AU2001234027A1 - TRH-like peptide derivatives as inhibitors of the TRH-degrading ectoenzyme - Google Patents

Description

TRH-LIKE PEPTIDE DERIVATIVES AS INHIBITORS OF THE TRH-DEGRADING ECTOENZYME

Field of the invention

The present invention relates to certain peptide derivatives and uses thereof. In particular it relates to inhibitors of the enzyme Thyrotropin-releasing hormone-degrading ectoenzyme

(TRH-DE), also known as pyrogluta yl aminopeptidase II (PAP-II) (EC 3.4.19.6). TRH-DE

catalyses the removal of the N-terminal pyroglutamyl (Glp or pGlu) residue of the

neuropeptide thyrotropin-releasing hormone (TRH) (1-4). TRH is a tripeptide with the

primary structure L-pyroglutamyl-L-histidyl-L-prolineamide (Glp-His-ProΝH₂). Compounds

ofthe invention are useful as novel inhibitors of activity ofthe ectoenzyme TRH-DE and find

application in investigating the role of the enzyme and the biological functions of its

substrate, with potential therapeutic use as enzyme inhibitors in the field of medicine,

particularly the treatment of brain and spinal injuries and central nervous system disorders.

Background of the Invention

TRH has the structure:

The nomenclature of Schechter and Berger (27) is used to describe the positions of the peptide substrate residues (P) relative to the scissile Pi -Pi' bond and the corresponding

subsets (S) in the active site of the enzyme. In other literature, the right portion of the molecule is called the "prolineamide" or "C-terminal" portion; the centre portion of the

molecule is called the "histidyl" portion; and the left portion of the molecule is called the

"pyroglutamyl", "COOH-terminal" or "N-terminal" portion.

TRH was the first hypothalamic regulatory hormone to be characterised and as such plays a

central role in regulating the pituitary-thyroid axis. In addition, TRH displays a broad

spectrum of stimulatory CNS actions that are independent of the hypothalamic-pituitary-

thyroid axis (5-7) and it is also now believed to function as a neurotransmitter and/or

neuromodulator within the central nervous system (CNS) (5,6). Based on its CNS-mediated

effects, TRH has been shown to have potential use in the treatment of brain and spinal injury

and certain CNS disorders including cognitive deficits, epilepsy, and shock (7,8). The

mechanisms by which TRH improves these clinical conditions are not yet clear but appear to

be mediated, in part, by various other neurotransmitter systems (7,8). The full extent of the

potential clinical usefulness of TRH cannot be realised until its functions in the CNS are

entirely understood.

Unfortunately, the therapeutic efficacy of TRH is limited by its susceptibility to enzymatic

degradation (8). Current evidence strongly indicates that TRH-DE is the principal enzyme

responsible for the degradation of neuronally released TRH (9, 10-12). TRH-DE is located

on synaptosomal membranes in the central nervous system (CNS) (13,14) and thus, it is strategically placed to play a significant role in controlling TRH signals within the CNS,

much like that of acetylcholinesterase in regulating the neurotransmitter actions of

acetylcholine. TRH-DE is the only ectoenzyme known to degrade TRH (15) and has been

shown to exhibit a remarkably high specificity for TRH. Thus, TRH-DE appears to be an exceptional example of a neuropeptide-specific peptidase (16).

In general, potent and selective enzyme inhibitors are required for establishing the exact role

of a particular enzyme and are also powerful tools for investigating the biological functions of the enzyme's substrate. In addition to providing valuable insights into the functional roles of

enzymes and their substrates, enzyme inhibitors may be used therapeutically to enhance the clinical effects of an enzyme's substrate either by (a) potentiating the endogenous levels ofthe

substrate and or by (b) protecting endogenously administered substrate from degradation.

Thus, compounds that potently and selectively inhibit TRH-DE could be used to determine

the role of TRH-DE in regulating TRH signals. Because TRH-DE displays a strict specificity

for TRH, administration of TRH-DE inhibitors should only affect TRH'S neurotransmitter

and/or neuromodulator actions. Therefore, TRH-DE inhibitors would also be particularly

attractive for investigating the actions of TRH in the CNS. Furthermore, this exclusivity may

offer a therapeutic advantage in cases where TRH-DE inhibitors are used to potentiate TRH's

CNS actions. The design of TRH-DE inhibitors, however, is made difficult by TRH-DE's

restricted specificity and by the lack of a 3D-structure for TRH-DE.

US Patent No. 4, 608, 365 Engel describes a treatment for the amelioration of symptoms of

amyotrophic lateral sclerosis and other conditions which result from dysfunction of lower or upper motor neurons by the administration of doses of thyrotropin-releasing hormone by intravenous infusion or subcutaneous injection.

Specificity studies thus far indicate that TRH-DE substrates require a five-membered

pyπolidinone, thiazolidinone or butyrolactone ring in the Pi position (4,17). In addition,

several studies indicate that TRH-DE specificity is restricted to tri- or tetra-peptides

containing the sequence Glp-His and that the presence of a histidine residue in the Pi' position

is essential for TRH-DE activity (2,4,18-20). Recently, the inventor was the first to publish

the observation that the naturally occuπing TRH-like peptide Glp-Phe-ProNH₂ is also a

substrate for TRH-DE whereas Glp-Glu-ProNH₂ is not (21). This finding has since been

confirmed by Gallagher et al. (22). To date, no reports of other TRH-like peptides, with the

general structure Glp-X-ProNH₂, acting as substrates or inhibitors of TRH-DE have been published.

Some tolerance in the P ' position is suggested by the observations that both pyroglutamyl -

histidyl-prolyl-β-naphthylamide (Glp-His-ProβNA) and pyroglutamyl-histidyl-prolylamido-4-

methyl coumarin (Glp-His-ProAMC) are substrates for TRH-DE (4, 20, 22, 23). Glp-His-Trp

is hydrolysed by TRH-DE (18) and substitution of the Pro residue in Glp-His-ProβNA by Ala

or Trp does not appear to reduce turnover (4).

TRH and the C-terminally amidated peptides Glp-His-Pro-GlyNH₂ and Glp-His-GlyNH₂, have all been found to have lower K, values than their coπesponding acids when examined as

competitive substrates (24, 19, 22) leading to the suggestion that TRH-DE prefers an amide group at the carboxyl-terminus of peptide substrates (22). In addition, because the K_m value observed for Glp-His-ProAMC was approximately ten times less than that for Glp-His-

ProNH₂ Gallagher et al. (22) have proposed that TRH-DE has a preference for large

hydrophobic groups at the carboxyl-terminus of substrates. On the contrary, using

compounds present in this application, the inventor has now discovered that the addition of a

large hydrophobic group to the C-terminus of TRH and TRH-like peptides causes a reduction

in both the catalytic rate of hydrolysis and the specificity constant and that this is a useful

feature to incorporate into an inhibitor not a substrate.

Only a few inhibitors have been synthesised that exhibit a significant effect on TRH-DE

activity and none of these have been shown to be sufficiently effective for pharmacological

studies in vivo (11,25). The most potent of these is N-[l(R,S)-carboxy-2-phenylethyl]-N-

imidazole benzyl-histidyl β-naphthylamide (CPHNA) (26). Because CPHNA is not a TRH-

DE substrate analogue, the specificity of the interactions of CPHNA with TRH-DE has been

questioned (11). Nevertheless, CPHNA appears to reversibly inhibit TRH-DE with an

inhibition constant (Kj) of 8 μM and to increase the recovery of TRH released from rat brain

slices (26). These results indicate that TRH-DE inhibitors can be used to increase local TRH

concentrations and that it may be possible to modulate TRH function in vivo via inhibition of

TRH-DE activity.

US Patent No. 4, 906, 614 Giertz et a describes a method of preventing or treating

posttraumatic nervous injuries by administering a compound ofthe formula:

wherein R is hydrogen, a lower alkyl group, cyclohexyl or benzyl; Z is one ofthe groups

(a) (b) if Z is a group (a), R² and R³ together represent an additional bond between the carbon atoms

bearing them, or if Z represents a group (b), R³ is hydrogen; R⁴ is hydrogen or lower alkyl; R⁵

is hydrogen, lower alkyl or phenyl, R⁶ is hydrogen or methyl.

US Patent No. 5, 244, 884 Spatola et al. describes thionated analogues of thyrotropin

releasing hormone, having the formula:

wherein,

Q, W, X and Y, same or different, are oxygen or sulphur, with the proviso that at least one of

Q, W, X and Y is always sulphur; Z is lower alkyl or (4-imidazo yl)methyl; and the pharmaceutically acceptable salts thereof.

The disclosed compounds are stated to highly and selectively bind to TRH binding sites in animal tissues, and their utility in treating a variety of diverse physical conditions is disclosed.

US Patent No. 5, 686, 420 Faden describes a series of novel thyrotropin-releasing hormone

analogs wherein the C-terminal prolineamide moiety has been preserved, the N-terminal

moiety comprises one of five different ring structures and the histidyl moiety is substituted

with CF₃, NO₂ or a halogen. A method of use of the analog for the treatment of neurologic

disorders in also provided.

The contents of each ofthe above-mentioned U.S. Patents is incorporated herein by reference.

The present invention relates to compounds that competitively inhibit TRH-DE and display

greater apparent binding affinities (i.e. lower Kj values) for TRH-DE than the endogenous

substrate, TRH. These compounds have not been reported to occur naturally. Searches carried out after the priority date of this application have revealed that one of the compounds

has been named in published papers.

Burt D.R et al., Brain Research, 93 (1975) 309-328 mention pGlu-Asn-ProNH₂ as one of 25

analogues of TRH used in tests of inhibition of TRH binding in the cerebral cortex and

pituitary.

Bissette G. et al., Neuropharmacology (1978) 17 (45), 229-37 list pGlu-Asn-Pro-NH₂ (Abbott

43689) as a TRH analogue used in tests of analeptic effect in mice. Mazurov A.A. et al., Russian Chemical Bulletin (1998) 47 (10) 19601964 describe the

preparation of (inter alia) Glp-Asn-ProNH₂ and the measurement of the antidepressant

activity ofthe compound in rats.

Mazurov A.A. et al., Int. J. Peptide Proteins Res. (1993) 42, 14-19 describe the synthesis of Glp-Asn-ProNH₂.

Oliver C. et al., Biochem Biophys. Res. Commun. (1978) 84 (4) 1097-1102 refer to pGlu-Asn-

Pro-NH₂ as one of 30 TRH analogues subjected to enzymic degradation by rat serum or brain homogenate.

None of these papers discloses or suggests the use of Glp-Asn-ProNH₂ as an inhibitor of

activity of TRH-DE and there is nothing in their teaching to indicate the line of research

leading to the present invention.

All ofthe other compounds ofthe invention are believed to be novel.

Summary of the invention

In one aspect the present invention provides novel compounds ofthe formula I:

wherein: R¹ is an optionally substituted 4-, 5- or 6-membered heterocyclic ring having one or more

heteroatoms, in which at least one carbon atom ofthe ring is substituted with O or S;

X¹ is -CO- or -CS- or -CH₂CO- or CH(R⁴) wherein R⁴ is H or optionally substituted alkyl or

-COOH or -COOR¹¹ wherein R¹¹ is optionally substituted alkyl; X and X (which may be the same or different) are -CO- or -CS- ;

Z is -CH₂- or -S- or -O- or -NH-;

Q is O or S;

R is H or optionally substituted alkyl or an optionally substituted carbocyclic ring;

R³ is H or optionally substituted alkyl or an optionally substituted mono- or polycychc ring,

optionally having one or more heteroatoms in the ring(s) and optionally being a fused ring; or

R and R together form an optionally substituted mono- or polycychc ring optionally having

one or more heteroatoms in the ring(s) and optionally being a fused ring;

R⁵ and R⁶ (which may be the same or different) are H, or lower alkyl;

R⁷ and R⁸ (which may be the same or different) are H, or optionally substituted lower alkyl;

R⁹ and R¹⁰ (which may be the same or different) are H, or optionally substituted alkyl, or an

optionally substituted carbocyclic ring;

Y is -(CH₂)_n- where n is 0, 1, 2 or 3 provided that when R² and R³ form part ofthe ring n is 0;

provided that when R¹ is:

and X -1', vX2^z a „„ndj vX3^J are -CO-

and R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ are H and Z is CH₂

and Q is O

and Y is -(CH₂)_n- where n is 0,

then R² and R³ are not both H;

and pharmaceutically acceptable salts thereof.

In another aspect the present invention provides compounds of the formula I as defined above

and pharmaceutically acceptable salts thereof for use in a method for treatment of the human

or animal body by therapy or a diagnostic method practised on the human or animal body.

Glp-Asn-ProNH₂ is disclaimed in formula I.

In a further aspect the present invention provides compounds of formula I^a:

wherein:

R¹ is an optionally substituted 4-, 5- or 6-membered heterocyclic ring having one or more

heteroatoms, in which at least one carbon atom ofthe ring is substituted with O or S; X¹ is -CO- or -CS- or -CH₂CO- or CH(R⁴) wherein R⁴ is H or optionally substituted alkyl or -COOH or -COOR¹ ' wherein R¹ ' is optionally substituted alkyl; X and X (which may be the sa e or different) are -CO- or -CS- ;

Z is -CH₂- or -S- or -O- or -NH-;

Q is O or S;

R is H or optionally substituted alkyl or an optionally substituted carbocyclic ring;

R³ is H or optionally substituted alkyl or an optionally substituted mono- or polycychc ring,

optionally having one or more heteroatoms in the ring(s) and optionally being a fused ring; or

R and R together form an optionally substituted mono- or polycychc ring optionally having

one or more heteroatoms in the ring(s) and optionally being a fused ring;

R⁵ and R⁶ (which may be the same or different) are H, or lower alkyl; R⁷ and R⁸ (which may be the same or different) are H, or optionally substituted lower alkyl;

R⁹ and R¹⁰ (which may be the same or different) are H or optionally substituted alkyl or an

optionally substituted carbocyclic ring;

Y is -(CH₂)_n- where n is 0, 1 , 2 or 3 provided that when R² and R³ form part of the ring n is

0;

and pharmaceutically acceptable salts thereof, for use as an inhibitor of activity of TRH-DE.

The invention also provides compounds of formula I^a as defined above and pharmaceutically

acceptable salts thereof for use in potentiating endogenous TRH and/or in protecting

exogenously administered TRH or TRH analogues from degradation by TRH-DE.

In particular embodiments, R⁵ and R⁶ are H and Q is O_, so that the TRH derivatives have L-

asparagine residue (Asn) in the Pi' position. According to one aspect, the invention relates to a series of novel TRH derivatives having L-

asparagine (Asn) in the Pi' position ofthe peptide with the general formula:

R¹-X¹-L-Asn-L-Pro-NR²Y R³,

where R , X , R , Y and R are as defined above.

Suitably, Z is -CH_2- and R⁷ and R⁸ are H.

In preferred embodiments X¹, X² and X³ are -CO-

In any ofthe optionally substituted derivatives defined above, suitable substituents may be

present which do not interfere substantially with the function ofthe compounds as inhibitors

of TRH-DE activity. Examples of ring substituents include oxo, thioxo, alkyl, alkenyl,

alkynyl, aryl, alkoxy, halo, haloalkyl, nitro, azido, cyano, hydroxyl, hydroxyalkyl, SO_nR¹⁴

where R¹⁴ is alkyl and n = 0, 1 or 2, or a carboxyl or ester group ofthe formula -COOR¹⁵ where R¹⁵ is H or alkyl and which may be in ionic form -COO^". Examples of substituents on

alkyl groups (including alkyl groups in ring substituents mentioned in the preceding sentence)

include halo, nitro or cyano. Optional hetero atoms in the ring(s) of R , or R and R together, include N, O or S. Suitably there may be from 1-3 hetero atoms per ring, and the

hetero atoms in any ring may be the same or different.

An alkyl, alkenyl, alkynyl, or alkoxy group may be straight chain or branched and suitably

contains from 1 to 20, more suitably from 1 to 10, most suitably from 1 to 5 carbon atoms. A

lower alkyl group suitably contains 1 to 5 carbon atoms. Halo includes iodo, bromo, chloro

or fluoro. A carbocyclic ring or a mono- or polycychc ring suitably contains from 4 to 20 ring

atoms, more suitably 4 to 8 ring atoms per ring, most suitably in the case of a polycychc ring

a total of 8 to 16 ring atoms, any ring atoms which are not hetero atoms being carbon atoms. Suitably R² is H or C1-C5 alkyl . Suitably Y is -(CH₂)_n- where n is 0 or 1. Desirably R³ is: optionally substituted alkyl, more particularly Cι-C₅ alkyl , such as propyl or isopropyl; an

optionally substituted monocyclic ring, such as optionally substituted phenyl or cyclohexyl;

an optionally substituted monocyclic ring having one or two heteroatoms in the ring, such as

optionally substituted thiazolyl; an optionally substituted polycychc ring, such as optionally

substituted naphthyl or tetrahydronaphthyl; an optionally substituted polycychc ring having one or two heteroatoms in the ring, such as optionally substituted chromene (particularly optionally substituted coumarin), quinoline or isoquinoline; any ofthe foregoing rings

optionally having a benzo ring fused thereto (particularly benzocoumarin). Among optional

substituents, particular substituents include C i -C₅ alkyl or trihaloalkyl, C i -C₅ alkoxy

(particularly methoxy), hydroxyl, oxo or halo.

o

Alternatively R and R together may form an optionally substituted monocyclic or polycychc

ring such as piperazinyl.

Suitably R² or R³, or R² and R³ together, represent a hydrophobic group, particularly a large

hydrophobic group such as methyl coumarin.

In particular prefeπed embodiments R³ is phenyl, optionally having 1 or 2 substituents

selected from Cι-C₅ alkyl or trihaloalkyl, C₁-C₅ alkoxy, hydroxyl, and/or halo; or coumarin

optionally substituted with Cj-C₅ alkyl or trihaloalkyl; or naphthyl; or tetrahydronaphthyl.

Suitably R³ is an optionally substituted mono- or polycychc ring having up to 10 ring atoms. Most suitably R includes an optionally substituted phenyl ring. In preferred embodiments,

-N(R²) Y R³ has a group R³ which includes O spaced from the N by 1, 2 or 3 C atoms,

particularly 3 atoms as in 7-amido coumarin.

Certain prefeπed compounds ofthe invention include the secondary amine structure

-NH Y R^J

where Y and R are as defined above.

The most prefeπed compounds have an N-substituted amide group at the C-terminus of Glp-

Asn-Pro.

R¹ may suitably be:

wherein R , 12 is hydrogen, lower alkyl or phenyl,

R is hydrogen or lower alkyl,

Q is O or S.

In prefeπed embodiments, Q is O. Most suitably R¹ is a five-membered heterocyclic ring,

particularly a pyπolidinone, thiazolidinone or butyrolactone ring.

In particular prefeπed embodiments, R¹ is

The following are examples of compounds of formula I"

Glp-L-Asn-L-ProNH₂

Glp-L-Asn-L-ProAMC (Glp-Asn-Pro-7-amido-4-methylcoumarin)

Glp-Asn-Pro-isopropylamide

Glp-Asn-Pro-cyclohexamide

Glp-Asn-Pro-piperazide

Glp-Asn-Pro-5amidoquinoline (or όamidoquinoline or 8amidoquinoline)

Glp-Asn-Pro-5-amido-isoquinoline

Glp-Asn-Pro-Anilide

Glp-Asn-Pro-7-amido(trifluoromethyl)coumarin

10

Glp-Asn-Pro-6-amido-3,4-benzocoumarin

Glp-Asn-Pro- 1 -Naphthy Imethylamide

Glp-Asn-Pro-p-Anisidide (-p-methoxyanilide)

35 Glp-Asn-Pro-para amidobenzoic acid

Glp-Asn-Pro- 1,2,3, 4-tetrahydro-l -naphthy lamide

Glp-Asn-Pro-5,6,7,8-tetrahydro-l -naphthy lamide

Glp-Asn-Pro-2-thiazolamide

15

Glp-Asn-Pro-w-anisidide (m-methoxy anilide)

Glp-Asn-Pro-o-anisidide (o-methoxy anilide)

25 Glp-Asn-Pro-5-chloro-2-methoxy anilide

Glp-Asn-Pro-3-hydroxy-4-methoxy anilide (i.e. 5-amino-2-methoxyphenol)

Glp-Asn-Pro-2-hydroxy anilide (i.e. 2-aminophenol)

Glp-Asn-Pro-2-(hydroxymethyl-) anilide i.e. 2-aminobenzyl alcohol

Glp-Asn-Pro-4-trifluoromethyl anilide

(S) - (-) - 4-oxo-2-azetidyl asparaginyl prolineamide Detailed description of the invention

To investigate the influence of Pi' (see Ref 27) residues on ligand binding to and catalytic activity of TRH-DE, kinetic studies were conducted on a library of 25 peptides in which the

central histidine residue of TRH was replaced by a series of other amino acids. Kinetic

parameters for the library peptides were measured either by continuous or discontinuous

fluorometric assays (23) or by a quantitative HPLC assay (28). All of the assays were

developed in the inventor's laboratory and were published recently (23,28). The data

collected in this investigation represent the first comprehensive structure-activity study for

TRH-DE and the first description of TRH-DE specificity at the Pi' residue.

The abbreviations used are: TRH-DE, Thyrotropin-releasing hormone-degrading ectoenzyme;

CNS, central nervous system; TRH, thyrotropin-releasing hormone; Cbz, Benzyloxycarbonyl;

βNA, β-naphthylamide; AMC, 7-amino-4-methyl coumarin; TRHAMC, pyroglutamyl-

histidyl-prolylamido-4-methyl coumarin; Fmoc, 9-fluoromethyloxycarbonyl; tBu, t-butyl

ether; Trt, triphenylmethyl; Pmc, 2,2,5,7,8-pentamethylchromane-6-sulfonyl; Boc, N-α-t-

butyloxycarbonyl; OtBu, t-butylester; HPLC, high pressure liquid chromatography; BSA,

bovine serum albumin; MBHA, 4-methylbenzylhydrylamine; DMF, Ν,Ν-

Dimethylformamide; HBTU, 2-(lH-benztriazole-l-yl)-l,l,3,3-tetramethyluronium

hexafluorophospahate; HOBt, N-hydroxybenzotriazole; DIPEA, N,N-diisopropylethylamine;

DCM, dichloromethane; TFA, trifluoroacetic acid; EDT, 1,2-ethanedithiol; DPP-IV,

Dipeptidyl peptidase IV; His-ProDKP, His-Pro diketopiperazine; Glp, pyroglutamic acid; Thi,

thienylalanine; RT, retention time; DTT, dithiothreitol; ACN, acetonitrile; Boc, butoxycarbonyl; BOP, benzotriazole-l-yl-oxy-tris(dimethylamino) - phosphonium hexafluorophosphate. All amino acids are in the L-configuration unless otherwise stated.

i.p. = intraperitoneal, i.c.v. = intra-cerebroventricular, min = minutes, s = seconds.

The structures of non-standard amino acids used in the construction ofthe peptide library are shown below:

L-Thienylalanine (Thi) L-Homoproline (HomoPro)

L-Homophenylalanine (HomoPhe) L-α-Phenylglycine (Phg)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. HPLC traces obtained following the incubation (18h) of representative library

peptides with TRH-DE. Trace B represents the control sample for TRH. In trace C the products formed from TRH by the action of TRH-DE are shown and it can be seen that no

TRH (RT 8.566 min) remained. In contrast, poor substrates, such as Glp-Ala-ProNH₂

(Ala TRH), shown in trace A, were not completely degraded under these conditions and the

amount of Glp produced was small in comparison to that formed from TRH. Other library

peptides, for example Glp-Gln-ProNH₂ (Gln²TRH), were not hydrolyzed at all. Thus, in trace

D, which depicts a sample of Gln²TRH incubated for 18 h with TRH-DE, there was no

evidence of Glp.

FIG. 2. Lineweaver-Burk plot for TRHAMC degradation by TRH-DE in the presence of

increasing concentrations of Glp-Thi-ProNH . Data were obtained using the discontinuous

fluorometric assay and represent the mean ± SEM (n=3). Eπor bars can be seen where the

SEM is greater than the size ofthe symbol.

FIG 3. Lineweaver-Burk plot for inhibition of TRH-DE hydrolysis of TRHAMC by Glp-

Asn-ProNH₂. Initial rates were determined using the continuous fluorometric assay. Data

are shown as a Lineweaver-Burk plot for illustrative purposes and represent the mean ± SEM

(3). Eπor bars can be seen where the SEM is greater than the size ofthe symbol.

FIG 4. Lineweaver-Burk plot for inhibition of TRH-DE hydrolysis of TRHAMC by

Glp-Asn-ProNH₂. Initial rates were determined using a continuous fluorometric assay. Data

are shown as a Lineweaver-Burk plot for illustrative purposes and represent the mean ± SEM

(3). Eπor bars can be seen where the SEM is greater than the size ofthe symbol.

Figure 5. Diagram of test results showing effect of i.c.v. administration of TRH ± Glp- Asn-ProAMC on Wet Dog Shake (WDS) behaviour in rats. Male Wistar rats received an injection of either vehicle (1 μl 0.9% saline) + vehicle; Glp-Asn-ProAMC (5μg in lμl) + vehicle; TRH (5μg in lμl) + ve icle; or TRH (5μg in lμl) + Glp-Asn-ProAMC (5μg in lμl). Animals were then placed in indπ idual cages and observed for 30 min. Data are expressed as mean ± S.E. of WDS/30 min (n=6). ***p<0.001 vs TRH, one-way ANOVA followed by a post-ANOVA Bonfeπoni test.

Figure 6. Diagram of test results showing time course of effect of i.c.v. administration of TRH (5μg in lμl) ± Glp-Asn-ProAMC (5μg in lμl) on WDS in rats. Male Wistar rats received an injection of either vehicle + vehicle (■); Glp-Asn-ProAMC + vehicle (O); TRH + vehicle (D); or TRH + Glp-Asn-ProAMC (•) and were placed in individual cages and observed for 30 min. Data are expressed as mean ± S.E. of WDS every 5 min for 30 min (n=6). *p<0.01 vs TRH, one-way ANOVA followed by a post-ANOVA Bonfeπoni test.

Figure 7. Diagram of test results showing effects of TRH (10 mg/kg) and TRH-DE inhibitors (10 mg/kg), alone and in combination, on rat activity scores. Male Wistar rats were placed in individual cages and observed 10 and 5 min prior to i.p. injection with saline (■), Glp-Asn-ProNH₂ (V), Glp-Asn-ProAMC (O), TRH (D) or TRH + Glp-Asn-ProAMC (•). They were then observed for 30 sec every 5 min for an additional 40 min. The occuπence of individual behaviours were noted using a behavioural check list and these behaviours were summed to yield an overall activity count. Points are the mean ± S.E. of 5 experiments. Arrow indicates time of injection.

Figure 8. Diagram of test results showing time course of effect of TRH ± Glp-Asn- ProAMC on WDS behaviour in rats. Male Wistar rats were observed for 40 min following i.p. injection with TRH 10 mg/kg (D) or TRH 10 mg/kg + Glp-Asn-ProAMC 10 mg kg (•). Points are the mean ± S.E. of wet dog shakes/5 min (n=5). Saline treated animals exhibited no wet dog shakes at any time point.

Purification of enzymes used in the study

The inventor purified TRH-DE from porcine brain according to purification procedures

previously published by Dr. K. Bauer, (Max-Planck-Institut, Hannover, Germany) (1). TRH- DE was purified almost 20,000 fold. The preparation was free of other TRH-degrading

enzyme activities and was found to have a protein concentration of 0.8 mg ml^'1 using a

modification of the Lowry method with BSA as a standard. This preparation had a specific activity of 0.17 U mg^'1 with pyroglutamyl-histidyl-prolylamido-4-methyl coumarin

(TRHAMC) as substrate under standard conditions of a continuous assay (23). Dipeptidyl

peptidase IV (DPP-IV) (EC 3.4.14.5), purified from bovine kidney (7.7 mg ml^"1, specific activity of 17.5 U mg^"1 with Gly-ProAMC as substrate), was obtained as a gift from Dr. C. H.

Williams (Queen's University Belfast, U.K). (DPP-IV was used as the coupling enzyme used in the fluorometric assays).

One unit (U) of enzyme activity was defined as that amount catalysing the formation of one

μmol of product in one minute under the standard conditions employed. All incubations in

the assays described below were caπied out in 20 mM potassium phosphate buffer, pH 7.5, at

37 °C.

Standard Preparation ofthe compounds ofthe invention and comparative compounds:

Compounds ofthe invention may be prepared by standard Fmoc peptide chemistry (29).

Of the 25 library peptides studied, Glp-His-ProNH₂, Glp-Glu-ProNH₂ and Glp-His-ProOH were purchased from Sigma- Aldrich (Ireland). Glp-Gln-ProNH₂ and Glp-Phe-ProNH₂ were

bought from Peninsula Laboratories Inc., (UK). Glp-Asn-ProNH₂, Glp-Tyr-ProNH₂ and Glp-

D-Asn-ProNH₂ were custom synthesised by the American Peptide Company (Sunnyvale,

California, U.S.A.) at the request of the inventor under conditions of confidentiality, in

addition to being synthesised by the method described below. All other peptides in the library

were synthesised either manually using a bubbler system (for details, see Ref 29) or using a

Synergy Peptide Synthesiser (Applied Biosystems, UK). In both cases, standard solid-phase Fmoc chemistry was employed (29). Pyroglutamic acid and Fmoc amino acid derivatives

were purchased from Calbiochem-Novabiochem UK Ltd. Tri-functional amino acids were

obtained with side-chain protecting groups as follows: Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-

OH, Fmoc-Asn(Trt)-OH, Fmoc-D-Asn(Trt)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Lys(Boc)-OH,

Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH, and Fmoc-Cys(Trt)-OH. In general, synthesis was

caπied out on Rink amide MBHA resin (Calbiochem-Novabiochem UK Ltd.) with a loading

capacity 0.64 mmol gm^"1. The resin was swollen using DMF and deprotected with 20%

piperidine in DMF. Coupling was performed twice for each amino acid using the Synergy

Peptide Synthesizer and once when the bubbler system was employed. Three equivalents (i.e. 3-fold excess over the resin loading capacity) of each amino acid were coupled onto the resin

with HBTU/HOBt/DIPEA (1 :1 :2 equivalents) at each step. No deprotection step was

necessary after the coupling of pyroglutamic acid. On completion of peptide assembly, the

resin was washed thoroughly with DCM followed by methanol and allowed to dry overnight.

In the absence of labile amino acids and side-protection groups, cleavage of the peptide from

the resin was achieved by placing the dry resin in a round-bottomed flask and adding 95%)

(v/v) TFA in water (10 ml per 1 gm dry resin). This reaction mixture was stiπed at room

temperature for approximately one hour before filtering the suspension through a sintered

glass funnel. Sequences containing Asn were deprotected and cleaved using a TFA solution

containing 95% TFA, 2.5% water and 2.5% triisopropylsilane (v/v/v). For sequences

containing serine, tryptophan or arginine, cleavage/deprotection was achieved using Reagent

K (82.5% TFA / 5% water / 5% thioanisole / 5% phenol / 2.5% EDT, v/v/v/v/v) in place of

TFA (28, 29). These small peptides proved difficult to precipitate directly from the filtrate using diethyl ether, so the TFA and scavengers were first removed by rotary evaporation. The residue was

washed with petroleum ether and diethyl ether was then added to precipitate the peptide. A

steady stream of nitrogen was used to evaporate the diethyl ether and dry the peptide pellet

simultaneously. Because of the hygroscopic nature of these peptides, it was essential to dry

the peptide pellets thoroughly under nitrogen before transfeπing them to glass containers for

storage in a dessicator at -20°C.

Glp-Asn-ProAMC was custom synthesised by the American Peptide Company (Sunnyvale, California, U.S. A) at the request ofthe inventor under conditions of confidentiality.

Example I

Preparation of Exemplary Compound used in the Invention: Glp-Asn-ProNH?

Synthesis of the title compound was caπied out using a manual bubbler system incorporating a scintered glass funnel with a capacity of approximately 150 ml and a three-way tap system as described in the Catalogue & Peptide Synthesis Handbook supplied by Calbiochem- Novabiochem UK Ltd. 2g of MB HA Rink amide resin (Calbiochem-Novabiochem UK Ltd.) with a loading capacity of 0.55 mmol/gm were weighed out and transfeπed to the bubbler system. (Note the loading capacity of the resin varied slightly with each batch). The resin was swelled in approximately 20 ml of DMF for 10 min and deprotected using 20% piperidine in DMF for 60 min. The resin was then washed three times with DMF and a Kaiser-ninhydrin test was performed on a small quantity of the resin to ensure complete deprotection had occuπed. 1.1 lg of Fmoc-Pro-OH were dissolved in 20 ml DMF and to this solution 1.25g of HBTU and 0.5g of HOBt were added and mixed. 1.23 ml of DIPEA was then added to the mixture. This activated amino acid derivative was then added to the deprotected resin and coupling was allowed to proceed for 60 min. After this time the resin was drained and washed 3 times with DMF and the Kaiser-ninhydrin test was repeated. The resin bound fmoc-Pro was the 1 deprotected using 20% piperidine in DMF for one hour. The resin was again drained and \ /ashed 3 times with DMF. 1.96g of fmoc-Asn(Trt)-OH were dissolved in 20ml DMF. This second amino acid was then coupled to the resin and deprotected as described above. A Kaiser-ninhydrin test was carried out to ensure complete deprotection ofthe Asn residue had occuπed before coupling the final Glp residue. 0.42 g of Pyr-OH (Glp) was dissolved in 20 ml of DMF and coupled to the resin as described above. No deprotection was necessary after the coupling of Glp. The resin was then washed 3 times with DMF, 3 times with DCM and 3 times with methanol before being allowed to dry overnight. The following day, the resin was placed in a 100 ml round-bottomed flask to which was added 20 ml of a TFA solution containing 95% TFA, 2.5% water, and 2.5% triisopropylsilane. The flask was stoppered and cleavage of the peptide from the resin was allowed to proceed for 60 min at room temperature with constant stiπing. The resin was removed by filtration under reduced pressure and washed twice with the TFA solution. The TFA and scavenger were removed by rotary evaporation. The residue was washed with petroleum ether and diethyl ether was added to precipitate the peptide. Excess diethyl ether was removed by pipette. A steady stream of nitrogen was used to evaporate off the remaining diethyl ether and to thoroughly dry the peptide pellet. The peptide was then transfeπed to a glass container for storage in a dessicator at -20° C.

HPLC analysis of a 1 mM solution of the peptide in 20 mM potassium phosphate buffer pH 7.5 was conducted as described in Table I.

Preparation of Glp-Asn-Pro-NH₂ by American Peptide Company Cι₄H₂ιN₅O₅ MW 339.4 Peptide Preparation Process

The synthesis is performed by incorporating the c-terminal end ofthe amino acid to an amino group in an appropriate support, MBHA resin. The peptide chain is then formed by coupling the C-terminal of another amino acid to the N-terminal of the previous amino acid that was previously coupled to the solid support.

American Peptide Company provides Boc amino acids and resin. Biograde DCM, DMF and related solvents are obtained from Fisher Scientific.

Boc-Pro; Boc-Asn; Glp; HBTU and MBHA resin

x g of peptide resin was transfeπed into a ~x ml size cleavage vessel. HF was filled in and a low-high HF cleavage was conducted.

After the HF cleavage, extract the CAN/Η O. Crude peptide is purified with RP-HPLC.

Preparative HPLC, Shimadzu 8-LC Analytical HPLC Shimadzu 10-LC Analytical column YMC 5 micron C 18 Preparative column 3 inch Varian 10 micron C18 RP-HPLC

Collect those fraction >95. Dry it over Virtis lyophilizer and white powder was obtained with good yield.

The material was finally tested and release by QC with that parameter specified in COA.

Glp-Asn-Pro-NH₂ prepared by this process exhibited the coπect molecular weight in Mass Spectral analysis (MALDI-TOF). It had a solubility of lmg/ml in water.

RP-HPLC Analysis

Column Vydac C18 5u

Flow rate: 1.0 ml/min Gradient type: Linear

Buffer A: 0.1% TFA i H O Buffer B: 0.1% TFA in Acetonitrile

From 0 % To 10 % During 20 min

Wavelength 215 nm Sens 0.16 Paper Speed ....5... mm min Retention Time 10.627 min Preparation of Glp-Asn-ProAMC by American Peptide Company C₂₄H₂₇N₅O₇ MW 497.5

Peptide Preparation Process

This peptide was prepared by solution phase chemistry. American Peptide Company provides Boc amino acids and resin. Biograde DCM, DMF and related solvents were obtained from Fisher Scientific.

Boc-Pro; Boc-Asn; Glp; BOP and AMC

x g of AMC was dissolved in DMF. The BOP reagent and Boc-Pro were added to the reaction mixture for a period of two hours. Let the reaction react for ~2 hours. Use standard work-up procedure to generate Boc-ProAMC analog.

Following removal of Boc group of Boc-ProAMC, Boc-Asn was added along with coupling agent. Noc-Asn-ProAMC was obtained.

Repeat same process, Glp was coupled to the sequence. Since no protection was employed, HF cleavage step was avoided. However, if Boc-Asn(Xan) had been used, HF step would have been necessary.

After the HF cleavage, extract the ACN/H O. Crude peptide is purified with RP-HPLC.

Preparative HPLC, Shimadzu 8-LC Analytical HPLC Shimadzu 10-LC

Analytical column YMC 5 micron C18

Preparative column 3 inch Varian 10 micron C18 RP-HPLC

Collect those fraction >95. Dry it over Virtis lyophilizer and white powder was obtained with good yield.

The material was finally tested and released by QC with that parameter specified in COA. Glp-Asn-ProAMC prepared by this process exhibited the coπect molecular weight in Mass Spectral analysis. It had a solubility of 0.5mg in 0.5ml water.

RP-HPLC Analysis

Column: 4.6m i.d. x 250: vydac, cl 8, 5 micron

Others: F:1.5ml/min

Buffer A: 0.1% TFA in water Buffer B : 0.1% TFA in ACN Wavelength 215 ran

C:\CLASS-VP\METHODS\5-35% 20 25. met Retention Time 13.7 min

Derivatives of this type have been previously synthesised by well known solution solid phase

procedures using Boc chemistry (Zimmerman et al.1977 (30), (Fujiwara & Tsuru, 1978 (31)).

Glp-Asn-ProAMC and other compounds of the invention can be prepared utilising such

procedures which are readily understood by those of ordinary skill in the art. As such, the

above experimental procedure utilised by APC to provide Glp-Asn-ProAMC herein is only

exemplary of suitable methods and this should not be considered to limit the present

invention.

Preparation of Glp-Asn-Proamides by Solution Phase Chemistry

Carboxamides of the invention may be prepared by standard solution phase coupling chemistry using Glp-Asn-Pro and the appropriate amine. Glp-Asn-Pro was obtained from the American Peptide Company (Sunnyvale, California, U.S.A.). 2-Aminobenzyl alcohol, o- anisidine, m-anisidine, p-anisidine, 5-amino-2-methoxyphenol, 2-aminophenol, 1,2,3,4 tetrahydro-1-naphthylamine, 5,6,7,8 tetrahydro-1-naphthylamine, benzylamine, 5-chloro-2- methoxy aniline were purchased from the Sigma-Aldrich Chemical Company. Glp-Asn-Pro was transfeπed to a flask equipped with a magnetic stiπer. DMF was introduced followed by the amine, HOBt and DCC '1 :1 :1 equivalents). The reaction mixture was stiπed for 24 hours, filtered, and the solve t evaporated at room temperature. The residue was washed successively with small volumes of ethyl acetate, diethyl ether, and petroleum ether. The amides were further purified by preparative HPLC using a Waters WAE-84176 semi prep C18 HPLC column. Two isomers of the 1,2,3,4-tetrahydro-l-naphthylamide were isolated by HPLC. These were tested separately. (See Tables VI-VII below).

Analysis of Peptides

All ofthe library peptides were analysed and judged to be homogeneous by HPLC (see Table

10 I). HPLC analysis was conducted using a Thermo Separation Products Inc., Spectra System

HPLC. Standard 1 mM solution of peptide in 20mM potassium phosphate buffer pH 7.5 was

analysed on a C-18 reverse-phase column (Hypersil UK) using a linear gradient of 0-70% acetonitrile in 0.08% TFA as previously described (21). In addition, the American Peptide

Company provided Mass Spectral analysis and HPLC analysis to confirm the identity of the

15 peptides synthesised by them. Similarly, Sigma- Aldrich (Ireland) and Peninsula Laboratories

Inc., (UK) provided amino acid analysis and HPLC analysis. HPLC traces obtained for

peptides synthesised in the laboratory were similar to those obtained for peptides purchased

from the American Peptide Company (Sunnyvale, California, U.S.A.).

Table I - HPLC Analysis

Table I. HPLC analysis of the peptide library -retention times (min) are shown for each peptide. Retention times are compared for peptides synthesised either by the inventor, the American Peptide Company (APC; Sunnyvale, California, U.S.A.), or other commercial companies (for details see text above ). HPLC analysis was conducted employing a Thermo Separation Products Spectra System HPLC. Peptides were eluted from a C-18 reverse-phase HPLC column using a linear gradient of 0-70% acetonitrile in 0.08% trifluoroacetic acid over a period of 30 min. UV absorbance was measured at 206nm. 0

Kinetic Studies

Kinetic analysis of the peptide library using HPLC — The ability of each peptide in the library

to act as a TRH-DE substrate was assessed using HPLC. As an initial screen, 1 mM peptide

was incubated with 0.8 μg TRH-DE in a total volume of 1 ml for 18 h at 37 °C. Control

5 samples were included in which peptide (1 mM) or Glp (0.2 mM - 0.8 mM) were incubated

under identical conditions in the absence of TRH-DE. TRH-DE activity was terminated by

the addition of TFA (0.15% v/v) and samples were then analysed using HPLC as described

previously (24). Products resulting from TRH-DE hydrolysis could be separated on a C-18 reverse-phase column (Hypersil, UK) using a linear gradient of 0-70% acetonitrile in 0.08%

(v/v) TFA. The concentration of Glp formed by the action of TRH-DE on the peptide library

was measured by UV absorbance at 206 nm with a quantitative detection limit of 0.1 mM for

a 40 μl injection volume, employing a signal to noise ratio of 10. Following the incubation of

each peptide with TRH-DE, evidence of Glp in the sample was taken to indicate that the peptide was a TRH-DE substrate.

The rates of hydrolysis for each of those peptides identified as substrates were then compared

by measuring Glp production using microassays with shorter incubation times. In these

assays peptide (1 mM) was incubated at 37 °C in a total volume of 100 μl. 0.8-3.2 μg TRH-

DE and incubations times of 5min to 18h were used routinely. Measurements were made in

triplicate. The rate of TRH-OH hydrolysis was also measured by this method.

Determination of inhibitor constants for selected TRH-DE substrates — The HPLC assay

lacked sufficient sensitivity for determining kinetic constants. Therefore, those peptides

undergoing significant hydrolysis (i.e. those that exhibited rates of hydrolysis > 0.5 U mg^"1)

were examined as competitive substrates of TRH-DE, using a recently published

discontinuous fluorometric TRH-DE assay (23). This assay employs TRHAMC as the

substrate and depends on the measurement of the fluorescence of 7-amino-4 methyl coumarin

(AMC) produced as follows: -

Glp-His-Pro-AMC ^TRH-^DE> His-Pro-AMC

Λ Of) o

His-Pro-AMC ³ⁿ' ^su > His-ProDKP + AMC (cyclisation) We have shown that the amount of AMC formed under these conditions is a quantitative measure of TRHAMC cleavage (23). Initial rates for the hydrolysis of TRHAMC by TRH-

DE were determined in triplicate at five different substrate concentrations both in the absence and presence of at least three concentrations of peptide. K, values were obtained by non-

linear regression analysis of the data collected. Determined in this way (i.e. by treating the

peptide substrates as inhibitors of TRHAMC hydrolysis), these K* values coπespond to the

Michaelis constants for those library peptides hydrolysed by TRH-DE (32).

Kinetic analysis of peptides that are not hydrolysed by TRH-DE — A recently-developed

continuous coupled fluorometric assay (23) was used to investigate the ability of those library

peptides that were not hydrolysed by TRH-DE to inhibit TRHAMC degradation by TRH-DE.

In this assay, TRHAMC is the substrate and dipeptidyl peptidase IV (DPP-IV) (EC 3.4.14.5)

is the coupling enzyme. The reaction sequence is: -

Gb-H is-Pro-AM C TRH-PE ». H is-Pro-A M C ^{P PP 'IV} *- H is-Pro + AM C

The reaction was monitored continuously by measuring the increase in AMC fluorescence. A

linear progress curve with no discernible lag period was observed when the reaction was

monitored over a period of 10 minutes. Nevertheless, data sampling was not commenced until 100 s from the start of the reaction to ensure measurements were taken after a steady

state had been reached (see Ref. 23). In a preliminary experiment, tl e peptides were screened for their ability to inhibit TRH-DE activity. The initial rate of TR AMC hydrolysis by TRH-DE was measured by incubating 5

μM TRHAMC with 1.23 μg DPP-IV and 0.32 μg TRH-DE, both in the absence and presence

of each library peptide (1 mM final concentration) under standard assay conditions (23). In

each case the percentage inhibition (i_%) was calculated using the equation i»_o = 100 (1 - v/v₀),

where Vj and v₀ are the initial velocities in the presence and absence of peptide, respectively.

Kj values for peptides giving < 20% inhibition, under these conditions, were calculated to be

greater than 1 mM using the relationship for competitive inhibition i = [I]/ {[I]+Kj

(1+[S]/K_m)} where i is the fractional amount of inhibition observed, [I] is the concentration of

the peptide, [S] is the concentration of substrate and K_m is the Michaelis constant for

TRHAMC, which was found to be 3.5±0.4 μM (n-=6) (see below). Since these peptides were

poor inhibitors they were not examined further. The Kj values for the remainder of the

peptides were determined by measuring their effects on TRHAMC degradation by TRH-DE

using the continuous assay. Data were collected in duplicate at five different substrate

concentrations and at least three different concentrations of each peptide.

All peptides that were observed to inhibit AMC production in the continuous coupled assay

were assessed for their ability to inhibit the coupling enzyme, DPP-IV, using a direct

continuous assay for DPP-IV that employed Gly-ProAMC as the substrate (23). Peptide

concentrations similar to those used to determine the Kj values above were employed in the

DPP-IV assay. None of these peptides were found to inhibit DPP-IV hydrolysis of Gly-

ProAMC. Thus, the effects produced by the peptides can be attributed solely to their

inhibition of TRH-DE. Reversibility and time dependence of the inhibition produced by Glp-Gln-ProNH₂, Glp-Asn- ProNH₂ and Glp-Asn-ProAMC were examined by initially preincubating TRH-DE with each peptide at 37 °C for various periods up to 75 min. To investigate time dependence, the enzyme-peptide solution was subsequently added to the reaction mixture containing TRHAMC, DPP-IV, buffer and peptide, and TRH-DE activity was measured using the continuous assay. The final concentration of Glp-Gln-ProNH , Glp-Asn-ProNH₂ and Glp- Asn-ProAMC used was 400, 160 and 1 μM, respectively. To test for reversibility, the enzyme-peptide solution was added to a reaction mixture that did not contain peptide. TRH- DE activity was not affected by pre-incubation at 37 °C.

Fluorescence measurements were made using a Perkin Elmer LS 50B Luminescence

Spectrometer fitted with a thermostated cell holder. Wavelengths for excitation and emission

were set at 370 nm and 440 nm, respectively, with slit widths of 10 nm and 5 nm,

respectively.

Analyses of results — All kinetic parameters were determined by non-linear regression

analysis using the computer program Prism (Graph Pad Software Inc. U.S.A). Linear

regression analysis employing proportional weighting was used to fit data to linear plots for

display purposes only. Unless otherwise stated, all values are shown as the mean ± SD.

RESULTS

HPLC analysis of the peptide library — HPLC analysis revealed that TRH-DE catalyzed the

removal of the N-terminal Glp residue from 15 out of the 25 members of the peptide library, including TRH. It can be seen from the representative HPLC traces shown in Fig. 1 that

following overnight incubation with TRH-DE, 1 mM Glp-His-ProNH₂ (TRH) was degraded

fully. In contrast, Glp-Ala-ProNH was only partially degraded to produce a relatively small amount of Glp (0.2 mM). No detectable Glp was released from Glp-Gln-ProNH₂ (Fig. 1) or

from those peptides where the Pi' position was occupied by D-Asn, Gly or the L-amino acids

Asn, Tip, Phg, HomoPro, Glu, Asp, and Pro (not shown). The HPLC-based assay also showed that there was no detectable cleavage of Glp-Cys-ProNH₂ by TRH-DE. This peptide,

however, was observed to undergo oxidation with disulfide bond formation during incubation

and it was not examined further. Table II shows the rates of hydrolysis of those peptides that

were found to be substrates for TRH-DE.

Inhibitor constants for selected TRH-DE substrates — K, values for those library peptides that

were significantly hydrolyzed by TRH-DE are shown in Table III. All of these peptides were

found to act as simple competitive inhibitors of the degradation of TRHAMC by TRH-DE

(example shown in Fig. 2). Non-linear regression analysis of the data collected in this study

gave a K_m value for TRHAMC of 3.1 ± 0.5 μM (n=8). V_maχ/K_m values (Table II) were

calculated assuming that the K, values for these peptides coπespond to Michaelis constants

(33).

Kinetic Analysis of peptides not hydrolyzed by TRH-DE. As noted above, 15 out of the 25

members of the peptide library, including TRH, were found to be TRH-DE substrates. Only

one of these peptides, Glp-Tyr-ProNH₂, displayed a K_m value that was lower than that of

TRH. Of the remaining 10 peptides that were not hydrolysed by TRH-DE, 5 were found to

inhibit TRH-DE with K, values of < 1000 μM. Of these, only one, Glp-Asn-ProNH₂, was found to display a binding affinity greater than TRH. Table IV shows the percent inhibition

of TRH-DE by each of the enzyme-resistant peptides measured by the continuous coupled

assay under standard conditions. Also presented are the Kj values obtained for those peptides exhibiting greater than 20% inhibition in the initial screening. The latter peptides were all

found to act in a simple competitive manner, as illustrated by a Lineweaver-Burk plot of data

obtained for Glp-Asn-ProNH₂ (Fig.3). Inhibition by Glp-Gln-ProNH₂, Glp-Asn-ProNH₂ and

Glp-Asn-ProAMC was found to be fully reversible and not time-dependent. Non-linear

regression analysis of data from the continuous assay gave a K_m value of 3.5 ± 0.4μM (n=6)

for TRHAMC.

Table II

Glp-His-ProOH 3.38 ± 0.18 (3)

Glp-His-ProNH₂ 2.54 ± 0.25 (11)

Glp-Thi-ProNH₂ 2.12 ± 0.16 (6)

Glp-Phe-ProNH₂ 1.56 ± 0.13 (5)

Glp-Tyr-ProNH₂ 0.66 ± 0.04 (5)

Glp-Arg-ProNH₂ 0.41 ± 0.02 (4)

Glp-Lys-ProNH₂ 0.46 ± 0.02 (3)

Glp-Met-ProNH₂ 0.21 ± 0.03 (4)

Glp-Leu-ProNH₂ 0.14 ± 0.01 (5)

Glp-Thr-ProNH₂ 0.14 ± 0.01 (5)

Glp-Ileu-ProNH₂ 0.06 ± 0.00 (4)

Glp-homoPhe-ProNH₂ 0.05+ 0.00 (3)

Glp-Val-ProNH₂ 0.05 ± 0.00 (3)

Glp-Ser-ProNH₂ 0.05 ± 0.00 (3)

Glp-NorVal-ProNH₂ 0.04 ± 0.00 (6)

Glp-Ala-ProNH₂ 0.02 ± 0.00 (3) Table II. Comparison of hydrolysis rates for library peptides (ImM) found to be TRH-DE substrates. Rates of hydrolysis were determined as outlined under "Experimental Procedures" Each value represents the mean ± S.D. The number of determinations is indicated in brackets.

Table III

Peptide V_max K_m V_max/K_m (U mg ¹) (μM) (U m ' μM-¹)

Glp-His-ProNH₂ 2.63 ± 0.26 (1 1) ^*35 ± 4 (3) 0.08 ± 0.01

Glp-Thi-ProNH₂ 2.19 ± 0.16 (6) 34 ± 6 (3) 0.06 ± 0.01

Glp-Tyr-ProNH₂ 0.67 ± 0.04 (5) 15 ± 3 (4) 0.05 ± 0.01

Glp-His-ProAMC 0.1 1 ± 0.01 (4) 3.1 ± 0.4 (9) 0.04 ± 0.00

Glp-Phe-ProNH₂ 1.64 ± 0.14 (5) 55 ± 8 (3) 0.03 ± 0.00

Glp-His-ProOH 4.43 ± 0.24 (3) ^*31 1 ± 31 (5) 0.01 ± 0.00

^*From Ref. 23

Table III. Comparison of kinetic parameters for selected library peptides hydrolyzed by TRH-DE. The Michaelis constant for Glp-His-ProAMC (TRHAMC) was measured directly, whereas the K_m values for the library peptides were measured indirectly by treating them as competitive inhibitors of TRHAMC hydrolysis by TRH-DE. The K_m values were all determined by non-linear regression analysis of data obtained from the discontinuous fluorometric TRH-DE assay, as detailed in the text, and represent the mean ± S.D. The number of determinations is shown in brackets. Included for comparison is the coπesponding K_m values for TRH and TRH-OH which the inventor recently published (23). V_maχ values were estimated from rates of hydrolysis measured by HPLC using the relationship V_maχ ⁼ v₀ {(K_m + [S])/[S]}. Table IV

Peptide % inhibition K, (μM)

Glp-Asn-ProNH₂ 97 17.5 ± 1.4

Glp-Gln-ProNH₂ 83 69.0 ± 4.4

Glρ-Trp-ProNH₂ 71 232 ± 26

Glp-Phg-ProNH₂ 57 356 ± 19

Glp-homoPro-ProNH2 40 500 ± 133

Glp-Glu-ProNH₂ 17 > 1000

Glp-Gly-ProNH₂ 8 > 1000

Glρ-Pro-ProNH₂ 3 > 1000

Glp-D-Asn-ProNH₂ 2 > 1000

Glp-Asp-ProNH₂ 1 > 1000

Table IV.

Inhibitory effects of library peptides that were not hydrolyzed by TRH-DE. Data were obtained using TRHMCA as substrate in the continuous coupled assay. The % inhibition produced by each peptide (ImM) was determined in the presence of 5 μM substrate. K, values (mean ± S.D (n=3)) were measured by the continuous coupled assay at five different TRHAMC concentrations and at least three different concentrations of peptide. K, values for peptides displaying < 20% inhibition were estimated to be > ImM as described in the text. The K, for Glp-Asn-ProNH₂ represents the mean ± S.D of three separate experiments using three different batches of Glp-Asn-ProNH - one batch was obtained from the American Peptide Company (U.S. A) and the other two batches were synthesized as described in Example 1 herein. No significant difference was found in the K, between the three different batches. Improvement in binding affinity of Glp-Asn-ProNH₂ was achieved by substituting the amino

group at the C-terminus with 7-amino-4-methyl coumarin. Comparison of the binding affinities of these two compounds is shown below:

Table V. Inhibition of TRH-DE activity by Glp-Asn-ProNH₂ and Glp-Asn-ProAMC

Peptide % inhibition K_j (μM)

Glp-Asn-ProAMC (1 μM) 50 0.97 ± 0.08

Glp-Asn-ProNH₂ (1 mM) 97 17.5 ± 1.4

Data were obtained using TRHAMC as substrate in a continuous coupled assay (23). The %

inhibition produced by 1 μM Glp-Asn-ProAMC and 1 mM Glp-Asn-ProNH₂ was determined

in the presence of 5 μM substrate. Kj values (mean ± S.D (n-=3)) were measured by the

continuous coupled assay at five different TRHAMC concentrations and at least three

different concentrations of peptide. The Kj for Glp-Asn-ProNH₂ represents the mean ± S.D

of three separate experiments using three different batches of Glp-Asn-ProNH₂.

Lineweaver-Burk plots of the data obtained, shown in Figures 3 and 4, demonstrate that both

Glp-Asn-ProAMC and Glp-Asn-ProNH act as a simple competitive inhibitors of TRH-DE

activity, in vitro.

Having regard to the literature references cited herein, including in particular the patent

documents whose contents are incorporated herein by reference, the peptide derivatives of

formula I and pharmaceutically acceptable salts thereof would be expected to inhibit TRH-DE

activity, in vitro, in a manner similar to that described and may be synthesized by adapting

the standard preparations presented, as appropriate. The properties of Asn in the Pi' position of TRH were found to be unique as this was the only

analogue that displayed a greater binding affinity for TRH-DE than TRH and was not

hydrolysed by TRH-DE. It is not obvious why Glp-Asn-ProNH2 and Glp-Gln-ProNH2 are not TRH-DE substrates, but it might be postulated that binding of these peptides to the enzyme is distorted, thus preventing catalysis. Although this invention is not limited by any theory, and the test results were not predictable, it is possible to two-dimensionally

superimpose the side chain of L-Asn onto that of L-His such that the amide NH group of L- Asn overlaps with the τN of L-His. Similarly, the side chain of L-Gln can be two-

dimensionally superimposed onto that of L-His such that the amide NH group of L-Gln

overlaps with the πN of L-His. However, the binding of Glp-Gln-ProNH₂ is not as favourable

as that for Glp-Asn-ProNH₂ and Glp-His-ProNH₂. Since the TRH-like peptides which

contain Asn, Gin and His in the Pi' position all bind relatively well to TRH-DE, it could be

suggested that the nitrogen atoms in these side chains represent recognition moieties for the

Si' subsite of the enzyme and facilitate binding. The more favourable inhibitory properties of

Glp-Asn-ProNH compared to Glp-Gln-ProNH₂ may be related to the position of the nitrogen

atom in the side chain and to the position of amide carbonyl group relative to the Si' subsite

on the enzyme. The enhanced binding of the coumarin derivative of Glp-Asn-ProNH₂ could be due to more favourable hydrophobic interactions occuπing between the C-teπninus of the

peptide and the enzyme. Indications that this substitution would improve inhibitor binding

were deduced from comparison of the kinetic parameters, which were determined in the

inventor's laboratory, for TRH, TRHAMC and TRH-OH (Table III). From these kinetic

parameters it was concluded that the addition of a large hydrophobic group, such as AMC, to

the C-terminus of TRH would be a useful feature to incorporate into an TRH-DE inhibitor. The resulting compound, Glp-Λsn-ProAMC, was found to have a Kj of 0.97 ± 0.08 μM and is

the most potent, competitive TRH-DE inhibitor described to date. Previously, the most

potent TRH-DE inhibitor to be reported was CPHNA (26). This compound, though substantially structurally different from TRH and Glp-Asn-Pro AMC, was found to inhibit

TRH-DE in a competitive manner with a Kj of 8 μM (21).

The influence of the C-terminal residue of Glp-Asn-Pro on binding to TRH-DE was examined further by synthesising and testing several compounds having an N-substituted amide group at the C-terminus of Glp-Asn-Pro similar to AMC. These compounds were

synthesised using solution phase peptide chemistry as described above.

Identity of C-terminal amide Retention times (min) on HPLC

Benzylamide 14.31 p-anisidide 14.99

5,6,7,8 -tetrahydro-1 -naphthylamide 19.31

5-chloro-2-methoxy anilide 19.59 m-anisidide 15.87 o-anisidide 15.41

2-aminophenol 13.20

5-amino-2-methoxyphenol 12.84

1 , 2, 3,4-tetrahydro-1 -naphthylamide isomer 1 18.07

1 , 2, 3,4-tetrahydro-1 -naphthylamide isomer 2 18.36

2-aminobenzyl alcohol 11.87 Table VI. HPLC analysis of Glp-Asn-Pro amides.

All of the peptides shown above were synthesised in the inventor's laboratory. All were

analysed and judged to be homogeneous by HP LC. HPLC analysis was conducted employing a Thermo Separation Products Spectra System HPLC. Peptides were eluted from

a C-18 reverse-phase HPLC column using a linear gradient of 0-70% acetonitrile in 0.08%

trifluoroacetic acid over a period of 30 min. UV absorbance was measured at 206nm.

Retention times (min) are shown for each of the peptides. Each of the Glp-Asn-Pro amides

were tested for their ability to act as TRH-DE substrates using the HPLC assay described previously. None of these peptides were hydrolysed by TRH-DE. The ability of the Glp-

Asn-Pro amides to inhibit TRH-DE activity was determined using the continuous coupled

assay described previously (Table VI). Most of the carboxamides of Glp-Asn-Pro that were

tested were found to display Kj values for TRH-DE in the low μM range (Table VI) below

that of Glp-Asn-ProNH . Nevertheless, all of the carboxamides of Glp-Asn-Pro that were

tested displayed greater affinity for TRH-DE than TRH.

Identity of C-terminal amide Kj (μM)

Benzylamide 11.87

p-anisidide 3.24

5,6,7,8 -tetrahydro-1 -naphthylamide 9.31

5-chloro-2-methoxy anilide 9.95 m-anisidide 0.86 o-anisidide 11.59

2-aminophenol 6.62

5-amino-2-methoxyphenol 1.72

1 , 2, 3,4-tetrahydro-1 -naphthylamide isomer 1 63.24 1 ,2,3,4-tetrahydro-1 -naphthylamide isomer 2 22.37

2-aminobenzyl alcohol 20.65

7-amido-4-methyl coumarin 0.41

Table VII. Inhibition of TRH-DE activity by Glp-Asn-Pro amides.

Data were obtained using TRH-AMC as substrate in the continuous coupled assay (23). The percent inhibition (i) produced by each peptide was measured in duplicate in the presence of 5μM substrate. K, values were calculated using the equation: i = [I] / [I] + K, (1 + [S]/K_m) where K_m is the Michaelis constant for the substrate TRH-AMC (23). The K, value of Glp-Asn-Pro-AMC was determined also by non-linear regression analysis of inhibition data from the continuous assay performed at five different TRH-AMC concentrations and three different concentrations of Glp-Asn-Pro-AMC (see Table V). HPLC analysis revealed that none of the peptides shown in the table above were hydrolysed by TRH-DE.

Preliminary evaluation of the functional effects of two TRH-DE inhibitors of the

present invention in vivo. A preliminary assessment of the ability of pyroglutamyl-asparaginyl-prolineamide (Glp-Asn-

ProNH₂, K, = 17.5 μM) and Glp-Asn-Pro-7amido-4-methyl coumarin (Glp-Asn-ProAMC, K,

= 0.97 μM) to enhance TRH central actions in vivo was caπied out using two different modes

of administration. Charli et al. found that CPHNA increased the recovery of TRH from rat

brain slices in vitro (26), indicating that TRH levels can be altered by inhibition of TRH-DE

activity. The biological effects of TRH-DE inhibitors in vivo, however, have never been

reported.

TRH has been shown to penetrate mouse brain after intravenous, intraperitoneal (i.p.), intramuscular, oral or rectal administration (33). Systemic administration of TRH has been

shown to produce several distinctive behavioural responses in rats. These include body

shaking behaviour, often refeπed to as 'wet dog shakes' (WDS) (34, 35) and increased grooming activity (36). The mechanism by which TRH produces these effects is not fully

understood. It has been suggested that the manifestation of such behaviours in the rat

following intrathecal administration of TRH results from an action at the level of the spinal

cord and brainstem and is facilitated by a tonically active noradrenergic pathway via cti-

adrenoreceptors, (37). Also, that WDS in response to i.c.v injection of TRH are dependent on

brain DA (38) and that the raphe-spinal 5-HTergic projection system may serve to modulate

WDS elicited by systemic administration of TRH (39).

By protecting endogenous TRH or exogenously administered TRH from degradation, TRH-

DE inhibitors would be expected to amplify these effects. Materials

Glp-Asn-ProNH₂ and Glp-Asn-ProAMC, were custom synthesized by the American Peptide

Company (U.S.A.). TRH (Glp-His-ProNH₂) was obtained from Sigma-Aldrich. ³[H]-3-

MeHis-TRH was purchased from New England Nuclear.

Methods

Behavioural studies were conducted using experimentally naϊve Male Wistar rats (200-250 g)

maintained in a temperature controlled (21 °C), artificially-lighted (12 hr light cycle) room

with free access to food and water.

Behavioural effects of TRH ± Glp-Asn-Pro AMC following intra-cerebroventricular (i.c.v.) administration:

Animals were implanted with guide cannula aimed at the lateral cerebral ventricles (AP -

0.92mm, ML -1.5mm, DV -2.6mm relative to Bregma, atlas of Paxinos & Watson) under halothane anaesthesia. One week following surgery placement of the cannulae was assessed

by the ability of a microinjection of angiotensin II (200ng in lμl) to induce drinking

behaviour. Seven days later animals that exhibited a drinking response were placed in a

perspex cage for 20 minutes. They were then administered either vehicle (lμl 0.9%) saline) or

Glp-Asn-ProAMC (5μg in lμl) followed 1 minute later by vehicle (lμl 0.9% saline) or TRH

(5μg in lμl). One minute after the last i.c.v. injection animals were placed back into the

perspex cage and their behaviour monitored by a computer automated tracking system for 30

minutes (Ethovision Pro, Noldus) to provide measures of locomotor activity. Animals were

videotaped for subsequent blind analysis of WDS behaviour by a trained observer. A

threshold dose of 5 μg TRH was chosen for i.c.v. administration so that if Glp-Asn-ProAMC

enhanced responses to TRH, this would be clearly visible.

Behavioural effects of TRH ± TRH-DE inhibitor following intraperitoneal (i.p.)

administration:

On the day ofthe experiment, animals were singly housed in perspex cages (30 x 20 x 20 cm)

and allowed to acclimatize for 20 min. After this period, two baseline measurements of

behaviour were made at 10 and 5 min prior to administration of drugs. At time 0, animals

were injected i.p. with either vehicle (0.9% saline), 10 mg/kg TRH, 10 mg/kg Glp-Asn-

ProNH₂, 10 mg/kg Glp-Asn-ProAMC or 10 mg/kg TRH + 10 mg/kg Glp-Asn-Pro AMC.

Animals were then observed by two independent observers who were blind to treatment, every 5 min for 30 sec. Blocks of 5 animals were treated at a time, 1 per drug group. Blocks were repeated 5 times.

The occuπence of individual behaviours were noted using a behavioural check list, which included grooming, locomotion, rearing, chewing, sniffing, head weave, tail elevation, and/or straub tail and WDS behaviours. These behaviours were summed to yield an overall activity

count. Animals were videotaped for separate subsequent analysis of WDS. All WDS

behaviours occuπing during each 5 min interval after drug administration were counted.

Since Glp-Asn-ProAMC was the more potent of the two TRH-DE inhibitors it was selected

for evaluation alone and in combination with TRH for both modes of administration in this preliminary study.

Evaluation of the ability of TRH, Glp-Asn-ProNH₂, and Glp-Asn-ProAMC to bind to ³H-

3MeHis-TRH-labelled receptors in rat cortex:

Radioligand binding assays were conducted to investigate the possibility that the two TRH-

DE inhibitors might exert their effects by acting independently at TRH receptors in the brain.

Cortex tissue from male Sprague-Dawley rats (250 g) was dissected out, weighed and

homogenised in 30 volumes of ice-cold sodium phosphate buffer (0.02 M, pH 7.5). The homogenates were centrifuged at 30,000 g at 4°C for 30 min. The supernatant was discarded

and the pellet was resuspended at 100 mg wet wt/ml in buffer containing 177 μM bacitracin.

The affinities of TRH and each of the TRH-DE inhibitors for rat cortical membranes were

determined in competition binding experiments essentially as described previously by Vonhof

et al., (40) using 6-8 nM ³H-3MeHis-TRH. Non-specific binding was defined in the presence of 10 μM TRH.

Analysis of data:

The statistical significance of differences between behavioural effects of treatments was

determined using the Student's t-test or ANOVA, followed by a post-ANOVA test, as

indicated. Radioligand binding data were analysed using non-linear curve fitting software (Prism, Graph Pad Software Inc.). IC o values calculated from competition experiments were

converted to K, values using the Cheng-Prusoff (41) equation K, = IC₅<J(1+L/K_d)_. where L =

ligand concentration and K = the apparent dissociation constant for [³H]-[3-MeHis]TRH. Results And Discussion

From Figure 5 it can be seen that co-administration of Glp-Asn-ProAMC with TRH resulted

in a significant (3.33 ± 1.23 vs 56.83 ± 11.06, n=6, p<0.001 one-way ANOVA followed by

Bonferπoni test), 18-fold increase in the number of WDS observed during the observation

period. Neither TRH nor Glp-Asn-ProAMC alone produced a significantly greater number of

WDS than vehicle during the 30 min observation period (Figure 5). Time dependent analysis

revealed that the maximum increase in WDS occurred 10 min post administration (p<0.01,

one-way ANOVA followed by a post-ANOVA Bonferπoni test) and steadily declined with

time, but still remained significant after 30 min (Figure 6). At all time points the number of

WDS observed in response to TRH + Glp-Asn-ProAMC was significantly greater than those

observed in response to TRH alone (p<0.01, one-way ANOVA followed by post-ANOVA Bonfeπoni test) (Figure 6). These data show that the central effects of exogenously

administered TRH can be significantly enhanced by the presence of Glp-Asn-ProAMC and

are consistent with exogenously administered TRH being protected from degradation by this

TRH-DE inhibitor. A significant increase in locomotion was not detected in response to either i.c.v. or i.p

administration of TRH ± TRH-DE inhibitors or Glp-Asn-ProNH₂ alone i.p. These results are

consistent with Ervin et al., (36) who noted that groups reporting locomotor hyperactivity in

response to either peripheral or intracerebral administration of TRH assess activity with

photocells or electronic devices, which measure other behaviours in addition to locomotion.

Figure 7 shows that following i.p. administration, TRH caused a significant (pO.Ol,

Repeated Measures ANOVA followed by post-ANOVA Dunnett test) increase in rat activity

scores throughout the 40 min period post injection. Analysis of the individual behaviours contributing to the activity score revealed that, in addition to WDS, TRH primarily promoted

grooming, sniffing, and chewing behaviours. The activity scores of the animals treated with

TRH together with Glp-Asn-ProAMC were also significantly higher than saline-treated rats

(p<0.01, Repeated Measures ANOVA followed by post-ANOVA Dunnett test). It can also be

seen from Fig. 7 that behavioural responses to TRH alone tended to decline 35 min after i.p.

administration, whereas animals treated with TRH plus Glp-Asn-ProAMC showed no such

decrease. Again, these results would be consistent with Glp-Asn-ProAMC protecting

exogenously administered TRH from degradation. Animals treated with Glp-Asn-ProNH₂

i.p. showed significantly increased activity counts (p<0.05, Repeated Measures ANOVA followed by post-ANOVA Dunnett test) for the last 10 min of the observation period

consistent with this inhibitor enhancing the actions of endogenous TRH in the central nervous

system. Administration of TRH i.p. caused an immediate induction of WDS in all animals with the

response gradually declining to zero after 40 min (Figure 8). Induction of WDS in animals

treated with TRH in combination with Glp-Asn-ProAMC was delayed by 5 min in

comparison with TRH alone (p<0.05, Student's t-test) (Figure 8). Nevertheless, administration of Glp-Asn-ProAMC with TRH resulted in a statistically significant

enhancement of responses compared to TRH alone over the last 25 min of the observation

period (p<0.05, 2-way ANOVA, Figure 8).

Overall, the data show that the number of WDS observed in response to either i.c.v or i.p

administration of TRH can be significantly enhanced by the presence of Glp- Asn-ProAMC

and are consistent with exogenously administered TRH being protected from degradation by this TRH-DE inhibitor.

To investigate the possibility that the two TRH-DE inhibitors might exert their effects by

acting independently at TRH receptors in the brain, radioligand binding assays were conducted to evaluate their ability to bind to high affinity receptors in rat brain cortex. In

saturation binding experiments, [³H]-3MeHis-TRH labelled a single population of sites in rat

cortical membranes with an apparent dissociation constant (K_d) of 4.54 ± 0.62 nM (n=5).

TRH competed for binding to [³H]-3-MeHis-TRH-labelled TRH receptors in rat cortical

membranes with a pKj of 7.65 ± 0.17 (n=7). In comparison, the pKj values for Glp-Asn-

ProNH₂ and Glp- Asn-ProAMC were 5.15 ± 0.24 (n=6) and 4.32 ± 0.24 (n=3), respectively.

Thus, both TRH-DE inhibitors were found to have low affinity compared to TRH for [ H]-

3MeHis-TRH-labelled receptors in rat cortical membranes, supporting the interpretation that

inhibition of TRH-DE underpins the observed behavioural effects. In conclusion, these studies demonstrate that, i.c.v. or i.p. administration of these novel,

potent TRH-DE inhibitors potentiate the biological actions of TRH in the central nervous system in vivo. The results provide the first demonstration that TRH-DE inhibitors can

potentiate the central biological actions of TRH in vivo.

Significance of these studies

The results from these preliminary studies: (i) demonstrate that these novel TRH-DE inhibitors are parentally active in vivo at doses of 10 mg/kg i.p., and (ii) show that TRH-DE inhibitors are active in vivo when administered centrally (i.c.v.) at a dose of 5μg indicating central sites of action.

It is considered, with support from the in vivo test results, that the TRH-DE inhibitors ofthe

present invention will be valuable tools for investigating the biological roles of TRH-DE, and

TRH, in the CNS and that they possess pharmacologically advantageous properties which

will allow them to be used to potentiate the endogenous levels of TRH and /or to protect exogenously administered TRH or TRH analogues from degradation. This effect should have

the potential to enhance therapeutic effectiveness of TRH or TRH analogues, particularly in

the treatment of brain and spinal injury and certain CNS disorders.

Desirably, the invention will provide for use of a compound of Formula I^a or a

pharmaceutically acceptable salt thereof in the preparation of a medicament, particularly for

the treatment of brain or spinal injuries or other central nervous system disorders or other

TRH dependent disorders in tissues in which TRH-DE is the principal enzyme influencing

TRH levels. Desirably also, the invention will provide a method of treatment of brain or spinal injuries or

other central nervous system disorders or other TRH-dependent disorders in tissues in which TRH-DE is the principal enzyme influencing TRH levels, which comprises administering to a

patient suffering from such injuries or disorders an amount of a compound of Formula I^a or a

pharmaceutically acceptable salt thereof effective to potentiate endogenous TRH and /or protect exogenously administered TRH or TRH analogues from degradation by TRH-DE.

According to one aspect, the present invention provides pharmaceutical compositions

comprising an effective amount, particularly a TRH-DE inhibiting effective amount, of a

compound of Formula I or a pharmaceutically acceptable salt thereof. Normally the

composition will also comprise a pharmaceutically acceptable caπier, particularly an inert caπier.

The compounds ofthe present invention may be administered by oral, parenteral, intramuscular (i.m.), intraperitioneal (i.p.), intravenous (i.v.) or subcutaneous (s.c.) injection, nasal, vaginal, rectal or sublingual routes of administration and can be formulated in dosage

forms appropriate for each route of administration. Suitable dosage forms are known to those

skilled in the art and are described, for example in US Patent No. 4, 906, 614 Giertz et a] or

US Patent No, 5, 244, 884 Spatola et al Dosage levels should be sufficient to achieve the TRH-DE inhibiting effect required for treatment ofthe particular physical condition being

treated.

REFERENCES

1. Bauer, K. (1994) Eur. J. Biochem. 224, 387-396. 2. O'Connor, B., and O Cuinn, G. (1985) Eur. J. Biochem. 150, 47-52.

3. Griffiths, E. C, Kelly, J. A., Ashcroft, A., and Robson, B. (1989) Ann. N Y. Acad.Sci.

553, 217-231.

4. Wilk, S., and Wilk, E. K., (1989) Neurochem. Int. 15, 81-89.

5. Griffiths, E. C, (1985) Psychoneuroendocrinol. 10, 225-235.

6. Horita, A., Carino, M. A., and Lai, H. (1986) Rev. Pharmacol. Toxicol. 26, 31 1-332.

7. Horita, A. (1998) Life Sci. 62, 1443-1448.

8. Kelly, J. A. (1995) Essays in Biochem. 30, 133-149.

9. Bauer, K. (1995) Metabolism of Brain Peptides (O'Cuinn G., Ed.), pp. 201-213, CRC

Press.

10. Charli, J. L., Cruz, C, Vargas, M. A., and Joseph-Bravo, P. (1988) Neurochem. Int. 13,

237-242.

11. Bauer, K., Heuer, H., Ifflander, F., Schmitmeier, S., Shomburg, L., Turwitt, S., and

Wilkins, M. (1997) Cell-Surface Peptidases in Health and Disease (Kenny, A. J., and

Boustead, CM., Eds.), pp 239-248, BIOS Scientific Publishers Limited, Oxford, UK.

12. Charli, J. L., Vargas, M. A., Cisneros, M., De Gortari, P., Baeza, M. A., Jasso, P.,

Bourdais, J., Perez, L., Uribe, R.M., and Joseph-Bravo, P. (1998) Neurobiology 6, 45-

57.

13. Bauer, K., Carmeliet, P., Schulz, M., Baes, M., and Denef, C. (1990) Endocrinology

127, 1224-1233.

14. Cruz, C, Charli, J. L., Vargas, M. A., and Joseph-Bravo, P. (1991) J. Neurochem. 56,

1594-1601.

15. Heuer, H., Schafer, M, K-H., and Bauer, K. (1998) Thyroid. 8, 915-920. 16. Turner, A. J. (1997) Cell-Surface Peptidases in Health and Disease (Kenny, A. J., and

Boustead, CM., Eds.), pp 239-248, BIOS Scientific Publishers Limited, Oxford, UK.

17. Lanzara, R., Liebman, M., and Wilk, S. (1989) Ann. NY. Acad. Sci. 553, 559-562.

18. O'Connor, B., and O'Cuinn, G. (1987) J. Neurochem. 48, 676-680. 19. Elmore, M. A., Griffiths, E. C, O'Connor, B., and O'Cuinn, G. O. (1990) Neuropeptides

15, 31-36.

20. O'Leary, R. M., and O'Connor, B. (1995) Int. J. Biochem. Cell. Biol. 27, 881-890.

21. Kelly, J. A., Loscher, C. E., Gallagher, S., and O'Connor, B. (1997) Biochem. Soc.

Trans. 25, 114S.

22. Gallagher, S. P., and O'Connor, B. (1998) Int. J. Biochem. Cell Biol. 30, 115-133.

23. Kelly, J. A., Slator, G. R., Tipton, K.F., Williams, C. H., and Bauer, K. Anal. Biochem.

274, 195-202 (1999)

24. O'Cuinn, G., O'Connor, B., and Elmore, M. (1990) J. Neurochem. 54, 1-13.

25. Wilk, S. (1989) Ann. N Y. Acad.Sci. 553, 252-264.

26. Charli, J. L., Mendez, M., Vargas, M-A., Cisneros, M., Assai, M., Joseph-Bravo, P., and

Wilk, S. (1989) Neuropeptides. 14, 191-196.

27. Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res Commun. 27, 157-162.

28. Kelly J.A., Slator G.R., and Bauer K. (1999) J. Neurochem. Suppl. 73, S45.

29. Walker B (1994) Peptide antigens - a practical approach (Wisdom, G. B., Ed) pp 27-

81 , IRL Press, Oxford, U.K.

30. Zimmerman M. Ashe B., Yurewicz E.C and Patel G., Anal. Biochem. (1977) 78, 47-51.

31. Fujiwara K. and Tsuru D., J. Biochem. (1978) 83, 1145-1149.

32. Dixon, M. and Webb, E.C (1979) Enzymes, 3rd Ed., pp 72-75, Longman, London and

Academic Press, New York, U.S.A. 33. Mitsuma , T and Nagimori, T(l 983) Experientia 39, 620-622.

34. Yamada, K., Demarest, K. T., and Moore K. E., (1984) Neuropharmacol. 23, 735-739.

35. Bennett, G. W., Marsden, C. A., Fone, K. C. F., Johnson, J. V., and Heal, D. J. (1989) Ann. N. Y. Acad. Sci. 553, 106-120.

36. Ervin, G. N., Schmitz, S. A., Nemeroff, C. B. and Prange, A. J. Jr., (1981) Eur. J. Pharmacol. 72, 35-43.

37. Johnson, J. V., Fone, K. C. F., Havler, M. E., Tulloch, I. F., Bennett, G. W. and Marsden, C. A. (1989) Neuroscience 29, 463-470.

38. Drust E. G., and Connor, J. D. (1983) J. Pharmacol. Exp. Ther. 224, 148-154.

39. Funk, D., Post, R. M., and Pert, A. (1997) Psychopharmacology 133, 356-362.

40. Vonhof, S., Feuerstein, G., Cohen, L. A., and Labroo, V. M., (1990) Eur. J. Pharmacol. 180, 1-12.

41. Cheng, Y. C. and Prusoff, W. H., (1973) Biochem. Pharmacol. 22, 3099-3108.

Claims

1. Novel compounds of the formula I:

wherein:

R is an optionally substituted 4-, 5- or 6-membered heterocyclic ring having one or more

heteroatoms, in which at least one carbon atom ofthe ring is substituted with O or S;

X¹ is -CO- or -CS- or -CH₂CO- or CH(R⁴) wherein R⁴ is H or optionally substituted alkyl or

-COOH or -COOR¹ ' wherein R¹ ' is optionally substituted alkyl;

X and X (which may be the same or different) are -CO- or -CS- ;

Z is -CH₂- or -S- or -O- or -ΝH-;

Q is O or S;

R is H or optionally substituted alkyl or an optionally substituted carbocyclic ring;

R is H or optionally substituted alkyl or an optionally substituted mono- or polycychc ring,

optionally having one or more heteroatoms in the ring(s) and optionally being a fused ring; or

R² and R³ together form an optionally substituted mono- or polycychc ring optionally having

one or more heteroatoms in the ring(s) and optionally being a fused ring;

R⁵ and R⁶ (which may be the same or different) are H, or lower alkyl; R⁷ and R⁸ (which may be the same or different) are H, or optionally substituted lower alkyl; R⁹ and R¹⁰ (which may be the same or different) are H, or optionally substituted alkyl, or an optionally substituted carbocyclic ring;

Y is -(CH )_n- where n is 0, 1, 2 or 3 provided that when R² and R³ form part ofthe ring n is 0;

provided that when R¹ is:

and X¹, X² and X³ are -CO-

and R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ are H

and Z is -CH₂-

and Q is O

and Y is -(CH₂)_n- where n is 0,

then R² and R³ are not both H;

and pharmaceutically acceptable salts thereof.

2. Compounds of the formula I as defined in claim 1, wherein R¹, X¹, X², X³, Z, Q, R⁵,

R⁶, R⁷, R⁸, R⁹, R¹⁰ and Y are as defined in claim 1,

R is H or optionally substituted alkyl or an optionally substituted carbocyclic ring,

R³ is optionally substituted alkyl or an optionally substituted mono- or polycychc ring,

optionally having one or more heteroatoms in the ring(s) and optionally being a fused ring;

or R² and R³ together form an optionally substituted mono- or polycychc ring optionally

having one or more heteroatoms in the ring(s) and optionally being a fused ring; and

pharmaceutically acceptable salts thereof.

3. Compounds according to either of the preceding claims wherein R or R , or R and

R together, represent an optionally substituted mono- or polycychc ring.

4. Compounds according to any ofthe preceding claims wherein R⁵ and R⁶ are H and Q is O_, so that the compounds are thyrotropin-releasing hormone (TRH) derivatives having L-

asparagine residue (Asn) in the Pi' position.

5. Compounds according to claim 2 having L-asparagine (Asn) in the Pi' position of the

peptide with the general formula:

R¹-X¹-L-Asn-L-Pro-NR²Y R³,

where R , X , R , Y and R are as defined in claim 1.

7 8

6. Compounds according to any of claims 1-4 wherein Z is -CH - and R and R are H.

1 9 "-Ϊ

7. Compounds according to any of claims 1-4 or 6 wherein X , X and X are -CO-

8. Compounds according to any ofthe preceding claims wherein R or R , or R and R

together, represent a large hydrophobic group.

9. Compounds according to any ofthe preceding claims wherein R¹ is selected from any

ofthe following:

wherein R , 12 is hydrogen, lower alkyl or phenyl, R¹³ is hydrogen or lower alkyl,

Q is O or S.

10. Compounds according to claim 9 wherein Q is O.

11. Compounds according to any ofthe preceding claims wherein R¹ is a five-membered

pyπolidinone, thiazolidinone or butyrolactone ring.

12. Compounds according to any ofthe preceding claims wherein R¹ is:

13. Compounds according to any of the preceding claims having substituents present which do not interfere substantially with the function of the compounds as inhibitors of

activity of thyrotropin-releasing hormone-degrading ectoenzyme (TRH-DE).

14. Glp-L-Asn-L-ProAMC

15. A novel compound selected from:

Glp-Asn-Pro-isopropylamide, Glp-Asn-Pro-cyclohexamide, Glp-Asn-Pro-

piperazide, Glp-Asn-Pro-5-amidoquinoline (or 6-amidoquinoline or 8-amidoquinoline), Glp-

Asn-Pro-5 -amido-isoquinoline, Glp-Asn-Pro-Anilide, Glp- Asn-Pro-7- amido(trifluoromethyl)coumarin, Glp-Asn-Pro-6-amido-3,4-benzocoumarin, Glp-Asn-Pro-

1,2,3, 4-tetrahydro-l -naphthylamide, Glp-Asn-Pro-5,6,7,8- tetrahydro-1 -naphthylamide, Glp- Asn-Pro-benzylamide, Glp-Asn-Pro-2-thiazolamide, Glp-Asn-Pro- 1 -naphthylmethylamide,

Glp-Asn-Pro-p-Anisidide, Glp-Asn-Pro-para amidobenzoic acid, Glp-Asn-Pro-w-anisidide,

Glp-Asn-Pro-o-anisidide, Glp-Asn-Pro-5-chloro-2-methoxy anilide, Glp-Asn-Pro-3-hydroxy-

4- methoxy anilide, Glp-Asn-Pro-2-hydroxy anilide, Glp-Asn-Pro-2-(hydroxymethyl-)

anilide, Glp-Asn-Pro-4- trifluoromethyl anilide,

and pharmaceutically acceptable salts thereof.

16. A compound according to claim 15 selected from:

Glp-Asn-Pro- 1,2,3, 4-tetrahydro-l -naphthylamide, Glp-Asn-Pro-5,6,7,8- tetrahydro-1 -

naphthylamide, Glp-Asn-Pro-benzylamide, Glp-Asn-Pro- ?-anisidide, Glp-Asn-Pro-w-

anisidide, Glp-Asn-Pro-ø-anisidide, Glp-Asn-Pro-5-chloro-2-methoxy anilide, Glp-Asn-Pro-

3-hydroxy-4-methoxy anilide, Glp-Asn-Pro-2-hydroxy anilide, Glp-Asn-Pro-2-

(hydroxymethyl-) anilide,

and pharmaceutically acceptable salts thereof.

17. A compound of formula I as defined in claim 1 or a compound according to claim 15

or 16, or a pharmaceutically acceptable salt of any of the foregoing, for use in a method for

treatment of the human or animal body by therapy or a diagnostic method optionally practised

on the human or animal body.

18. A compound of formula l^a:

wherein:

R¹ is an optionally substituted 4-, 5- or 6-membered heterocyclic ring having one or more heteroatoms, in which at least one carbon atom ofthe ring is substituted with O or S;

X¹ is -CO- or -CS- or -CH₂CO- or CH(R⁴) wherein R⁴ is H or optionally substituted alkyl or

-COOH or -COOR¹¹ wherein R¹¹ is optionally substituted alkyl;

X² and X³ (which may be the same or different) are -CO- or -CS- ;

Z is -CH₂- or -S- or -O- or -ΝH-;

Q is O or S;

R² is H or optionally substituted alkyl or an optionally substituted carbocyclic ring;

R³ is H or optionally substituted alkyl or an optionally substituted mono- or polycychc ring,

optionally having one or more heteroatoms in the ring(s) and optionally being a fused ring; or

R² and R³ together form an optionally substituted mono- or polycychc ring optionally having

one or more heteroatoms in the ring(s) and optionally being a fused ring;

R⁵ and R⁶ (which may be the same or different) are H, or lower alkyl;

R⁷ and R⁸ (which may be the same or different) are H, or optionally substituted lower alkyl;

R⁹ and R¹⁰ (which may be the same or different) are H, or optionally substituted alkyl, or an

optionally substituted carbocyclic ring;

Y is -(CH₂)_n- where n is 0, 1, 2 or 3 provided that when R² and R³ form part ofthe ring n is 0; and pharmaceutically acceptable salts thereo

for use as an inhibitor of activity of thyrotropin-releasing hormone-degrading ectoenzyme

(TRH-DE).

19. A compound of formula I^a as defined in claim 18, or a pharmaceutically acceptable

salt thereof, for use in potentiating thyrotropin-releasing hormone.

20. Glp-L-Asn-L-ProNH₂

or a pharmaceutically acceptable salt thereof, for use as an inhibitor of activity of thyrotropin- releasing hormone-degrading ectoenzyme (TRH-DE).

21. Use of a compound of formula I^a as defined in claim 18, or a pharmaceutically

acceptable salt thereof, in inhibiting activity of TRH-DE.

22. Use according to claim 21 in potentiating endogenous thyrotropin-releasing hormone

(TRH) and/or in protecting exogenously administered TRH or TRH analogues from

degradation by TRH-DE.

23. A pharmaceutical composition comprising an effective amount of a compound of the

formula I^a as defined in claim 18, or a pharmaceutically acceptable salt thereof, together with

a pharmaceutically acceptable caπier.

24. A pharmaceutical composition according to claim 23 which also contains TRH or a

TRH analogue.

25. Use of a compound of formula I^a as defined in claim 18, or a pharmaceutically

acceptable salt thereof, in the manufacture of a medicament for potentiating endogenous

thyrotropin-releasing hormone (TRH) and/or in protecting exogenously administered TRH or

TRH analogues from degradation by TRH-DE.

26. Use of a compound of formula I^a as defined in claim 18, or a pharmaceutically

acceptable salt thereof, in the manufacture of a medicament for the treatment of brain or

spinal injuries, or other central nervous systems disorders, or other TRH dependent disorders

in tissues in which TRH-DE is the principal enzyme influencing TRH levels.

27. A method of treatment of brain or spinal injuries, or other central nervous systems

disorders, or other TRH dependent disorders in tissues in which TRH-DE is the principal

enzyme influencing TRH levels, which comprises administering to a patient suffering from

such injuries or disorders an amount of a compound of formula 1 ^a as defined in claim 18, or a

pharmaceutically acceptable salt thereof, effective to potentiate endogenous TRH and /or

protect exogenously administered TRH or TRH analogues from degradation by TRH-DE.