the alternative, the neurotensin analog may include a Lysine (D or L) at position 9. Additionally, or in the alternative, the neurotensin analog may include a tert-leucine at position 12. 
         [0023]    In another embodiment, neurotensin analogs that are pentapeptides designated NT(9-13) having a D-neo-Tryptophan at position 11 are described. Additionally, or in the alternative, the neurotensin analog may include a diaminobutyric acid at position 9. Additionally, or in the alternative, the neurotensin analog may include a Lysine (D or L) at position 9. Additionally, or in the alternative, the neurotensin analog may include a tert-leucine at position 12. 
         [0024]    In another embodiment, neurotensin analogs that are hexapeptides designated NT(8-13) having a D-neo-Tryptophan at position 11 are described. Additionally, the neurotensin analog may include an Ornithine (D or L), a diaminobutyric acid, or a Lysine (D or L) at position 9. Additionally, or in the alternative, the neurotensin analog may include an N-methyl-arginine at position 8. Additionally, or in the alternative, the neurotensin analog may include a Lysine (D or L) at position 8. Additionally, or in the alternative, the neurotensin analog may include a tert-leucine at position 12. 
         [0025]    In an alternative embodiment, methods for treating pain using any of the above-described analogs are described. The neurotensin analog is provided and administered to a patient in need of pain management. Administration of the neurotensin analog does not substantially reduce the patient&#39;s blood pressure. The dosage range for the neurotensin analog could be about 5 to about 20 mg/kg, alternatively about 7 to about 18 mg/kg, alternatively about 10 to about 15 mg/kg, alternatively about 12 to about 15 mg/kg. Alternatively, the dosage may be about 5 mg, alternatively about 7.5 mg, alternatively about 10 mg, alternatively about 12.5 mg, alternatively about 15 mg, alternatively about 17.5 mg, alternatively about 20 mg. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0026]      FIG. 1  depicts the structures of unnatural, i.e., synthetic and/or modified, amino acids that were used to make the NT analogs. 
           [0027]      FIG. 2  is a graph of a competition binding between radio-labeled NT and NT analogs at NTS2. 
           [0028]      FIG. 3  depicts the K d &#39;s for NT(8-13) and NT(9-13) analogs at human NTS1 vs. human NTS2. 
           [0029]      FIG. 4  is a graph showing degradation of NT(8-13) and NT(9-13) peptides in human plasma in vitro. 
           [0030]      FIG. 5  is a graph of body temperature lowering effects of neurotensin agonists in mice. 
           [0031]      FIG. 6  is a graph of the effect of NT79 (20 mg/kg intraperitoneally) on the tail flick and on the hot plate antinociceptive models in rats. 
           [0032]      FIG. 7  is a graph of the effect of NT79 (20 mg/kg intraperitoneally) in the acetic acid-induced writhing test in rats. 
           [0033]      FIG. 8  is a graph of the effect of saline, NT69 (2 mg/kg intraperitoneally), and NT79 (20 mg/kg intraperitoneally) on plasma prostaglandin levels in mice 30 min after injection. Blood samples from 3 mice were pooled for each condition. 
       
    
    
     DETAILED DESCRIPTION 
       [0034]    Because of the evidence from animal and human studies suggesting that NT is an endogenous neuroleptic (Bissette G and Nemeroff C B. “The neurobiology of neurotensin.” In: P SYCHOPHARMACOLOGY: THE  F OURTH  G ENERATION OF  P ROGRESS  (Eds. Kupfer D and Bloom F), pp. 573-83. Raven Press, New York (1995); Wolf, S. S. et al. J N EURAL  T RANSM  102: 55-65 (1995); Lahti, R. A. et al. J N EURAL  T RANSM  105: 507-16 (1998); and Cusack, B. et al. B RAIN  R ES  856: 48-54 (2000)), Dr. Richelson and colleagues have studied NT and its receptors, with the goal of developing a drug that mimics the effects of this neuropeptide. Such a compound possibly could have antipsychotic effects and represent a novel means of treating psychoses. Since the last 6 amino acids of the parent NT, namely NT(8-13) (Arg 8 ,Arg 9 ,Pro 10 ,Tyr 11 ,Ile 12 ,Leu 13 ), are sufficient for biological activity at NTS1, these researchers have focused their efforts on analogs of this hexapeptide and analogs of the pentapeptide NT(9-13). Thus, a large number of NT analogs were synthesized that are mostly based on NT(8-13). (See Cusack, B. et al. J B IOL  C HEM  270: 18359-66 (1995); Cusack, B. et al. J B IOL  C HEM  271: 15054-59 (1996); and Tyler, B. M. et al. N EUROPHARMACOLOGY  38: 1027-34 (1999)) 
         [0035]    With the availability of this peptide library and the molecularly cloned hNTS1 and hNTS2, the selectivity of these peptides for these receptors was determined from their affinities derived in radioligand binding studies. Most of the compounds tested showed no selectivity for either receptor. A few compounds, however, were both relatively potent and selective (&gt;30 fold higher affinity) at one or the other receptor. 
       Peptide Analogs 
       [0036]    The peptides, which contain unnatural, i.e., synthetic or modified, amino acids, used here and listed in Table 1, were synthesized in the Mayo Peptide Synthesis Facility of the Mayo Proteomics Research Center, formerly known as the Mayo Protein Core Facility (Mayo Clinic, Rochester Minn.), as described in previous publications. (See Morbeck, D. E. et al. “Analysis of hormone-receptor interaction sites using synthetic peptides: receptor binding regions of the alpha-subunit of human choriogonadotropin.” In:  Methods: A Companion to Methods in Enzymology,  Vol. 5, pp. 191-200. Academic Press, Inc., New York (1993)). The structures of the unnatural amino acids are depicted in  FIG. 1 . Briefly, all NT peptides were synthesized on automated 433A peptide synthesizers using orthogonal 9-fluorenyl-methoxycarbonyl (Fmoc) protection chemistry with tert-butyl (tBut), tert-butyloxycarbonyl (Boc), 4-methoxy-2,3,6-trimethylbenzenesulphonyl (Mtr) or 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc)-protected side chains. Protocols concerning activation coupling times, amino acid dissolution, coupling solvents and synthesis scale were followed according to the manufacturer&#39;s instructions (Applied Biosystems). All peptides were purified by reverse-phase HPLC on silica-bonded C 18  columns (Phenomenex or Vydac) in aqueous gradients of 0.1% trifluoroacetic acid (v/v) containing 5% to 80% acetonitrile (v/v) as an organic modifier. The methods of analytical reverse-phase HPLC and ESI-mass spectrometry (ThermoFischer Scientific, MSQ instrument) were used to analyze peptide homogeneity and peptide mass weight, respectively. To prepare the analogs for binding, they were dissolved as 10 mM stock solutions in deionized H 2 O, aliquoted in 20-80 μl quantities, and frozen at −30° C. A small number of less hydrophilic compounds were dissolved in DMSO (Sigma Chemical Co., St. Louis, Mo.). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Amino Acid Sequences of Selected Neurotensin (NT) Analogs 
               
             
          
           
               
                 Polypeptide 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
               
               
                   
               
               
                 NT 
                 p-Glu 
                 L-Leu 
                 L-Tyr 
                 L-Glu 
                 L-Asn 
                 L-Lys 
                 L-Pro 
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT02 
                   
                   
                   
                   
                   
                   
                   
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT03 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 D-Lys 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT04 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 D-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT06 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 D-Leu 
               
               
                 NT07 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 Gly 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT08 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-Ala 
                 L-Ile 
                 L-Leu 
               
               
                 NT09 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Leu 
                 L-Leu 
               
               
                 NT10 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Val 
                 L-Leu 
               
               
                 NT13 
                   
                   
                   
                   
                   
                   
                   
                 D-Arg 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT14 
                   
                   
                   
                   
                   
                   
                   
                 D-Arg 
                 D-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT15 
                   
                   
                   
                   
                   
                   
                   
                 D-Arg 
                 L-Lys 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT16 
                   
                   
                   
                   
                   
                   
                   
                 L-Lys 
                 D-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT17 
                   
                   
                   
                   
                   
                   
                   
                 L-Lys 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT18 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Lys 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT19 
                   
                   
                   
                   
                   
                   
                   
                 L-Lys 
                 L-Lys 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT20 
                   
                   
                   
                   
                   
                   
                   
                 D-Lys 
                 D-Lys 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT21 
                   
                   
                   
                   
                   
                   
                   
                 L-Orn 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT22 
                   
                   
                   
                   
                   
                   
                   
                 D-Orn 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT23 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Orn 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT24 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT25 
                   
                   
                   
                   
                   
                   
                   
                 L-Orn 
                 L-Orn 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT26 
                   
                   
                   
                   
                   
                   
                   
                 L-Orn 
                 D-Orn 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT27 
                   
                   
                   
                   
                   
                   
                   
                 D-Orn 
                 L-Orn 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT28 
                   
                   
                   
                   
                   
                   
                   
                 D-Orn 
                 D-Orn 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT29 
                   
                   
                   
                   
                   
                   
                   
                 DAB 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT30 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 DAB 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT31 
                   
                   
                   
                   
                   
                   
                   
                 DAB 
                 DAB 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT32 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 CHA 
                 L-Ile 
                 L-Leu 
               
               
                 NT33 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-3,2- 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Nal 
               
               
                 NT34 
                   
                   
                   
                   
                   
                   
                   
                   
                 L-Orn 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT35 
                   
                   
                   
                   
                   
                   
                   
                   
                 D-Orn 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT36 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Orn 
                 L-Pro 
                 D-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT37 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 D-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT38 
                   
                   
                   
                   
                   
                   
                   
                 DAP 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT39 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 DAP 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT40 
                   
                   
                   
                   
                   
                   
                   
                 DAP 
                 DAP 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT44 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L- 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 homoArg 
               
               
                 NT45 
                   
                   
                   
                   
                   
                   
                   
                 L- 
                 L- 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 homoArg 
                 homoArg 
               
               
                 NT46 
                   
                   
                   
                   
                   
                   
                   
                 L- 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 homoArg 
               
               
                 NT47 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-TIC 
                 L-Ile 
                 L-Leu 
               
               
                 NT48 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 D-TIC 
                 L-Ile 
                 L-Leu 
               
               
                 NT49 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-3,1- 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Nal 
               
               
                 NT50 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 D-3,1- 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Nal 
               
               
                 NT51 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 D-3,2- 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Nal 
               
               
                 NT52 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pip 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT54 
                 p-Glu 
                 L-Leu 
                 L-Tyr 
                 L-Glu 
                 L-Asn 
                 L-Lys 
                 L-Pro 
                 BPA 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT55 
                 p-Glu 
                 L-Leu 
                 L-Tyr 
                 L-Glu 
                 BPA 
                 L-Lys 
                 L-Pro 
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT56 
                 p-Glu 
                 L-Leu 
                 L-Tyr 
                 L-Glu 
                 L-Asn 
                 L-Lys 
                 L-Pro 
                 L-Arg 
                 BPA 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT59 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 DAB 
                 L-Pro 
                 L-3,1- 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Nal 
               
               
                 NT60 
                 p-Glu 
                 L-Leu 
                 L-Tyr 
                 L-Glu 
                 L-Asn 
                 L-Lys 
                 L-Pro 
                 L-Arg 
                 L-Orn 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT61 
                 p-Glu 
                 L-Leu 
                 L-Tyr 
                 L-Glu 
                 L-Asn 
                 L-Lys 
                 L-Pro 
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                 NT62 
                 p-Glu 
                 L-Leu 
                 L-Tyr 
                 L-Glu 
                 L-Asn 
                 L-Lys 
                 L-Pro 
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-3,1- 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Nal 
               
               
                 NT64L 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-neo- 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Trp 
               
               
                 NT65 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-neo- 
                 tert-Leu 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Trp 
               
               
                 NT66L 
                   
                   
                   
                   
                   
                   
                   
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 L-neo- 
                 tert-Leu 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Trp 
               
               
                 NT66T 
                   
                   
                   
                   
                   
                   
                   
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 L-Trp 
                 tert-Leu 
                 L-Leu 
               
               
                 NT67L 
                   
                   
                   
                   
                   
                   
                   
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 L-neo- 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Trp 
               
               
                 NT67T 
                   
                   
                   
                   
                   
                   
                   
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 L-Trp 
                 L-Ile 
                 L-Leu 
               
               
                 NT69L 
                   
                   
                   
                   
                   
                   
                   
                 N- 
                 L-Lys 
                 L-Pro 
                 L-neo- 
                 tert-Leu 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 methyl- 
                   
                   
                 Trp 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 Arg 
               
               
                 NT70 
                 p-Glu 
                 L-Leu 
                 L-iodo- 
                 L-Glu 
                 L-Asn 
                 L-Lys 
                 L-Pro 
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-Tyr 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                 Tyr 
               
               
                 NT71 
                   
                   
                   
                   
                   
                   
                   
                 N- 
                 DAB 
                 L-Pro 
                 L-neo- 
                 tert-Leu 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 methyl- 
                   
                   
                 Trp 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 Arg 
               
               
                 NT72 
                   
                   
                   
                   
                   
                   
                   
                   
                 D-Lys 
                 L-Pro 
                 L-neo- 
                 tert-Leu 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Trp 
               
               
                 NT73 
                   
                   
                   
                   
                   
                   
                   
                   
                 D-Lys 
                 L-Pro 
                 L-neo- 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Trp 
               
               
                 NT75 
                   
                   
                   
                   
                   
                   
                   
                 DAB 
                 L-Arg 
                 L-Pro 
                 L-neo- 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Trp 
               
               
                 NT77 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 L-neo- 
                 tert-Leu 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Trp 
               
               
                 NT77T 
                   
                   
                   
                   
                   
                   
                   
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 L-Trp 
                 tert-Leu 
                 L-Leu 
               
               
                 NT78 
                   
                   
                   
                   
                   
                   
                   
                 N- 
                 D-Orn 
                 L-Pro 
                 L-neo- 
                 tert-Leu 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 methyl- 
                   
                   
                 Trp 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 Arg 
               
               
                 NT78T 
                   
                   
                   
                   
                   
                   
                   
                 N- 
                 D-Orn 
                 L-Pro 
                 L-Trp 
                 tert-Leu 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 methyl- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 Arg 
               
               
                 NT79 
                   
                   
                   
                   
                   
                   
                   
                 N- 
                 L-Arg 
                 L-Pro 
                 D-3,1- 
                 tert-Leu 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 methyl- 
                   
                   
                 Nal 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 Arg 
               
               
                 NT80 
                   
                   
                   
                   
                   
                   
                   
                 N- 
                 L-Arg 
                 L-Pro 
                 D-3,1- 
                 L-Ile 
                 L-Leu 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 methyl- 
                   
                   
                 Nal 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 Arg 
               
               
                   
               
               
                 Abbreviations: 
               
               
                 BPA = benzoylphenylalanine; 
               
               
                 CHA = cyclohexylalanine; 
               
               
                 DAB = diaminobutyric acid; 
               
               
                 DAP = diaminoproprionic acid; 
               
               
                 homoArg = homoarginine; 
               
               
                 Orn = ornithine; 
               
               
                 Nal = naphthyl-alanine; 
               
               
                 NT = neurotensin; 
               
               
                 Pip = 1-pipecolinic acid; 
               
               
                 neo-Trp = a regio-isomer of the native tryptophan (See Fauq, A. H. et al. “Synthesis of (2S)-2-amino-3-(1H-4-indolyl)propanoic acid, a novel tryptophan analog for structural modification of bioactive peptides.” Tetrahedron: Asymmetry 9: 4127-34 (1998)); 
               
               
                 TIC = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) 
               
             
          
         
       
     
       Cell Culture 
       [0037]    CHO—K1 cells that had been stably transfected separately with the hNTS1 or hNTS2 genes were cultured in 150 mm (500 cm 2 ) Petri plates with 35 ml of Dulbecco&#39;s modified Eagle&#39;s medium containing 100 μM minimal essential medium nonessential amino acids (Life Technologies, Inc.) supplemented with 5% (v/v) FetalClone II bovine serum product (Hyclone Labs, Logan, Utah). CHO cells (subculture 7-15) were harvested at confluence by aspiration of the medium, followed by a wash with ice-cold 50 mM Tris-HCl buffer, pH=7.4, which was discarded, resuspension in 5-15 ml of Tris-HCl, scraping the cells with a plastic spatula into a centrifuge tube, and collection of cells by centrifugation at 300×g for 5 min at 4° C., in a GPR centrifuge (Beckman Instruments, Fullerton, Calif.). The cellular pellet (in Tris-HCl buffer) was stored at −180° C. until the radioligand binding was performed. 
         [0038]    For use in binding assays, crude membrane preparations were prepared by centrifugation of the cellular pellet at 35,600×g for 10 min. The supernatant was decanted and discarded, and the cellular pellet was resuspended in 1 ml of Tris-HCl buffer followed by homogenization with a Brinkmann Polytron at setting 5.5 for 15 s. Centrifugation was repeated as above, the supernatant was decanted and discarded, and the cellular pellet was resuspended in 1 ml of Tris-HCl buffer followed by homogenization. Centrifugation was repeated a third time, the supernatant was discarded, and the final cellular pellet was suspended in 0.5-2.5 ml of Tris-HCl buffer. Protein concentration of the membrane preparation was estimated by the method of Lowry et al. using bovine serum albumin as a standard. (Lowry O. H. et al. J B IOL  C HEM  193: 265-75 (1951)). 
       Radioligand Binding Assays 
       [0039]    A Biomek 1000 robotic workstation (Beckman Instruments) performed all pipetting steps in the radioligand binding assays as described previously by Cusack et al. J R ECEPT  R ES  13: 123-134, 1993. Competition binding assays with [ 3 H]NT (1 nM), varying concentrations of unlabeled NT, and varying concentrations of peptide analogs were carried out in duplicate in at least three independent experiments with membrane preparations from the appropriate cell lines. Nonspecific binding was determined with 1 μM unlabeled NT in assay tubes with a total volume of 1 ml. Incubation was at 20° C. for 40 min. The assay was routinely terminated by addition of ice-cold 0.9% NaCl (5×1.5 ml), followed by rapid filtration through a GF/B filter strip that had been pretreated with 0.2% or 2% polyethyleneimine. Details of binding assays have been described previously. (See Cusack, B. et al. E UR  J P HARMACOL  206: 339-42 (1991)). Data were analyzed using the LIGAND program. (Munson, P. J. and Rodbard, D. A NALYTICAL  B IOCHEMISTRY  107: 220-39 (1980)). 
       Statistical Analysis 
       [0040]    The values presented for equilibrium dissociation constants are expressed as the geometric means±S.E.M. (See Fleming, W. W. et al. J P HARMACOL  E XP  T HER  181: 339-45 (1972) and De Lean, A. M OL  P HARMACOL  21: 5-16 (1982)). 
       Results 
     Radioligand Binding Studies 
       [0041]    Results from the radioligand binding studies are listed in Table 2. All the peptides tested had Hill coefficients close to unity (data not shown), indicating binding to a single class of receptors. The most potent compound at both receptors was [L-neo-Trp 11 ]NT(8-13), abbreviated as NT64, with a K d =0.09 nM at hNTS1 and 0.32 nM at hNTS2. Nine analogs had sub-nanomolar K d &#39;s at hNTS1, the data for some of which were reported previously (Table 2). (See Cusack, B. et al. J B IOL  C HEM  270: 18359-66 (1995) and Tyler, B. M. et al. N EUROPHARMACOLOGY  38: 1027-34 (1999)). Six analogs had sub-nanomolar K d &#39;s at hNTS2 (Table 2), all but one of which (NT44) also had sub-nanomolar K d &#39;s at hNTS1. Two compounds, [L-Om 9 ,D-Tyr 11 ]NT(8-13) (NT36) and [D-Orn 9 ,D-Tyr 11 ]NT(8-13) (NT37), had no detectable binding to hNTS1, but had micromolar K d &#39;s at hNTS2. The least potent compounds at hNTS2 were [D-Orn 9 ]NT(1-13) (NT61, K d =6.6 μM) and [D-Orn 9 ]NT(9-13) (NT35, K d =10 μM). 
         [0042]    An example of some competition binding curves for compounds at hNTS2, expressed by CHO—K1 cells, is shown in  FIG. 2 . Assays were performed with membrane preparations, 1 nM [ 3 H]NT, and varying concentrations of compounds as described in the text. Curves were generated using the LIGAND program. (See Munson, P. J. and Rodbard, D. A NALYTICAL  B IOCHEMISTRY  107: 220-39 (1980)). Data are the means of duplicate determinations and are representative results from one of at least three independent experiments. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Radioligand Binding Data for Neurotensin and Analogs at the Human NTS1 and NTS2. 
               
             
          
           
               
                   
                   
                 hNTS1 
                   
                 hNTS2 
                   
               
               
                 Reference 
                   
                 Geometric 
                 hNTS1 
                 Geometric 
                 hNTS2 
               
               
                 Name 
                 Compound Sequence 
                 Mean ∀ SEM 
                 Selectivity 
                 Mean ∀ SEM 
                 Selectivity 
               
               
                   
               
             
          
           
               
                 NT 
                 Neurotensin 
                 1.94 ± 0.07 
                 3.4 
                 6.5 ± 0.1 
                 0.3 
               
               
                 NT02 
                 [D-Lys 8 ]NT(8-13) 
                  1.0 ± 0.1† 
                 4.6 
                 4.6 ± 0.5 
                 0.2 
               
               
                 NT03 
                 [D-Lys 9 ]NT(8-13) 
                 690 ± 30  
                 0.4 
                 280 ± 30  
                 2.5 
               
               
                 NT04 
                 [D-Arg 9 ]NT(8-13) 
                 158 ± 7  
                 0.2 
                 24 ± 2  
                 6.5 
               
               
                 NT06 
                 [D-Leu 13 ]NT(8-13) 
                 4200 ± 100  
                 0.8 
                 3300 ± 300  
                 1.3 
               
               
                 NT07 
                 [Gly 10 ]NT(8-13) 
                 1380 ± 50  
                 0.7 
                 970 ± 40  
                 1.4 
               
               
                 NT08 
                 [Ala 11 ]NT(8-13) 
                 2500 ± 200  
                 0.02 
                 58 ± 5  
                 43 
               
               
                 NT09 
                 [L-Leu 12 ]NT(8-13) 
                 7.2 ± 0.6 
                 0.3 
                 2.4 ± 0.3 
                 2.9 
               
               
                 NT10 
                 [L-Val 12 ]NT(8-13) 
                 11.3 ± 0.6  
                 0.8 
                 8.8 ± 0.4 
                 1.3 
               
               
                 NT13 
                 [D-Arg 8 ]NT(8-13) 
                  0.50 ± 0.03† 
                 5.7 
                 2.9 ± 0.2 
                 0.2 
               
               
                 NT14 
                 [D-Arg 8 ,D-Arg 9 ]NT(8-13) 
                 28 ± 3† 
                 0.7 
                 20 ± 2  
                 1.4 
               
               
                 NT15 
                 [D-Arg 8 ,L-Lys 9 ]NT(8-13) 
                  3.5 ± 0.5‡ 
                 4.0 
                 18 ± 2  
                 0.2 
               
               
                 NT16 
                 [L-Lys 8 ,D-Arg 9 ]NT(8-13) 
                 33 ± 6† 
                 1.2 
                 39.6 ± 0.6  
                 0.8 
               
               
                 NT17 
                 [L-Lys 8 ]NT(8-13) 
                  0.25 ± 0.02† 
                 4.0 
                 1.2 ± 0.2 
                 0.2 
               
               
                 NT18 
                 [L-Lys 9 ]NT(8-13) 
                  1.49 ± 0.09‡ 
                 0.8 
                 1.18 ± 0.09 
                 1.3 
               
               
                 NT19 
                 [L-Lys 8 ,L-Lys 9 ]NT(8-13) 
                  1.4 ± 0.2‡ 
                 1.7 
                 2.4 ± 0.3 
                 0.6 
               
               
                 NT20 
                 [D-Lys 8 ,D-Lys 9 ]NT(8-13) 
                 185 ± 5†  
                 4.0 
                 730 ± 60  
                 0.3 
               
               
                 NT21 
                 [L-Orn 8 ]NT(8-13) 
                  0.41 ± 0.03† 
                 5.2 
                 2.2 ± 0.1 
                 0.2 
               
               
                 NT22 
                 [D-Orn 8 ]NT(8-13) 
                  1.9 ± 0.2‡ 
                 3.2 
                 5.9 ± 0.2 
                 0.3 
               
               
                 NT23 
                 [L-Orn 9 ]NT(8-13) 
                  0.94 ± 0.06‡ 
                 1.6 
                 1.5 ± 0.1 
                 0.6 
               
               
                 NT24 
                 [D-Orn 9 ]NT(8-13) 
                 120 ± 10‡ 
                 6.6 
                 790 ± 20  
                 0.2 
               
               
                 NT25 
                 [L-Orn 8 ,L-Orn 9 ]NT(8-13) 
                  3.0 ± 0.3‡ 
                 1.3 
                 3.9 ± 0.2 
                 0.8 
               
               
                 NT26 
                 [L-Orn 8 ,D-Orn 9 ]NT(8-13) 
                 360 ± 40‡ 
                 3.0 
                 1082 ± 6   
                 0.3 
               
               
                 NT27 
                 [D-Orn 8 ,L-Orn 9 ]NT(8-13) 
                  3.6 ± 0.2† 
                 6.6 
                 24 ± 2  
                 0.2 
               
               
                 NT28 
                 [D-Orn 8 ,D-Orn 9 ]NT(8-13) 
                 600 ± 20† 
                 3.2 
                 1900 ± 100  
                 0.3 
               
               
                 NT29 
                 [DAB 8 ]NT(8-13) 
                  1.2 ± 0.1‡ 
                 5.6 
                 6.5 ± 0.3 
                 0.2 
               
               
                 NT30 
                 [DAB 9 ]NT(8-13) 
                  0.41 ± 0.05‡ 
                 2.2 
                 0.90 ± 0.04 
                 0.5 
               
               
                 NT31 
                 [DAB 8 ,DAB 9 ]NT(8-13) 
                  2.1 ± 0.3‡ 
                 9.1 
                 19.5 ± 0.7  
                 0.1 
               
               
                 NT32 
                 [CHA 11 ]NT(8-13) 
                 1000 ± 200  
                 0.1 
                 99 ± 2  
                 10.1 
               
               
                 NT33 
                 [L-3,2-Nal 11 ]NT(8-13) 
                 89 ± 9  
                 0.2 
                 18 ± 1  
                 5.0 
               
               
                 NT34 
                 [L-Orn 9 ]NT(9-13) 
                 300 ± 50† 
                 4.0 
                 1190 ± 40  
                 0.3 
               
               
                 NT35 
                 [D-Orn 9 ]NT(9-13) 
                 550 ± 80  
                 19.1 
                 10500 ± 200  
                 0.1 
               
               
                 NT36 
                 [L-Orn 9 ,D-Tyr 11 ]NT(8-13) 
                 n.d.** 
                 — 
                 1160 ± 20  
                 — 
               
               
                 NT37 
                 [D-Orn 9 ,D-Tyr 11 ]NT(8-13) 
                 n.d. 
                 — 
                 1800 ± 100  
                 — 
               
               
                 NT38 
                 [DAP 8 ]NT(8-13) 
                 5.8 ± 0.7 
                 4.3 
                 25 ± 1  
                 0.2 
               
               
                 NT39 
                 [DAP 9 ]NT(8-13) 
                 8.6 ± 0.8 
                 3.0 
                 17.0 ± 0.2  
                 0.5 
               
               
                 NT40 
                 [DAP 8 ,DAP 9 ]NT(8-13) 
                 175 ± 10  
                 6.3 
                 1100 ± 30  
                 0.2 
               
               
                 NT44 
                 [L-Homoarg 9 ]NT(8-13) 
                 1.7 ± 0.1 
                 0.6 
                 0.96 ± 0.06 
                 1.8 
               
               
                 NT45 
                 [L-Homoarg 8 ,L-Homoarg 9 ]NT(8-13) 
                 1.4 ± 0.1 
                 0.4 
                 0.52 ± 0.02 
                 2.6 
               
               
                 NT46 
                 [L-Homoarg 8 ]NT(8-13) 
                 0.41 ± 0.05 
                 1.1 
                 0.45 ± 0.01 
                 0.9 
               
               
                 NT47*** 
                 [L-TIC 11 ]NT(8-13) 
                 720 
                 0.02 
                 14 
                 51.4 
               
               
                 NT48*** 
                 [D-TIC 11 ]NT(8-13) 
                 350 
                 0.73 
                 255  
                 1.4 
               
               
                 NT49 
                 [L-3,1-Nal 11 ]NT(8-13) 
                 6.4 ± 0.5 
                 0.2 
                 1.28 ± 0.05 
                 5.0 
               
               
                 NT50 
                 [D-3,1-Nal 11 ]NT(8-13) 
                 1800 ± 500  
                 0.01 
                 17 ± 3  
                 104 
               
               
                 NT51 
                 [D-3,2-Nal 11 ]NT(8-13) 
                 1080 ± 80  
                 0.03 
                 32.9 ± 0.6  
                 32.8 
               
               
                 NT52 
                 [L-Pip 10 ]NT(8-13) 
                 33 ± 6  
                 1.2 
                 38 ± 2  
                 0.9 
               
               
                 NT54 
                 [BPA 8 ]NT(1-13) 
                 18.6 ± 0.9  
                 35.5 
                 660 ± 50  
                 0.03 
               
               
                 NT55 
                 [BPA 5 ]NT(1-13) 
                 0.91 ± 0.09 
                 6.2 
                 5.7 ± 0.3 
                 0.2 
               
               
                 NT56 
                 [BPA 9 ]NT(1-13) 
                 72 ± 8  
                 4.6 
                 330 ± 40  
                 0.2 
               
               
                 NT59 
                 [DAB 9 ,L-3,1-Nal 11 ]NT(8-13) 
                 6.8 ± 0.2 
                 0.3 
                 1.73 ± 0.09 
                 3.9 
               
               
                 NT60 
                 [L-Orn 9 ]NT(1-13) 
                 3.2 ± 0.1 
                 5.4 
                 17 ± 2  
                 0.2 
               
               
                 NT61 
                 [D-Orn 9 ]NT(1-13) 
                 1500 ± 100  
                 4.4 
                 6600 ± 100  
                 0.2 
               
               
                 NT62 
                 [L-3,1 Nal 11 ]NT(1-13) 
                 8.4 ± 0.3 
                 1.7 
                 14.2 ± 0.5  
                 0.6 
               
               
                 NT64L 
                 [L-neo-Trp 11 ]NT(8-13) 
                  0.09 ± 0.01* 
                 3.4 
                 0.32 ± 0.02 
                 0.3 
               
               
                 NT65 
                 [neo-Trp 11 ,tert-Leu 12 ]NT(8-13) 
                 1.01 ± 0.05 
                 0.5 
                 0.52 ± 0.03 
                 1.9 
               
               
                 NT66L 
                 [D-Lys 8 ,L-neo-Trp 11 ,tert-Leu 12 ]NT(8-13) 
                 10.2 ± 0.6|| 
                 0.7 
                 7.1 ± 0.8 
                 1.4 
               
               
                 NT66T 
                 [D-Lys 8 ,L-Trp 11 ,tert-Leu 12 ]NT(8-13) 
                 140 ± 19  
                 0.1 
                 18.1 ± 0.7  
                 7.7 
               
               
                 NT67L 
                 [D-Lys 8 ,L-neo-Trp 11 ]NT(8-13) 
                  0.59 ± 0.05|| 
                 2.1 
                 1.23 ± 0.03 
                 0.5 
               
               
                 NT67T 
                 [D-Lys 8 ,L-Trp 11 ]NT(8-13) 
                 17 ± 2  
                 0.5 
                 8.0 ± 0.4 
                 2.2 
               
               
                 NT69L 
                 [N-methyl-Arg 8 , L-Lys 9 , L-neo-Trp 11 , tert-Leu 12 ]NT(8-13) 
                 3.1 ± 0.4 
                 0.7 
                 2.1 ± 0.2 
                 1.5 
               
               
                 NT70 
                 [L-iodo-Tyr 3 ]NT(1-13) 
                 2.52 ± 0.05 
                 1.7 
                 4.20 ± 0.04 
                 0.6 
               
               
                 NT71 
                 [N-methyl-Arg 8 , DAB 9 , L-neo-Trp 11 , tert-leu 12 ]NT(8-13) 
                 1.71 ± 0.06 
                 0.7 
                 1.11 ± 0.03 
                 1.5 
               
               
                 NT72 
                 [D-Lys 9 , L-neo-Trp 11 , tert-Leu 12 ]NT(9-13) 
                 34 ± 9  
                 41.0 
                 1400 ± 100  
                 0.02 
               
               
                 NT73 
                 [D-Lys 9 , L-neo-Trp 11 ]NT(9-13) 
                 30 ± 3  
                 5.5 
                 162 ± 3  
                 0.2 
               
               
                 NT75 
                 [DAB 9 , L-neo-Trp 11 ]NT(9-13) 
                 73 ± 5  
                 2.3 
                 169 ± 8  
                 0.4 
               
               
                 NT77 
                 [D-Orn 9 , L-neo-Trp 11 , tert-Leu 12 ]NT(8-13) 
                 1500 ± 100  
                 0.3 
                 460 ± 70  
                 3.3 
               
               
                 NT77T 
                 [D-Orn 9 , L-Trp 11 , tert-Leu 12 ]NT(8-13) 
                 1530 ± 80  
                 0.2 
                 320 ± 20  
                 4.8 
               
               
                 NT78 
                 [N-methyl-Arg 8 , D-Orn 9 , L-neo-Trp 11 , tert-Leu 12 ]NT(8-13) 
                 1300 ± 400  
                 0.3 
                 380 ± 40  
                 3.4 
               
               
                 NT78T 
                 [N-methyl-Arg 8 , D-Orn 9 , L-Trp 11 , tert-Leu 12 ]NT(8-13) 
                 1400 ± 300  
                 0.5 
                 660 ± 50  
                 2.1 
               
               
                 NT79 
                 [N-methyl-Arg 8 , D-3,1-Nal 11 , tert-Leu 12 ]NT(8-13) 
                  1800*** 
                 — 
                 22 ± 3  
                 82 
               
               
                 NT80*** 
                 [N-methyl-Arg 8 , D-3,1-Nal 11 ]NT(8-13) 
                 2000  
                 — 
                 30 
                 67 
               
               
                   
               
               
                 *published in Tyler, B. M. et al. “In vitro binding and CNS effects of novel neurotensin agonists that cross the blood-brain barrier.” Neuropharmacology 38: 1027-34 (1999); 
               
               
                 †published before in Cusack, B. et al. “Pharmacological and biochemical profiles of unique neurotensin 8-13 analogs exhibiting species selectivity, stereoselectivity, and superagonism.” J Biol Chem 270: 18359-66 (1995); 
               
               
                 ‡reported before, but numbers are now slightly different from previous numbers (See Cusack, B. et al. J Biol Chem 270: 18359-66 (1995)) because we added more values to obtain the mean; 
               
               
                 ||Published in Tyler et al. 1999, but these numbers are slightly different, because we added more values to obtain the mean. 
               
               
                 **no detectable binding at 1 μM. 
               
               
                 ***data are not sufficient to calculate geometric mean ± S.E.M. 
               
               
                 Abbreviations: 
               
               
                 BPA = benzoylphenylalanine; 
               
               
                 CHA = cyclohexylalanine; 
               
               
                 DAB = diaminobutyric acid; 
               
               
                 DAP = diaminoproprionic acid; 
               
               
                 Homoarg = homoarginine; 
               
               
                 Orn = ornithine; 
               
               
                 Nal = naphthyl-alanine; 
               
               
                 NT = neurotensin; 
               
               
                 Pip = 1-pipecolinic acid; 
               
               
                 neo-Trp = a regio-isomer of the native tryptophan (See Fauq, A. H. et al. “Synthesis of (2S)-2-amino-3-(1H-4-indolyl)propanoic acid, a novel tryptophan analog for structural modification of bioactive peptides.” Tetrahedron: Asymmetry 9: 4127-34 (1998)); 
               
               
                 TIC = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) 
               
             
          
         
       
     
         [0043]    There was a strong correlation between the log K d  at hNTS1 and the log K d  at hNTS2 (y=0.76x-1.75, R=0.84, P&lt;0.0001) for the peptides ( FIG. 3 ). The relationship between the log K d &#39;s at human NTS1 and human NTS2 is depicted in  FIG. 3 . The equation for the regression of the log K d  at hNTS1 versus the log K d  at hNTS2 was y=0.76x-1.75 (R=0.84, P&lt;0.0001). The dashed line is the line of identity. About one-half of the compounds fell at or around the line of identity. There were several compounds, however, that had at least a 10-fold selectivity for one or the other receptor. Thus, three compounds had 19-fold or greater (range 19 to 41 fold) selectivity for hNTS1: [D-Orn 9 ]NT(9-13) (NT35, K d =550 nM at hNTS1 and 10500 nM at hNTS2); [BPA 11 ]NT(1-13) (NT54, K d =18.6 nM at hNTS1 and 660 nM at hNTS2); [D-Lys 9 ,L-neo-Trp 11 ,tert-Leu  12 ]NT(9-13) (NT72, K d =34 nM at hNTS1 and 1400 nM at hNTS2). Five compounds had 10 fold or greater (range 10 to 104 fold) selectivity for hNTS2: [CHA 11 ]NT(8-13) (NT32, K d =1000 nM at hNTS1 and 99 nM at hNTS2); [D-3,2-Nal 11 ]NT(8-13) (NT51, K d =1080 nM at hNTS1 and 32.9 nM at hNTS2); [Ala 11 ]NT(8-13) (NT08, K d =2500 nM at hNTS1 and 58 nM at hNTS2); [L-TIC 11 ]NT(8-13) (NT47, K d =720 nM at hNTS1 and 14 nM at hNTS2); and [D-3,1-Nal 11 ]NT(8-13) (NT50, K d =1800 nM at hNTS1 and 17 nM at hNTS2). 
         [0044]    In the present series of peptides, about one-half of the compounds had essentially the same affinities for both hNTS1 and hNTS2 (see  FIG. 3 , line of identity). Furthermore, there is strong correlation between the log K d  at hNTS1 and the log K d  at hNTS2 for the peptides. Thus, the binding site for these peptides at the hNTS2 is likely in a region with high homology to the binding site in the hNTS1. 
       Receptors 
     Compounds Selective for NTS2 
       [0045]    In previous publications, Dr. Richelson and colleagues showed the importance of position 11 of NT(8-13) for high-affinity binding to hNTS1. (See Cusack, B. et al. J B IOL  C HEM  271: 15054-59 (1996); Pang, Y. P. et al. J B IOL  C HEM  271: 15060-68 (1996); and Cusack, B et al. B IOCHEM  P HARMACOL  60: 793-801 (2000)). Pi electrons in this position are critical for the cation-pi interactions that contribute to the binding of the ligand to the hNTS1. (See Cusack, B. et al. J B IOL  C HEM  271: 15054-59 (1996) and Pang, Y. P. et al. J B IOL  C HEM  271. 15060-68 (1996)). It is therefore interesting to note that the most selective compounds at the hNTS2 were compounds with substitutions in position 11: [L-Ala 11 ]NT(8-13), [D-3,1-Nal 11 ]NT(8-13), [L-TIC 11 ]NT(8-13), and [D-3,2-Nal 11 ]NT(8-13). At both receptors, these substitutions reduced the binding affinity, compared to that for NT, for example. The effect, however, was much greater at the hNTS1 than at the hNTS2, leaving very selective and relatively potent compounds at the second subtype. 
         [0046]    NT50, [D-3,1-Nal 11 ]NT(8-13), may be the agonist that is selective for NTS2 not only in vitro, but also in vivo based on studies with this compound. After direct injection into the brains of rats, NT50 caused little or no effects on body temperature, but caused behavioral activation (McMahon et al., unpublished observations), results different from those obtained with non-selective agonists. (See Cusack, B. et al. B RAIN  R ES  856: 48-54 (2000) and Tyler-McMahon, B. M. et al. E UR  J P HARMACOL  390: 107-11 (2000)). 
         [0047]    Of the many NT(8-13) and NT(9-13) peptide analogs that have been synthesized and tested, about 70 have been tested for their affinities at both hNTS1 and hNTS2. Few are selective for either NTS1 or NTS2. Table 3 lists several compounds having selectivity for hNTS2. Based on preliminary in vivo data, NT79 and NT80 have also been found to be selective for NTS2 (not listed in Table 3). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 hNTS2-Selective Compounds 
               
             
          
           
               
                   
                   
                 hNTS1 
                 hNTS2 
                 NTS2 
               
             
          
           
               
                   
                 Compound 
                   
                 K d  (nM) 
                 Selectivity 
               
               
                   
                   
               
             
          
           
               
                   
                 NT08 
                 2500 
                 58 
                 43 
               
               
                   
                 NT47 
                 720 
                 14 
                 51 
               
               
                   
                 NT50 
                 1800 
                 17.3 
                 104 
               
               
                   
                 NT51 
                 1080 
                 33 
                 33 
               
               
                   
                   
               
             
          
         
       
     
         [0048]    The sequences of these compounds are listed in Table 4, along with several other compounds. All compounds, except for NT72, are NT(8-13) analogs. NT72 is an analog of NT(9-13). The four compounds of Table 3 differ from the natural sequence by the single amino acid substitution in position 11. NT(8-13) has L-Tyr in this position. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Sequences of hNTS2-Selective and hNTS2-Non-Selective Compounds 
               
             
          
           
               
                   
                 Sequence 
                 hNTS2 
               
             
          
           
               
                 Compound 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
                 Selectivity 
               
               
                   
               
             
          
           
               
                 NT08 
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-Ala 
                 L-Ile 
                 L-Leu 
                 43 
               
               
                 NT47 
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-TIC 
                 L-Ile 
                 L-Leu 
                 51 
               
               
                 NT50 
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 D-3,1-Nal 
                 L-Ile 
                 L-Leu 
                 104 
               
               
                 NT51 
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 D-3,2-Nal 
                 L-Ile 
                 L-Leu 
                 33 
               
               
                 NT64 
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-neo-Trp 
                 L-Leu 
                 L-Leu 
                 — 
               
               
                 NT65 
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-neo-Trp 
                 Tert-Leu 
                 L-Leu 
                 1.7 
               
               
                 NT66 
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 L-neo-Trp 
                 Tert-Leu 
                 L-Leu 
                 2 
               
               
                 NT67 
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 L-neo-Trp 
                 L-Ile 
                 L-Leu 
                 — 
               
               
                 NT69 
                 N-Me-L-Arg 
                 L-Lys 
                 L-Pro 
                 L-neo-Trp 
                 tert-Leu 
                 L-Leu 
                 1.5 
               
               
                 NT72 
                   
                 D-Lys 
                 L-Pro 
                 L-neo-Trp 
                 tert-Leu 
                 L-Leu 
                 — 
               
               
                 NT77 
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 L-neo-Trp 
                 tert-Leu 
                 L-Leu 
                 3.3 
               
               
                 NT79 
                 N-Me-L-Arg 
                 L-Arg 
                 L-Pro 
                 D-3,1-Nal 
                 tert-Leu 
                 L-Leu 
                 82 
               
               
                 NT80 
                 N-Me-L-Arg 
                 L-Arg 
                 L-Pro 
                 D-3,1-Nal 
                 L-Ile 
                 L-Leu 
                 67 
               
               
                   
               
               
                 Nal = naphthyl-alanine; 
               
               
                 TIC = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; 
               
               
                 Orn = ornithine; 
               
               
                 “—” indicates higher affinity for hNTS1; 
               
               
                 “ND” indicates not yet determined 
               
             
          
         
       
     
         [0049]    Dubuc et al. described [3,2-Nal 11 ]NT(8-13) analogs (JMV509 and JMV510) that showed some selectivity for NTS2 receptors (non-human). (See Dubuc, I. et al. J N EUROSCI  19:503-10 (1999)) Their binding assays made use of the molecularly cloned rat NTS1 and the molecularly cloned mouse NTS2. The sequences and binding data are reported in Tables 5A-B below. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 5A 
               
             
             
               
                   
               
               
                 Sequences of some [3,2-Nal 11 ]NT(8-13) Analogs 
               
             
          
           
               
                   
                 Sequence 
               
             
          
           
               
                 Compound 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
               
               
                   
               
               
                 NT33 
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 L-3,2-Nal 
                 L-Ile 
                 L-Leu 
               
               
                 NT51 
                 L-Arg 
                 L-Arg 
                 L-Pro 
                 D-3,2-Nal 
                 L-Ile 
                 L-Leu 
               
               
                 JMV510 
                 Boc-L-Lys 
                 L-Lys 
                 L-Pro 
                 L-3,2-Nal 
                 L-Ile 
                 L-Leu 
               
               
                 JMV509 
                 Boc-L-Lys 
                 L-Lys 
                 L-Pro 
                 D-3,2-Nal 
                 L-Ile 
                 L-Leu 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5B 
               
             
             
               
                   
               
               
                 Binding Data of some [3,2-Nal 11 ]NT(8-13) Analogs 
               
             
          
           
               
                   
                 hNTS1 
                 hNTS2 
                 NTS2 
               
             
          
           
               
                   
                 Compound 
                 K d  (nM) 
                   
                 Selectivity 
               
               
                   
                   
               
             
          
           
               
                   
                 NT33 
                   89 (human) 
                  18 (human) 
                 5 
               
               
                   
                 NT51 
                  1080 (human) 
                  33 (human) 
                 33 
               
               
                   
                 JMV510 
                   13 (rat) 
                 215 (mouse) 
                 0.06 
               
               
                   
                 JMV509 
                 23000 (rat) 
                 910 (mouse) 
                 25 
               
               
                   
                   
               
             
          
         
       
     
         [0050]    There is relatively high homology between the rodent receptors and the human receptors. Specifically, BLAST protein alignment analysis of the deduced amino acid sequences for hNTS1 and rNTS1 indicates 83% identity 89% positives. For hNTS2 and mNTS2, this analysis-shows these receptors to have 75% identity and 83% positives. (See Tatusova, T. A. et al. FEMS M ICROBIOL  L ETT  174:247-50 (1999)) 
         [0051]    Despite the relatively high homology, Dr. Richelson and collaborators showed previously and unexpectedly that compounds could bind with much higher affinity to rat NTS1 than to human NTS1. (See Cusack, B. et al. J B IOL  C HEM  271:15054-9 (1996)) In fact, one compound that contained L-3,1-Nal in the 11 position bound to the rat receptor 126 fold better than to the human receptor. Additionally, Dr. Richelson and collaborators have never found a compound that bound significantly better to the human receptor than to the rodent receptor. (See Pang, Y. P. et al. J B IOL  C HEM  271:15060-8 (1996) and Cusack, B. et al. J B IOL  C HEM  270:18359-66 (1995)) Because the binding studies in Dubuc et al. were performed with the molecularly cloned rat NTS1 and the molecularly clone mouse NTS2, it would not have been obvious from their studies that their results would correlate to studies with human molecularly cloned NTS1 and NTS2. Therefore, although in the present case, data are for compounds binding to NTS2, it can be argued strongly that it could not be predicted from the results with murine NTS2 (see Dubuc, I. et al. J N EUROSCI  19:503-10 (1999)) that any of the compounds tested by Dr. Richelson and colleagues would bind with higher affinity to the human receptor than to the rodent receptor. 
         [0052]    Table 5B lists the binding data for JMV 509 and NT51, both of which have D-3,2-Nal 11 , and JMV 510 and NT 33, both of which have L-3,2-Nal 11 . As described above, previous work found that for all compounds tested, no compound bound significantly better to human NTS1 than to rodent NTS1. Therefore, the results with NT33 and NT51 obtained with human NTS2 could not have been predicted from the results of Dubuc et al. with murine NTS2 and their 3,2-Nal substituted compounds. As seen in Table 5B, the affinities of NT33 and NT51 are much higher at hNTS2 than the affinities of JMV 510 and JMV 509 at mNTS2 (12 and 28 fold higher affinities compared, respectively, to their D- and L-Nal peptides). Although the NTS2 selectivity over NTS1 of JMV 509 (25 fold) is similar to that for NT51 (33 fold), JMV 509 has nearly 1 μM affinity for mNTS2, while NT51 has an affinity of 33 nM, which is nearly 30 fold higher affinity. Furthermore, changing from L- to D-3,2-Nal in our peptides (NT33 compared to NT51) caused less than a 2 fold decrease in affinity at NTS2. In contrast, this change in Dubuc&#39;s peptides caused a decrease of more than 4 fold. Finally, changing from L- to D-3,2-Nal in our peptides did not reverse the selectivity of our compounds for hNTS2, as it did for Dubuc et al. That is, both NT33 and NT51 are selective for NTS2 over NTS1, while only JMV 509 has that selectivity. 
         [0053]    The single property that predicts whether one of the NT(8-13) or NT(9-13) peptides has pharmacological effects in vivo upon injection outside of the brain or spinal cord is stability to degradation by plasma peptidases. As seen in  FIG. 4 , the results from this simple assay in which peptide was incubated in a test tube with either human ( FIG. 4 ) or rat (data not shown) plasma show that some of the peptides were much more stable than others. All peptides that were stable (half-lives&gt;100 h), such as NT66, NT67, NT69, NT72, and NT73, have either a blocked amino group (N-Methyl-Arg) or a D-amino in the 8 or 9 position (Table 4). Those that lack this feature, such as NT64 and NT65 (Table 4 and  FIG. 4 ) were rapidly degraded. 
         [0054]    Virtually all the peptides that had long half-lives in this assay cause their pharmacological effects in brain after administration outside the brain. Likewise, virtually all the short half-life compounds required direct administration into the brain to cause their effects. On this basis, it can be predicted that none of the highly selective compounds at hNTS2 will work by injection outside the brain. Therefore, NT79 and NT80 were designed based on the most selective compound NT50, the sequences for all of which are shown in Table 4. In binding studies with membrane preparations from cells expressing hNTS2, NT79 had a K d  of 22 nM (Table 2), close to that found for NT50 (17.3 nM, Table 3), both of which contain D-3,1-Nal 11  (Table 4). Additionally, in a single experiment with membrane preparations from cells expressing hNTS1, NT79 had a K d  of about 1800 nM, giving it a selectivity for hNTS2 of 82 (Table 2). Also, in a single experiment with membrane preparations from cells expressing hNTS1, NT80 had a K d  of about 2000 nM, similar to that for NT79. Furthermore, in two separate experiments with membrane preparations from cells expressing hNTS2, NT80 had a K d  of about 30 nM, giving it a selectivity for hNTS2 of 67 (Table 2). 
       Antinociceptive Testing 
       [0055]    Preliminary data on the pharmacological effects of NT79 and NT80 after intraperitoneal administration to mice (NT79 and NT80,  FIG. 5 ) or to rats (NT79 only,  FIGS. 6 and 7 ) was obtained. 
       Body Temperature Lowering 
       [0056]    At time “0” baseline readings were made. Afterwards, the mice were injected with a neurotensin analog compound (NT69, NT79, or NT80) and the first reading was taken 30 min after the injection. The thermistor probe was inserted 2 cm into the rectum for the measurement of body temperature. 
         [0057]    When injected into the brain, NT causes hypothermia, which indicates a central effect of this peptide on thermal regulation. (See Martin, G. E. et al. P EPTIDES  1:333-9 (1980)) NTS1 mediates the hypothermic effects of NT. (See Boules, M. et al. P EPTIDES  27:2523-33 (2006)) NT69, an L-neo-Trp NT(8-13) analog is non-selective for the NT-receptor subtypes and has a hypothermic effect. As seen in  FIG. 5 , administration of NT69 to the mice resulted in a significant change in body temperature (about 10° C. decrease). In contrast, the minimal effects of NT79 and NT80, which were administered at 10 times the dosage of that for NT69, suggest that these compounds have low affinity for NTS1, as we have found in preliminary binding studies (Table 2). Although these results with NT79 and NT80 could also mean that these compounds did not penetrate into brain, this is not consistent with the results of the antinociceptive studies shown in  FIGS. 6 and 7 . Assuming that these peptides penetrate into brain, these data support the binding data and again suggest that NT79 and NT80 bind weakly to NTS1 and together with the antinociceptive data ( FIGS. 6 and 7 ) have selectivity for NTS2. 
       Hot Plate Test 
       [0058]    The rats were administered 20 mg/kg of NT79 intraperitoneally. A metal plate (15×20 cm) was heated to 52.5° C. and surrounded by a transparent plastic cage. Baseline testing for the hot plate was measured for each rat immediately prior to the experiment. The latency between the time the rat was placed on the surface and the time it licked either of its hind paws was measured. Failure to respond in a 30 s period resulted in ending the trial and removing the rat from the plate to prevent tissue damage. Hot plate tests were scored as the percentage of Maximal Possible Effect (% MPE) and was calculated according to the following equation: 
         [0000]      %  MPE= 100×(test latency-baseline latency)/(cutoff time {30 s}-baseline latency). 
         [0000]    Analgesic compounds will result in higher % MPE. 
       Tail Flick Test 
       [0059]    The tail flick test also measures changes in nociceptive threshold to thermal stimulus. The rats were administered 20 mg/kg of NT79 intraperitoneally. The rat was placed in a restrainer. Water was heated to 52° C. (52-54° C.). The rat&#39;s tail was immersed in the heated water. The latency to flick the tail was recorded. A 10 sec cutoff period was used to prevent tissue damage. Antinociception was expressed as a percentage of the Maximal Possible Effect (MPE) % MPE=100×(test latency-baseline latency)/(cutoff time {10 s}-baseline latency). Analgesic compounds will result in higher % MPE. 
       Writhing Test 
       [0060]    The writhing test was used to measure changes in the nociceptive threshold to a chemical stimulus. The rats were administered 20 mg/kg of NT79 intraperitoneally. The rats were also injected with 0.5 ml of a 2% (v/v) aqueous solution of acetic acid and placed individually in clear plastic containers for observation. 
         [0061]    Behavioral Measure: The number of writhes was counted during a 60 min observation period. A writhe was defined as stretching of the hind limbs accompanied by a contraction of abdominal muscles. Analgesic compounds will result in lower number of writhes. 
         [0062]    As seen in  FIG. 6 , NT79 demonstrated antinociceptive effects in the tail flick assay, but not the hot plate test. Additionally, NT79 had a robust antinociceptive effect in the writhing pain model in rodents (see  FIG. 7 ). 
       Prostaglandin Levels 
       [0063]    Furthermore, evidence suggests that NTS1 also mediates hypotension. (See Schaeffer, P. et al. E UR  J P HARMACOL  323:215-21 (1997)) Therefore, NT79 and NT80 would also be expected to have minimal effects on blood pressure. In this regard, the release of prostacyclins may be related in part to the mechanism whereby NT causes hypotension. (See Schaeffer, P. et al. E UR  J P HARMACOL  323:215-21 (1997) and Ertl, G. et al. A M  J P HYSIOL  264:H1062-8 (1993)) Consequently, measurement of plasma prostacylin (or its stable metabolite, 6-keto-prostaglandin F 1α ) may be a surrogate marker for hypotension caused by NT and related compounds. Therefore, in preliminary studies, levels of 6-keto-prostaglandin F 1α  immunoreactivity were measured after injection of saline, NT69, or NT79 into mice ( FIG. 8 ). Consistent with the literature (See Schaeffer, P. et al. E UR  J P HARMACOL  323:215-21 (1997) and Ertl, G. et al. A M  J P HYSIOL  264:H1062-8 (1993)) and because it causes hypotension, NT69 markedly elevated plasma levels of prostaglandin. On the other hand, as seen in  FIG. 8 , NT79 had no effect on these levels, compared to the saline-injected animal. These data suggest that NT79 did not cause hypotension. 
       Additional Compounds 
       [0064]    The peptides listed in Tables 6A-D were designed to provide hNTS2-selectivity and stability to degradation by peptidases. Rules for this latter feature have come from extensive studies on NT(8-13) and NT(9-13) peptide analogs (e.g.,  FIG. 4 ). Additionally, it has been observed in binding studies with hNTS1 and hNTS2 with these analogs that tert-Leu reduces affinity of peptides at both receptors, but more so at hNTS1 than at hNTS2. Radioligand binding studies on hNTS1 and hNTS2 are performed on all the compounds using the protocol described previously. Additional pharmacological studies, including antinociceptive tests, are performed on those analogs showing selectivity for hNTS2. 
         [0065]    Peptides (about 30 mg of peptide (&gt;95%) purity) are synthesized using Fmoc chemistry with tBut, Boc, Mtr, or Pmc protected side chains, on an automated 433A peptide synthesizer (Perkin-Elmer/Applied Biosystems, Foster City, Calif.) or by simultaneous methods on an APEX 396 multiple peptide synthesizer (AAPPTEC). Protocols for activation, coupling times, amino acid dissolution, coupling solvents, and synthesis scales at either 40 or 100 μmol are followed according to the manufacturer&#39;s programs. The NT peptides are purified by reverse-phase HPLC using a semi-preparative C 18  column (2.2 cm×25 cm, Phenomenex, Hesperia, Calif.) in aqueous solutions of 0.1% trifluoroacetic acid and an aqueous gradient of 10%-60% acetonitrile in 0.1% trifluoroacetic acid. A combination of analytical reverse-phase HPLC and electrospray ionization (ESI) mass spectrometry (MSQ, ThermoFischer Scientific) was used to analyze peptide homogeniety and to confirm peptide molecular weight, respectively. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 6A 
               
             
             
               
                   
               
               
                 NT(8-13) and NT(9-13) D-3,1-Napthylalanine 11  Analogs 
               
             
          
           
               
                 Com- 
                 Sequence 
               
             
          
           
               
                 pound 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
               
               
                   
               
             
          
           
               
                 1 
                   
                 DAB 
                 L-Pro 
                 D-3,1-Nal 
                 L-Ile 
                 L-Leu 
               
               
                 2 
                   
                 DAB 
                 L-Pro 
                 D-3,1-Nal 
                 tert-Leu 
                 L-Leu 
               
               
                 3 
                   
                 D-Lys 
                 L-Pro 
                 D-3,1-Nal 
                 L-Ile 
                 L-Leu 
               
               
                 4 
                   
                 D-Lys 
                 L-Pro 
                 D-3,1-Nal 
                 tert-Leu 
                 L-Leu 
               
               
                 5 
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 D-3,1-Nal 
                 L-Ile 
                 L-Leu 
               
               
                 6 
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 D-3,1-Nal 
                 tert-Leu 
                 L-Leu 
               
               
                 7 
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 D-3,1-Nal 
                 L-Ile 
                 L-Leu 
               
               
                 8 
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 D-3,1-Nal 
                 tert-Leu 
                 L-Leu 
               
               
                 9 
                 N-methyl-Arg 
                 DAB 
                 L-Pro 
                 D-3,1-Nal 
                 L-Ile 
                 L-Leu 
               
               
                 10 
                 N-methyl-Arg 
                 DAB 
                 L-Pro 
                 D-3,1-Nal 
                 tert-Leu 
                 L-Leu 
               
               
                 11 
                 N-methyl-Arg 
                 D-Orn 
                 L-Pro 
                 D-3,1-Nal 
                 L-Ile 
                 L-Leu 
               
               
                 12 
                 N-methyl-Arg 
                 D-Orn 
                 L-Pro 
                 D-3,1-Nal 
                 tert-Leu 
                 L-Leu 
               
               
                 13 
                 N-methyl-Arg 
                 L-Lys 
                 L-Pro 
                 D-3,1-Nal 
                 L-Ile 
                 L-Leu 
               
               
                 14 
                 N-methyl-Arg 
                 L-Lys 
                 L-Pro 
                 D-3,1-Nal 
                 tert-Leu 
                 L-Leu 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 6B 
               
             
             
               
                   
               
               
                 NT(8-13) and NT(9-13) 
               
               
                 L-1,2,3,4-Tetrahydroisoquinoline-3-Carboxylic Acid 11  Analogs 
               
             
          
           
               
                 Com- 
                 Sequence 
               
             
          
           
               
                 pound 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
               
               
                   
               
               
                 15 
                   
                 DAB 
                 L-Pro 
                 L-TIC 
                 L-Ile 
                 L-Leu 
               
               
                 16 
                   
                 DAB 
                 L-Pro 
                 L-TIC 
                 tert-Leu 
                 L-Leu 
               
               
                 17 
                   
                 D-Lys 
                 L-Pro 
                 L-TIC 
                 L-Ile 
                 L-Leu 
               
               
                 18 
                   
                 D-Lys 
                 L-Pro 
                 L-TIC 
                 tert-Leu 
                 L-Leu 
               
               
                 19 
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 L-TIC 
                 L-Ile 
                 L-Leu 
               
               
                 20 
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 L-TIC 
                 tert-Leu 
                 L-Leu 
               
               
                 21 
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 L-TIC 
                 L-Ile 
                 L-Leu 
               
               
                 22 
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 L-TIC 
                 tert-Leu 
                 L-Leu 
               
               
                 23 
                 N-methyl-Arg 
                 DAB 
                 L-Pro 
                 L-TIC 
                 L-Ile 
                 L-Leu 
               
               
                 24 
                 N-methyl-Arg 
                 DAB 
                 L-Pro 
                 L-TIC 
                 tert-Leu 
                 L-Leu 
               
               
                 25 
                 N-methyl-Arg 
                 D-Orn 
                 L-Pro 
                 L-TIC 
                 L-Ile 
                 L-Leu 
               
               
                 26 
                 N-methyl-Arg 
                 D-Orn 
                 L-Pro 
                 L-TIC 
                 tert-Leu 
                 L-Leu 
               
               
                 27 
                 N-methyl-Arg 
                 L-Lys 
                 L-Pro 
                 L-TIC 
                 L-Ile 
                 L-Leu 
               
               
                 28 
                 N-methyl-Arg 
                 L-Lys 
                 L-Pro 
                 L-TIC 
                 tert-Leu 
                 L-Leu 
               
               
                   
               
               
                 DAB = diaminobutyric acid; 
               
               
                 tert-Leu = tertiary leucine; 
               
               
                 D-Orn = D-Ornithine 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 6C 
               
             
             
               
                   
               
               
                 NT(8-13) and NT(9-13) L-Alanine 11  Analogs 
               
             
          
           
               
                   
                 Sequence 
               
             
          
           
               
                 Compound 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
               
               
                   
               
               
                 29 
                   
                 DAB 
                 L-Pro 
                 L-Ala 
                 L-Ile 
                 L-Leu 
               
               
                 30 
                   
                 DAB 
                 L-Pro 
                 L-Ala 
                 tert-Leu 
                 L-Leu 
               
               
                 31 
                   
                 D-Lys 
                 L-Pro 
                 L-Ala 
                 L-Ile 
                 L-Leu 
               
               
                 32 
                   
                 D-Lys 
                 L-Pro 
                 L-Ala 
                 tert-Leu 
                 L-Leu 
               
               
                 33 
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 L-Ala 
                 L-Ile 
                 L-Leu 
               
               
                 34 
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 L-Ala 
                 tert-Leu 
                 L-Leu 
               
               
                 35 
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 L-Ala 
                 L-Ile 
                 L-Leu 
               
               
                 36 
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 L-Ala 
                 tert-Leu 
                 L-Leu 
               
               
                 37 
                 N-methyl-Arg 
                 DAB 
                 L-Pro 
                 L-Ala 
                 L-Ile 
                 L-Leu 
               
               
                 38 
                 N-methyl-Arg 
                 DAB 
                 L-Pro 
                 L-Ala 
                 tert-Leu 
                 L-Leu 
               
               
                 39 
                 N-methyl-Arg 
                 D-Orn 
                 L-Pro 
                 L-Ala 
                 L-Ile 
                 L-Leu 
               
               
                 40 
                 N-methyl-Arg 
                 D-Orn 
                 L-Pro 
                 L-Ala 
                 tert-Leu 
                 L-Leu 
               
               
                 41 
                 N-methyl-Arg 
                 L-Lys 
                 L-Pro 
                 L-Ala 
                 L-Ile 
                 L-Leu 
               
               
                 42 
                 N-methyl-Arg 
                 L-Lys 
                 L-Pro 
                 L-Ala 
                 tert-Leu 
                 L-Leu 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 6D 
               
             
             
               
                   
               
               
                 NT(8-13) and NT(9-13) D-neo-Trp 11  Analogs 
               
             
          
           
               
                 Com- 
                 Sequence 
               
             
          
           
               
                 pound 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
               
               
                   
               
               
                 43 
                   
                 DAB 
                 L-Pro 
                 D-neo-Trp 
                 L-Ile 
                 L-Leu 
               
               
                 44 
                   
                 DAB 
                 L-Pro 
                 D-neo-Trp 
                 tert-Leu 
                 L-Leu 
               
               
                 45 
                   
                 D-Lys 
                 L-Pro 
                 D-neo-Trp 
                 L-Ile 
                 L-Leu 
               
               
                 46 
                   
                 D-Lys 
                 L-Pro 
                 D-neo-Trp 
                 tert-Leu 
                 L-Leu 
               
               
                 47 
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 D-neo-Trp 
                 L-Ile 
                 L-Leu 
               
               
                 48 
                 D-Lys 
                 L-Arg 
                 L-Pro 
                 D-neo-Trp 
                 tert-Leu 
                 L-Leu 
               
               
                 49 
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 D-neo-Trp 
                 L-Ile 
                 L-Leu 
               
               
                 50 
                 L-Arg 
                 D-Orn 
                 L-Pro 
                 D-neo-Trp 
                 tert-Leu 
                 L-Leu 
               
               
                 51 
                 N-methyl-Arg 
                 DAB 
                 L-Pro 
                 D-neo-Trp 
                 L-Ile 
                 L-Leu 
               
               
                 52 
                 N-methyl-Arg 
                 DAB 
                 L-Pro 
                 D-neo-Trp 
                 tert-Leu 
                 L-Leu 
               
               
                 53 
                 N-methyl-Arg 
                 D-Orn 
                 L-Pro 
                 D-neo-Trp 
                 L-Ile 
                 L-Leu 
               
               
                 54 
                 N-methyl-Arg 
                 D-Orn 
                 L-Pro 
                 D-neo-Trp 
                 tert-Leu 
                 L-Leu 
               
               
                 55 
                 N-methyl-Arg 
                 L-Lys 
                 L-Pro 
                 D-neo-Trp 
                 L-Ile 
                 L-Leu 
               
               
                 56 
                 N-methyl-Arg 
                 L-Lys 
                 L-Pro 
                 D-neo-Trp 
                 tert-Leu 
                 L-Leu 
               
               
                   
               
               
                 DAB = diaminobutyric acid; 
               
               
                 tert-Leu = tertiary leucine; 
               
               
                 D-Orn = D-Ornithine 
               
             
          
         
       
     
         [0066]    Radioligand binding studies are performed as detailed above to determine the equilibrium dissociation constants (K d ) for the additional compounds for NTS1 and NTS2 to determine which compounds have selectivity for NTS2. Additionally, stability tests with plasma peptidases, prostaglandin level tests, and antinociceptive tests are performed as described above. 
         [0067]    Although the foregoing invention has, for the purposes of clarity and understanding, been described in some detail by way of illustration and example, it will be obvious that certain changes and modifications may be practiced which will still fall within the scope of the appended claims.