Patent Application
20090062212
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.
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
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
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)).
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
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
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
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
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 Kd (equilibrium dissociation constant) at hNTS1 and the log Kd 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.
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
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
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
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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'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.
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
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 (>30 fold higher affinity) at one or the other receptor.
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
CHO—K1 cells that had been stably transfected separately with the hNTS1 or hNTS2 genes were cultured in 150 mm (500 cm2) Petri plates with 35 ml of Dulbecco's modified Eagle'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.
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
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
The values presented for equilibrium dissociation constants are expressed as the geometric means±S.E.M. (See Fleming, W. W. et al. J P
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-Trp11]NT(8-13), abbreviated as NT64, with a Kd=0.09 nM at hNTS1 and 0.32 nM at hNTS2. Nine analogs had sub-nanomolar Kd's at hNTS1, the data for some of which were reported previously (Table 2). (See Cusack, B. et al. J B
An example of some competition binding curves for compounds at hNTS2, expressed by CHO—K1 cells, is shown in
There was a strong correlation between the log Kd at hNTS1 and the log Kd at hNTS2 (y=0.76x-1.75, R=0.84, P<0.0001) for the peptides (
In the present series of peptides, about one-half of the compounds had essentially the same affinities for both hNTS1 and hNTS2 (see
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
NT50, [D-3,1-Nal11]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
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).
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.
Dubuc et al. described [3,2-Nal11]NT(8-13) analogs (JMV509 and JMV510) that showed some selectivity for NTS2 receptors (non-human). (See Dubuc, I. et al. J N
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
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
Table 5B lists the binding data for JMV 509 and NT51, both of which have D-3,2-Nal11, and JMV 510 and NT 33, both of which have L-3,2-Nal11. 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'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.
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
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 Kd of 22 nM (Table 2), close to that found for NT50 (17.3 nM, Table 3), both of which contain D-3,1-Nal11 (Table 4). Additionally, in a single experiment with membrane preparations from cells expressing hNTS1, NT79 had a Kd 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 Kd of about 2000 nM, similar to that for NT79. Furthermore, in two separate experiments with membrane preparations from cells expressing hNTS2, NT80 had a Kd of about 30 nM, giving it a selectivity for hNTS2 of 67 (Table 2).
Preliminary data on the pharmacological effects of NT79 and NT80 after intraperitoneal administration to mice (NT79 and NT80,
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.
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
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:
% MPE=100×(test latency-baseline latency)/(cutoff time {30 s}-baseline latency).
Analgesic compounds will result in higher % MPE.
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'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.
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.
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.
As seen in
Furthermore, evidence suggests that NTS1 also mediates hypotension. (See Schaeffer, P. et al. E
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.,
Peptides (about 30 mg of peptide (>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's programs. The NT peptides are purified by reverse-phase HPLC using a semi-preparative C18 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.
Radioligand binding studies are performed as detailed above to determine the equilibrium dissociation constants (Kd) 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.
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.
