Patent Application
20180127465
The present invention relates to ligands for mu, delta, and kappa opioid receptors, more particularly to multifunctional opioid peptides that function as mu opioid receptor (MOR), delta opioid receptor (DOR) agonists, and kappa opioid receptor (KOR) antagonists (or partial agonists). The present invention also relates to treating pain or other conditions using the multifunctional opioid peptides herein. The present invention also relates to pharmacophores for modifying C-terminal regions of opioid peptides (e.g., enkephalins, DALDA, EM-1, EM-2, etc.) for conferring particular KOR activity.
Opioids are commonly used in the treatment of severe pain. Opioids have analgesic activity through their interaction with the opioid receptors (e.g., mu (μ) opioid receptor (MOR), delta (δ) opioid receptor (DOR), kappa (κ) opioid receptor (KOR)), mostly with MOR. However, the clinical use of opioids is limited by associated side effects such as respiratory depression, constipation, development of tolerance, and addiction. Indeed, chronic pain and subsequence chronic administration of a MOR agonist can lead to KOR activation, which results in undesirable adverse and addictive behaviors. For this reason, a KOR antagonist (or partial agonist) could be used to reduce such undesirable effects of chronic MOR activation.
Inventors have surprisingly discovered opioid peptides, e.g., opioid receptor ligands (ORLs) that are multifunctional, e.g., acting as MOR agonists, DOR agonists, and KOR antagonists (or partial agonists). Without wishing to limit the present invention to any theory or mechanism, it is believed that this MOR/DOR agonist with KOR antagonist/partial agonist activity encompassed by a single molecule may be better and/or more effective than using co-administration of two or more molecules to achieve MOR/DOR agonist and KOR antagonist/partial agonist activity.
In some embodiments, the multifunctional ORLs may comprise peptide analogs derived from enkephalins. Enkephalins are pentapeptides (peptides containing 5 amino acids) that are endogenous ligands of the opioid receptors (e.g., MOR, and DOR). There are two known forms of enkephalins: leucine-containing enkephalin (Leu-Enk, or YGGFL (SEQ ID NO: 1)) and methionine-containing enkephalin (Met-Enk, or YGGFM (SEQ ID NO: 2)). In some embodiments, the multifunctional ORLs comprise peptide analogs derived from endomorphin-1 (EM-1), endomorphin-2 (EM-2) or other opioid ligands such as DALDA, FE20066, etc.
In some embodiments, the ORLs comprise a 4-anilidopiperidine moiety, e.g., fentanyl analog, an analog of a 4-anilidopiperidine, etc., e.g., N-phenyl-N-piperidin-4-ylpropionamide (Ppp).
The present invention also provides C-terminal modifications (pharmacophores) that confer KOR antagonist activity to opioid peptides. The present invention also provides modifications, such as halogenation of a phenylalanine of a Ppp moiety, that confer KOR antagonist or partial agonist activity. Those kappa activities may help reduce KOR- or MOR-related side effects. For example modifications of opioid ligands (such as DALDA, EM-1, EM-2, and FE20066) with pharmacophores (e.g., the C-terminal modifications of the aforementioned molecules) may generate the same KOR activity.
For example, the present invention provides ORLs with a Ppp tail, wherein the Ppp comprises an R group (e.g., a halogen).
The present invention features multifunctional opioid receptor ligands (ORLs) and methods of use of said multifunctional ORLs.
In certain embodiments, the ORLs herein have agonist activity at mu opioid receptor (MOR), agonist activity at delta opioid receptor (DOR), and antagonist activity at kappa opioid receptor (KOR). In certain embodiments, the ORLs herein have agonist activity at mu opioid receptor (MOR), agonist activity at delta opioid receptor (DOR), and partial agonist activity at kappa opioid receptor (KOR).
The present invention provides multifunctional opioid receptor ligands (ORLs) according to Formula 1: Aaa-Bbb-Ccc-Ddd(X)-Eee.
In some embodiments, Aaa is selected from 2′-6′-dimethyltyrosine (Dmt), Tyrosine (Tyr), Tmt, Phe, Dmp, and Mdp; Bbb is selected from D-Alanine (D-Ala), Alanine (Ala), D-Norleucine (D-Nle), Norleucine (Nle), Proline (Pro), Arginine (Arg), D-Arginine (D-Arg), tetrahydroisoquinoline-3-carboxylic acid (Tic), and D-Tic; Ccc is selected from Gly, Phenylalanine(X) (Phe(X)), Trp, and naphthylalanine (Nal) or is absent; Ddd(X) is Gly, Phe(X), Trp, Nal, or Lys; and Eee comprises N-phenyl-N-piperidin-4-ylpropionamide-R (Ppp(R)); wherein X and R both comprise a halogen. In some embodiments, X is selected from H, F, Cl, and Br. In some embodiments, X is selected from H, F, and Cl. In certain embodiments, R is selected from 3-Cl, 4-Cl, 3-F, 4-F, and 2,4-diCl.
In some embodiments, Aaa is selected from 2′-6′-dimethyltyrosine (Dmt), Tyrosine (Tyr), Tmt, Phe, Dmp, and Mdp; Bbb is selected from D-Alanine (D-Ala), D-Norleucine (D-Nle), and D-Tic; Ccc is selected from Gly, Phenylalanine(X) (Phe(X)), Trp, and naphthylalanine (Nal) or is absent; Ddd(X) is Gly, Phe(X), Trp, Nal, or Lys; and Eee comprises N-phenyl-N-piperidin-4-ylpropionamide-R (Ppp(R)); wherein X and R both comprise a halogen. In some embodiments, X is selected from H, F, Cl, and Br. In some embodiments, X is selected from H, F, and Cl. In certain embodiments, R is selected from 3-Cl, 4-Cl, 3-F, 4-F, and 2,4-diCl.
In some embodiments, Aaa is selected from Tmt, Phe, Dmp, and Mdp; Bbb is selected from D-Alanine (D-Ala), Alanine (Ala), D-Norleucine (D-Nle), Norleucine (Nle), Proline (Pro), Arginine (Arg), D-Arginine (D-Arg), tetrahydroisoquinoline-3-carboxylic acid (Tic), and D-Tic; Ccc is selected from Gly, Phenylalanine(X) (Phe(X)), Trp, and naphthylalanine (Nal) or is absent; Ddd(X) is Gly, Phe(X), Trp, Nal, or Lys; and Eee comprises N-phenyl-N-piperidin-4-ylpropionamide-R (Ppp(R)); wherein X and R both comprise a halogen. In some embodiments, X is selected from H, F, Cl, and Br. In some embodiments, X is selected from H, F, and Cl. In certain embodiments, R is selected from 3-Cl, 4-Cl, 3-F, 4-F, and 2,4-diCl.
In some embodiments, Aaa is selected from 2′-6′-dimethyltyrosine (Dmt), Tyrosine (Tyr), Tmt, Phe, Dmp, and Mdp; Bbb is selected from Alanine (Ala), Norleucine (Nle), Proline (Pro), Arginine (Arg), tetrahydroisoquinoline-3-carboxylic acid (Tic); Ccc is selected from Gly, Phenylalanine(X) (Phe(X)), Trp, and naphthylalanine (Nal) or is absent, wherein X is a halogen; Ddd(X) is Gly, Phe(X), Trp, Nal, or Lys, wherein X is a halogen; and Eee is a 4-anilidopiperidine moiety (e.g., Ppp). In some embodiments, Ppp comprises Ppp(R), wherein R comprises a halogen. In some embodiments, R is selected from 3-Cl, 4-Cl, 3-F, 4-F, and 2,4-diCl. In some embodiments, X is selected from H, F, Cl, and Br.
The present invention also provides multifunctional opioid receptor ligands (ORLs) according to a Formula 3: Aaa-Bbb-Ccc-Phe(Br)-Eee.
In certain embodiments, Aaa is selected from 2′-6′-dimethyltyrosine (Dmt), Tyrosine (Tyr), Tmt, Phe, Dmp, and Mdp; Bbb is selected from D-Alanine (D-Ala), Alanine (Ala), D-Norleucine (D-Nle), Norleucine (Nle), Proline (Pro), Arginine (Arg), D-Arginine (D-Arg), tetrahydroisoquinoline-3-carboxylic acid (Tic), and D-Tic; Ccc is selected from Gly, Phenylalanine(X) (Phe(X)), Trp, and naphthylalanine (Nal) or is absent, wherein X is a halogen; and Eee is a 4-anilidopiperidine moiety (e.g., Ppp). In some embodiments, Ppp comprises Ppp(R), wherein R comprises a halogen. In some embodiments, R is selected from 3-Cl, 4-Cl, 3-F, 4-F, and 2,4-diCl. In some embodiments, X is selected from H, F, Cl, and Br.
In certain embodiments, Aaa is selected from 2′-6′-dimethyltyrosine (Dmt), Tyrosine (Tyr); Bbb is selected from D-Alanine (D-Ala), Alanine (Ala), D-Norleucine (D-Nle), Norleucine (Nle), Proline (Pro), Arginine (Arg), D-Arginine (D-Arg), tetrahydroisoquinoline-3-carboxylic acid (Tic), and D-Tic; Ccc is selected from Gly, Phenylalanine(X) (Phe(X)), Trp, and naphthylalanine (Nal) or is absent, wherein X is a halogen; and Eee is a 4-anilidopiperidine moiety (e.g., Ppp). In some embodiments, Ppp comprises Ppp(R), wherein R comprises a halogen. In some embodiments, R is selected from 3-Cl, 4-Cl, 3-F, 4-F, and 2,4-diCl. In some embodiments, X is selected from H, F, Cl, and Br.
The present invention also provides multifunctional opioid receptor ligands (ORLs) according to Formula 4: Aaa-Bbb-Ccc-Ddd(X)Yyy(n)-Eee.
In some embodiments, Aaa is selected from 2′-6′-dimethyltyrosine (Dmt), Tyrosine (Tyr), Tmt, Phe, Dmp, and Mdp; Bbb is selected from D-Alanine (D-Ala), D-Norleucine (D-Nle), Proline (Pro), and D-Arginine (D-Arg), tetrahydroisoquinoline-3-carboxylic acid (Tic), D-Tic; Ccc is selected from Gly, Phenylalanine(X) (Phe(X)), Trp, and naphthylalanine (Nal) or is absent, wherein X is a halogen; Ddd(X) is Gly, Phe(X), Trp, Nal, or Lys, wherein X is a halogen; Yyy is selected from one or a combination of Leu, Arg, Met, Lys, or lie; and Eee is a 4-anilidopiperidine moiety (e.g., Ppp). In some embodiments, Ppp comprises Ppp(R), wherein R comprises a halogen. In some embodiments, R is selected from 3-Cl, 4-Cl, 3-F, 4-F, and 2,4-diCl. In some embodiments, X is selected from H, F, Cl, and Br. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In some embodiments, n is 8 or more, e.g., 9, 10, 11, 12, 13, etc.
The present invention also provides multifunctional opioid receptor ligands (ORLs) according to Formula 5: Aaa-DArg-Ccc-Ddd-Eee.
In some embodiments, Aaa is selected from Tyr or 2′-6′-dimethyltyrosine (Dmt); Ccc is selected from Phe, Phe(X), or 1-naphthylalanine (1Nal); Ddd is selected from Lys, Gly or is absent; Eee is a 4-anilidopiperidine moiety; and X is selected from F, Cl, or Br. In some embodiments, the 4-anilidopiperidine moiety comprises Ppp. In some embodiments, Ppp comprises Ppp(R), wherein R comprises a halogen. In some embodiments, R is selected from 3-Cl, 4-Cl, 3-F, 4-F, and 2,4-diCl. In some embodiments, the ORL is SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO; 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 42, or SEQ ID NO: 43.
The present invention also provides multifunctional opioid receptor ligands (ORLs) according to Formula 6: Aaa-Pro-Ccc-Phe(X)-Eee.
In some embodiments, Aaa is selected from Tyr or 2′-6′-dimethyltyrosine (Dmt); Ccc is selected from Trp, Phe, Gly, or Phe(X); Eee is a 4-anilidopiperidine moiety (e.g., Ppp), and X is selected from F, Cl, or Br. In some embodiments, Ppp comprises Ppp(R), wherein R comprises a halogen. In some embodiments, R is selected from 3-Cl, 4-Cl, 3-F, 4-F, and 2,4-diCl. In some embodiments, the ORL is selected from SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30.
The present invention also provides multifunctional opioid receptor ligands (ORLs) according to Formula 7: DPhe-DPhe-DNle-Ddd-Eee.
In some embodiments, Ddd is selected from D-Arg or D-Lys, and Eee is a 4-anilidopiperidine moiety (e.g., Ppp). For example, in some embodiments, Ddd is D-Arg and Eee is Ppp. In some embodiments, Ddd is D-Lys and Eee is Ppp. In some embodiments, Ppp comprises Ppp(R), wherein R comprises a halogen. For example, in some embodiments, Ddd is D-Arg and Eee is Ppp(R), wherein R comprises a halogen (e.g., Cl, F, Br). In some embodiments, Ddd is D-Lys and Eee is Ppp(R), wherein R comprises a halogen (e.g., Cl, F, Br). In some embodiments, R is selected from 3-Cl, 4-Cl, 3-F, 4-F, and 2,4-diCl. In some embodiments, the ORL is according to SEQ ID NO: 44.
The present invention also provides an opioid receptor ligand dimer according to SEQ ID NO: 19. The present invention also provides an opioid receptor ligand dimer according to SEQ ID NO: 20.
The present invention also provides methods of reducing pain, e.g., reducing pain in a subject in need of a KOR antagonist or KOR partial agonist. In some embodiments, the method comprises identifying a subject in need of a kappa opioid receptor (KOR) antagonist or partial agonist; and introducing to the subject a multifunctional ORL according the present invention, wherein the ORL is effective for reducing pain.
The present invention also features methods of blocking kappa opioid receptor. In some embodiments, the method comprises introducing to the KOR a multifunctional ORL according to the present invention.
The present invention also features methods of blocking KOR, activating MOR, and activating DOR in a subject. In some embodiments, the method comprises introducing to the subject a multifunctional ORL according to the present invention.
Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.
This patent application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The present invention features multifunctional opioid receptor ligands (ORLs), acting as MOR agonists, DOR agonists, and KOR antagonists (or partial agonists). The present invention also features methods of use of said multifunctional ORLs, e.g., methods of treating pain or other conditions using peptides of the present invention.
The ORLs of the present invention comprise a peptide portion, e.g., a peptide analog derived from enkephalins (e.g., Leu-Enk (YGGFL, SEQ ID NO: 1) or Met-Enk (YGGFM, SEQ ID NO: 2)) and a tail portion linked to the C-terminus of the peptide portion. In some embodiments, the peptide portion comprises four residues (e.g., amino acids, analogs or derivatives thereof), occupying position 1, 2, 3, and 4. In some embodiments, the peptide portion comprises three residues (e.g., amino acids, analogs or derivatives thereof), occupying position 1, 2, and 4. The peptide portion may be based on the enkephalin sequence e.g., Leu-Enk (YGGFL, SEQ ID NO: 1) or Met-Enk (YGGFM, SEQ ID NO: 2).
In some embodiments, the tail portion comprises a lipophilic molecule (e.g., a 4-anilidopiperidine moiety), e.g., the tail portion may comprise a residue or compound that increases the lipophilicity of the peptide portion. In some embodiments, the tail comprises a N-phenyl-N-piperidin-4-ylpropionamide (Ppp) moiety. In some embodiments, the tail comprises —NH2. Other non-limiting examples of tail portion molecules (tail compounds) are shown in
Various non-limiting examples of formulas are presented herein for ORLs. For example, the present invention provides ORLs according to Formula 1 (Aaa-DBbb-Ccc-Ddd(X)-Eee). In some embodiments, Aaa is selected from 2′-6′-dimethyltyrosine (Dmt) and Tyrosine (Tyr). In some embodiments, D-Bbb is selected from D-Alanine (D-Ala), D-Norleucine (D-Nle), Proline (Pro), and D-Arginine (D-Arg); In some embodiments, Ccc is selected from Gly, Phenylalanine(X) (Phe(X)), and naphthylalanine (Nal) or is absent. In some embodiments, Ddd(X) is Gly, Phe(X), or Lys. Eee is a tail portion, wherein the tail portion is lipophilic. In some embodiments, X is Br. In some embodiments, X is selected from H, F, Cl, and Br. In some embodiments, Eee is selected from —NH2 and a 4-anilidopiperidine moiety. In some embodiments, the 4-anilidopiperidine moiety comprises N-phenyl-N-piperidin-4-ylpropionamide (Ppp). The present invention is not limited to Formula 1. Dmt refers to 2′-6′-dimethyltyrosine, DXxx refers to a D amino acid, and X refers to a halogen or other appropriate compound, e.g., H, Cl, F, or a methyl group. N-phenyl-N-piperidin-4-ylpropionamide may be abbreviated as Ppp. In some embodiments, residue 1 (e.g., Dmt, Aaa, etc.) comprises Dmt or Tyr. In some embodiments, residue 2 (DXxx, Bbb, etc.) comprises DAla, DNle (D-norleucine), Pro, or DArg. In some embodiments, residue 3 (Gly, Ccc, etc.) comprises Gly, Phe, Phe(X), or Nal, wherein X may refer to H, Cl, F, methyl group, or any other appropriate modification of Phe. In some embodiments, residue 3 is absent. In some embodiments, residue 4 (Phe(X), Ddd, etc.) comprises Gly, Phe, Phe(X), wherein X may refer to H, Cl, F, methyl group, or any other appropriate modification of Phe. In some embodiments, the tail of the ORL comprises Ppp or NH2. The present invention is not limited to the aforementioned formula molecules. For reference, DTic refers to D-tetrahydroisoquinoline-3-carboxylic acid.
Table 1 below shows non-limiting examples of ORLs of the present invention. Note that the Phe residues in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 are halogenated with F, and the Phe residue in SEQ ID NO: 15 is halogenated with Cl.
Note MR111 comprises two units of SEQ ID NO; 19, e.g., MR111 comprises (Dmt-DNle-Homocys-Phe(4-F)-Ppp)2. MR112 comprises two units of SEQ ID NO: 20, e.g., MR112 comprises (Dmt-Homocys-Gly-Phe(4-F)-Ppp)2.
The ORLs of the present invention may be synthesized as appropriate (see, for example, Lee et al., 2011, J. Med. Chem. 54:382-886). For example, the ORLs of the present invention may be synthesized by a protocol for liquid phase peptide synthesis (LPPS), e.g., using Boc-chemistry in high yields. In some embodiments, halogen modification on the aromatic ring is on the para position, e.g., to help avoid unfavorable steric hindrance.
Table 2 shows analytical data of various multifunctional ORLs of the present invention with a Ppp group at the C-terminus. aFAB-MS (JEOL HX110 sector instrument) or MALDI-TOF. bPerformed on a Hewlett Packard 1100 [C-18, Vydac, 4.6 mm×250 mm, 5 μm, 10-100% of acetonitrile containing 0.1% TFA within 45 min, 1 mL/min]. chttp://www.vcclab.org/lab/alogps/. dLow resolution-Mass. ° Retention time. n.d. not determined.
As show n Table 3, Table 4.1, Table 4.2, and
a[total bound-basal]/[basal-nonspecific] × 100.
bRelative % of 10 μM U50,488 stimulation.
cRelative % of naloxone blocking 100 nM U50,488 stimulation,
a[total bound-basal]/[basal-nonspecific] × 100.
bRelative % of 10 μM U50,488 stimulation.
cRelative % of naloxone blocking 100 nM U50,488 stimulation.
Preliminary in vivo studies of LYS739 (SEQ ID NO: 10) showed that intrathecal (i.th.) administration of LYS739 (SEQ ID NO: 10) at 10 μg/5 μl in L5/L6 SNL-operated male SD rats can reverse thermal hyperalgesia in nerve injured animals and reverse tactile allodynia. For example,
For reference, Table 5 lists examples of ORLs with various tail portions (e.g., NH2 and Tail Compounds 1-5). Structures of the Tails (e.g., anilidopiperidine moieties) can be found in
The present invention also features ORLs that are derived from LYS739 (SEQ ID NO: 10), e.g., LYS739 analogs. In some embodiments, the ORLs are obtained by modifying LYS739 (SEQ ID NO: 10) by substitution, dimerization, and/or cyclization. Modifications may involve the incorporation of an unnatural amino acid and/or constrained amino acids. For example, in some embodiments, Dmt is substituted with trimethyltyrosine (Tmt). In some embodiments, the ORL comprises 2-methyl-3-(2′,6′-dimethyl-4′-hydroxyphenyl)-propionic acid (Mdp).
In some embodiments, the ORL comprises a bivalent ligand. In some embodiments, a disulfide bond is used to link two monomeric pharmacophores. For example, a disulfide bond may be used through a homocysteine residue at position 2 (or 3). In some embodiments, ORLs comprise cyclic structures, e.g., the ORLs are cyclic and retain the pharmacophoric structure for the receptors within a constrained structure, e.g., since linear peptide ligands can be flexible even with multiple modifications due to high flexibility of enkephalins. Cyclization may be through the formation of various bonds such as a disulfide and a lactam, but is not limited to these mechanisms.
In some embodiments, the ORLs are bifunctional ligands. In some embodiments, the ORLs are trifunctional ligands. In some embodiments, ORLs are constructing based on an enkephalin tetrapeptide (Tyr-Gly-Gly-Phe-NH2, SEQ ID NO: 46). In some embodiments, ORLs are constructed using endomorphin-1 (EM-1) and/or DALDA (D-Arg2, Lys4]dermorphin. The present invention features ORL designs using EM-1 (Tyr-Pro-Trp-Phe-NH2, SEQ ID NO: 35) and DALDA (Tyr-DArg-Phe-Lys-NH2, SEQ ID NO: 36). The present invention also features ORLs using endomorphin-2 (EM-2) (Tyr-DArg-Phe-Lys-NH2, SEQ ID NO: 45).
Various ORLs (e.g., analogs of LYS739 (SEQ ID NO: 10)) were tested for their binding affinities at MOR, DOR, and KOR using [3H]-Diprenorphine in the membranes of Chinese Hamster Ovary (CHO) cells expressing the relevant human opioid receptor. Analogues with particular binding affinity (Ki<10 nM for MOR and DOR; Ki<30 nM for KOR) as well as others were tested for receptor functional activity in the [3S]-GTPγS assay. In this assay, antagonist activity at all three receptors expressed in CHO cells were determined by the inhibition of stimulation caused by 100 nM of control agonist (DAMGO for MOR, SNC80 for DOR, U50,488 for KOR) in a 96-well plate. Table 6 summarized in vitro biological activities of multifunctional ligands at MOR, DOR, and KOR with a Ppp group at the C-terminus. (Note: a=Competition analyses were carried out using membrane preparations from transfected HNB9.10 cells that constitutively expressed the respective receptor types; b=[3H]DAMGO, Kd=0.85 nM; c=[3H]DPDPE, Kd=0.50 nM; d=[3H]U69,593, Kd=5.3 nM; e=Expressed in CHO cells; f=Mean±SEM of the % relative to 10 μM U50,488 stimulation; g=Mean±SEM of the % relative to 10 μM naloxone inhibition of 100 nM U50,488; h=at 10 μM.
70h
Analogues were tested for their activity at KOR, and GTPγS assays were performed at the MOR and DOR for LYS739 (SEQ ID NO: 10) and LYS744 (SEQ ID NO: 15) (see
The present invention also features ORLs having half lives longer than 4 hours. For example, in some embodiments, the ORL has a half life longer than 1 hour. In some embodiments, the ORL has a half life longer than 2 hours. In some embodiments, the ORL has a half life longer than 3 hours. In some embodiments, the ORL has a half life longer than 4 hours. In some embodiments, the ORL has a half life longer than 5 hours. In some embodiments, the ORL has a half life longer than 10 hours. In some embodiments, the ORL has a half life longer greater than 24 hours.
In some embodiments, the ORL is 4 amino acids in length. In some embodiments, the ORL is 5 amino acids in length. In some embodiments, the ORL is 6 amino acids in length. In some embodiments, the ORL is 7 amino acids in length. In some embodiments, the ORL is 8 amino acids in length. In some embodiments, the ORL is 9 amino acids in length. In some embodiments, the ORL is 10 amino acids in length. In some embodiments, the ORL is more than 10 amino acids in length.
In some embodiments, the ORL is between 4 to 6 amino acids in length. In some embodiments, the ORL is between 4 to 7 amino acids in length. In some embodiments, the ORL is between 4 to 8 amino acids in length. In some embodiments, the ORL is between 4 to 9 amino acids in length. In some embodiments, the ORL is between 4 to 10 amino acids in length. In some embodiments, the ORL is between 4 to 20 amino acids in length. In some embodiments, the ORL is between 4 to 30 amino acids in length. In some embodiments, the ORL is between 4 to 40 amino acids in length. In some embodiments, the ORL is between 4 to 50 amino acids in length.
As shown in
Examples of other enkephalin analogues may include but are not limited to those shown in
In some embodiments, competitive radioligand binding assays and cell based functional assays are performed. In some embodiments, compounds with a binding affinity of about Ki<100 nM for MOR, DOR and KOR may be tested for receptor functional activity in a cyclic AMP assay. Compounds that show partial agonist (EC50<100 nM, Emax<40%) or antagonist activity (IC50<100 nM, Imax>60%) at the KOR and agonist activity (EC50<100 nM, Emax>70%) at the MOR and DOR in the cyclic AMP assay may be used for off-target screening. In some embodiments, binding affinity (K) will be determined by radioligand competition analysis using [3H]Diprenorphine for MOR, DOR, and KOR, in cell membrane preparations from stably transfected CHO cells expressing respective receptor types.
In some embodiments, cAMP accumulation may be measured. As a non-limiting example, in some embodiments, MOR, DOR, and KOR-CHO cells as above may be plated in 96 well culture microplates, and recovered overnight. The cells may then be serum starved for 20 minutes in serum free medium with 500 μM IBMX, followed by 15 minutes of treatment with 500 μM IBMX, 100 μM forskolin, and concentration curves of experimental drug or reference agonist (DAMGO for MOR, SNC80 for DOR, U50,488 for KOR). Antagonist measurements may be performed using a concentration curve of experimental drug or reference antagonist combined with a fixed concentration of agonist (EC90). The incubation may be terminated, and lysates may be combined with ˜1 pmol of [3H]cAMP and 7 μg of recombinant PKA, and incubated for 1 hour at room temperature. The reaction may be harvested and analyzed to generate potency (EC50/IC50) and efficacy (Emax/IMax) values for each compound. In some embodiments, off-target activities of compounds selected from in vitro analysis may be confirmed by the screening offered by the National Institute of Mental Health's Psychoactive Drug Screening Program (contract # HHSN-271-2008-025C (51). Note LYS739 did not show any off-target activities. In some embodiments, compounds with binding affinity below 100-fold vs. MOR/DOR/KOR for the other off-target receptors may be excluded from further studies.
In some embodiments, NMR analysis and/or computer modeling experiments are used to help identify structural features of enkephalin that may be important for KOR antagonist activity.
Cyclization may be achieved through the formation of various bonds such as a disulfide and a lactam bond. Bivalent ligands may be built on the pharmacophore structure. In order to link two monomeric pharmacophores, a disulfide bond may be utilized, e.g., through a homocysteine residue at position 2 (or 3). In some embodiments, the C-terminal chain elongation may be applied to enhance the KOR activity. For example, this modification may feature attachment of Leu5, Arg6, Ile8, and Arg9 residues in the dynorphin structure to a tetrapeptide scaffold.
The present invention also provides modifications of several known opioid ligands, such as endomorphin-1 (EM-1) (Ki=0.36 nM for MOR with 4,000- and 15,000-fold preference over DOR and KOR, respectively) (70) and [D-Arg2, Lys4]-dermorphin (DALDA) (Ki=1.69 nM for MOR with 11,000- and 2,500-fold preference over DOR and KOR, respectively) (see
For example, in some embodiments, the ORL is derived from DALDA, e.g., according to Formula 5: Aaa-DArg-Ccc-Ddd-Eee. In some embodiments, Aaa is selected from Tyr or 2′-6′-dimethyltyrosine (Dmt); Ccc is selected from Phe, Phe(X), or 1-naphthylalanine (1Nal); Ddd is selected from Lys, Gly or is absent; Eee is a 4-anilidopiperidine moiety (e.g., Ppp); and X is selected from F, Cl, or Br. In some embodiments, Ppp comprises Ppp(R), wherein R comprises a halogen. In some embodiments, R is selected from 3-Cl, 4-Cl, 3-F, 4-F, and 2,4-diCl. In some embodiments, the ORL is SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO; 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 42, or SEQ ID NO: 43.
In some embodiments, the ORL is derived from EM-1 or EM-2, e.g., the ORL is according to Formula 6: Aaa-Pro-Cco-Phe(X)Eee. In some embodiments, Aaa is selected from Tyr or 2′-6′-dimethyltyrosine (Dmt); Ccc is selected from Trp, Phe, Gly, or Phe(X); Eee is a 4-anilidopiperidine moiety (e.g., Ppp), and X is selected from F, Cl, or Br. In some embodiments, Ppp comprises Ppp(R), wherein R comprises a halogen. In some embodiments, R is selected from 3-Cl, 4-Cl, 3-F, 4-F, and 2,4-diCl. In some embodiments, the ORL is selected from SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30.
In some embodiments, the ORL is derived from FE20066, e.g., according to Formula 7: DPhe-DPhe-DNle-Ddd-Eee. In some embodiments, Ddd is selected from D-Arg or D-Lys, and Eee is a 4-anilidopiperidine moiety (e.g., Ppp). For example, in some embodiments, Ddd is D-Arg and Eee is Ppp. In some embodiments, Ddd is D-Lys and Eee is Ppp. In some embodiments, Ppp comprises Ppp(R), wherein R comprises a halogen. For example, in some embodiments, Ddd is D-Arg and Eee is Ppp(R), wherein R comprises a halogen (e.g., Cl, F, Br). In some embodiments, Ddd is D-Lys and Eee is Ppp(R), wherein R comprises a halogen (e.g., Cl, F, Br). In some embodiments, R is selected from 3-Cl, 4-Cl, 3-F, 4-F, and 2,4-diCl. In some embodiments, the ORL is according to SEQ ID NO: 44.
The present invention also provides an opioid receptor ligand dimer according to SEQ ID NO: 19. The present invention also provides an opioid receptor ligand dimer according to SEQ ID NO: 20.
In some embodiments, modifications to the peptide ligand (e.g., incorporation of a Ppp group at the C-terminus) enhances metabolic stability and/or lipophilicity and/or blood brain barrier (BBB)/central nervous system (CNS) permeability.
Table 7 shows analytical data for several multifunctional opioid receptor ligands (ORLs) with a Ppp group at the C-terminus. For reference: aFAB-MS (JEOL HX110 sector instrument) or MALDI-TOF. bPerformed on a Hewlett Packard 1100 [C-18, Vydac, 4.6 mm×250 mm, 5 μm, 10-100% of acetonitrile containing 0.1% TFA within 45 min, 1 mL/min]. chttp://www.vcclab.org/lab/alogps/. dLow resolution-Mass. n.d. not determined.
Example 1 describes non-limiting approaches to designing ORLs.
Step 1: Discover pharmacophoric structures of EM-1 and DALDA for MOR agonist/KOR antagonist activities. The C-terminus of EM-1 and DALDA may be modified with Ppp(R) (the R group may be decided by SAR results). This modification may improve their lipophilicities (aLogP increase >2) and metabolic stabilities, and thus afford high potential of BBB penetration. This modification may cause a biological profile change. The Ppp(R) group may be kept at the C-terminus, and the other positions may be modified. Substitution of Tyr with 2′,6′-dimethyltyrosine (Dmt) in opioid peptides can increase opioid activities dramatically, thus a Tyr1 residue may be replaced in both ligands with a Dmt residue or a β-methyl-2,6-dimethyltyrosine (Tmt) residue, which is more sterically hindered due to an extra methyl group. EM-1 and DALDA have distinct primary structures in positions 2, 3, and 4 but a Phe residue in common. The Phe residue in both ligands may be substituted with Phe(p-X) for altering receptor selectivity and inducing KOR interactions. A Phe3 residue in DALDA may also be substituted with a Phe(p-X) residue to observe SAR. However, to conserve its MOR selectivity over DOR, positions 2 and 4 of DALDA may be limited to basic amino acid residues. Likewise, position 2 of EM-1 may be limited to turn making amino acid residue. A Trp3 residue in EM-1 may be modified with other aromatic amino acid residues.
Step 2: Build dimerized ligands of MOR agonist/KOR antagonist using pharmacophores discovered in the first step. Position 2 and 4 of EM-1 and DALDA, respectively, may be consumed. Two homo pharmacophores may be linked through a disulfide bond of homocysteine residue. Cyclic bifunctional ligands may be designed. Insertion (l, m, and/or n=1) or deletion (l, m, and/or n=0) of Bbb, Ccc, and Ddd may optimize the distance between two aromatic rings, which may be the most important factor of high potency and selectivity.
Ischemic stroke is one of the leading causes of mortality and morbidity in the world. Example 2 describes the evaluation of multifunctional ORLs, e.g., LYS436_(SEQ ID NO: 8), LYS739 (SEQ ID NO: 10) and LYS416 (YGGF-Ppp, SEQ ID NO: 37), for their neuroprotective potential using in vitro and in vivo ischemic models. In vitro, neuronal death and total reactive oxygen species level, upon exposure to hypoxia-aglycemia followed by reoxygenation or challenged with NMDA was significantly decreased when treated with non-selective opioid agonists compared to no drug treatment group. Fluorinated enkephalin-fentanyl conjugate, LYS739 (SEQ ID NO: 10) showed better neuroprotection in all in vitro ischemic models compared to biphalin. An in vivo mouse middle cerebral artery occlusion (MCAO) stroke model was utilized to screen biphalin and LYS739 (SEQ ID NO: 10). Both agonists significantly decreased brain infarct ratio and edema ration measured with TTC staining compared to saline treated group. Neuronal deficit was improved in terms of neurological score and locomotor activity with LYS739 (SEQ ID NO: 10) and biphalin treatment. All enkephalin fentanyl conjugates and biphalin demonstrated better neuroprotection compared to fentanyl treated groups. Neuroprotective effects of biphalin and multivalent analogs were reversed, in most cases, by naltrexone, a non-selective opioid antagonist. This suggests that LYS739 (SEQ ID NO: 10) is a potential neuroprotective agent for ischemic stroke.
Primary cortical neuron survival upon exposure to 3 hr H/A and 24 hr reperfusion in presence or absence of fentanyl analogs and biphalin (10 nM) was determined using MTT (see
The effect of three fentanyl analogs, LYS436, LYS739 and LYS416 and biphalin and fentanyl (10 nM) were evaluated in primary cortical neurons exposed to 50 μM NMDA for 3 hours followed by 24 hours normal condition media exposure. Relative neuronal survival and cytotoxicity were quantified using MTT (see
Generation of total ROS in primary cortical neuron exposed to 3 hr H/A and 24 hr reoxygenation in presence or absence of OR agonist fentanyl analogs and biphalin (10 nM) was assessed in this experiment (see
As shown in
Twenty-four hours after the reperfusion neurological score was evaluated in the experimental groups (see
Locomotor activity (horizontal activity, vertical activity, total distance, rest time, stereotype counts and number of movements) was evaluated 24 hr after reperfusion in experimental animals (Table 8). Before the start of surgery all animals went through locomotor evaluation to get the baseline.□ Both LYS739 and biphalin (5 mg/kg, 10 min post reperfusion, i.p.) statistically significantly improved all the locomotor parameters compared to saline treated control animals. When compared the effect of LYS739 to that of biphalin most of the parameter were improved although they were not statistically significant except for vertical activity (p<0.05). But, in comparison to fentanyl treated group, both LYS739 and biphalin showed better locomotor activity and the effects were statistically significant. Non-selective OR antagonist NTX did not improve any locomotor parameters.
Table 8 shows measurement of locomotor activity 24 h after stroke and drug treatments. Data represent the mean±S.E.M. of 4-5 independent determinations; numbers indicated in parenthesis in the line of the table columns donate to the number of experimental animals per group. ‘*’ Compared to Saline treated group—*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ‘#’ Compared to biphalin treated group—#p<0.05; ##p<0.01; ###p<0.001; ####p<0.0001; ‘ϕ’ Compared to fentanyl treated group—ϕp<0.05; ϕϕp<0.01; ϕϕϕp<0.001; ϕϕϕϕp<0.0001
Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.
Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. Reference numbers recited in the claims are exemplary and for ease of review by the patent office only, and are not limiting in any way. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting of”, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting of” is met.
The reference numbers recited in the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings.
This application claims priority to U.S. Patent Application No. 62/165,063 filed May 21, 2015, PCT/US16/33529 filed May 20, 2016, and U.S. Patent Application 62/476,980 filed Mar. 27, 2017, the specification(s) of which is/are incorporated herein in their entirety by reference.
This invention was made with government support under Grant No. P01 DA006284, awarded by NIH. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62165063 | May 2015 | US | |
| 62476980 | Mar 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US16/33529 | May 2016 | US |
| Child | 15820133 | US |
