WO1997018238A2

WO1997018238A2 - Crf analogs

Info

Publication number: WO1997018238A2
Application number: PCT/EP1996/005010
Authority: WO
Inventors: Joachim Spiess; Andreas RÜHMANN
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 1995-11-14
Filing date: 1996-11-14
Publication date: 1997-05-22
Also published as: WO1997018238A3

Abstract

The present invention relates to lipophilic CRF analogs which are useful as active ingredients in pharmaceutical or diagnostic compositions.

Description

CRF Analogs

Corticotropin-releasing factor (CRF) appears to integrate the endocrine, autonomic, immunologic and behavioral responses to stress in the CNS. The 41 residue polypeptide was originally characterized on the basis of its hypophysiotropic activity stimulating the release of adrenocorticotropic hormone (ACTH), which is known to stimulate the secretion of glucocorticoids from the adrenal cortex. It is generally accepted that CRF is the main regulator of the hypothalamus-pituitary-adrenal axis (HPA) leading to the release of glucocorticoids after exposure to stress whereby the signal transduction is mediated through receptors. Agents that can imitate or block the biological function of CRF (agonists or antagonists, respectively) may be useful in the investigation of peptidergic systems and also as therapeutic agents, e.g. in the prevention or treatment of stress, depression and anxiety and other psychosomatic diseases. An example of a CRF antagonist is astressin, cyclo(30-33) [D-Phe¹²,Nle^21,38,Glu³⁰,Lys³³]h/rCRF-(12-41).

In order to enhance their bioavailability and to produce the desired effect, useful compounds have to be able to bind to the respective receptors in the brain which requires passage through the blood-brain barrier. Thus, the technical problem underlying the present invention is to provide CRF analogs having high affinity to the CRF receptor which have been modified so that they can easily pass the blood-brain barrier.

The solution to said technical problem is provided by the embodiments characterized in the claims.

Accordingly, the present invention provides lipophilic CRF analogs wherein, preferably, several amino acids in the native CRF have been replaced by lipophilic amino acids, preferably alanine. The analogs may be straight-chained or cyclic.

In this context the term "analog" encompasses any variant or fragment of CRF which retains CRF ligand binding activity.

Specific examples of such CRF analogs are (Ala20,22,25,32,33) oCRF(1-41) and (D-Phe12, Ala20,22,

25,32,33) oCRF(12-41) as well as Ala32 - Astressin and 3Ala-Astressin. The amino acid sequences of some CRF analogs are shown in Table I.

Brief description of the figures;

Fig. 1 shows cAMP accumulation (fmol cAMP/tube) in Y79 cells (2.5 × 10⁸ cells/tube) stimulated by oCRF (1-41) [ ], h/rCRF (1-41) [ ], (Ala20,22,25,32,33) oCRF (1-41) [+] and Ala6 oCRF (1-41) [×] in a concentration dependent manner (c = 0-1 μM); incubation time = 30 min.

Fig. 2 shows cAMP accumulation (fmol cAMP/tube) in Y79 cells (2.5 × 10⁸ cells/tube) stimulated by oCRF (1-41) [ ] in a concentration dependent manner (c = 0-1 μM) and in the presence of either 1 μM (D-Phe12, Nle21,38) h/rCRF (12- 41) [ ] or 1 μM (D-Phe12, Ala20,22,25,32,33) oCRF (12-41) [×]; incubation time = 30 min.

The CRF analogs of the invention can be prepared by usual peptide synthesis methods, e.g. by the fmoc solid phase method, using a peptide synthesizer model 433A (Applied Biosystems), see, e.g. Barany, G. and Merrifield, R.B. 1980. The Peptides. Analysis, Synthesis, Biology (Gross, E. and Meienhofer, J. ed.), Vol, 2, pp. 1-284, Academic Press, New York or Atherloo, E. and Sheppard, R.C. 1987. The Peptides. Analysis, Synthesis, Biology (Underfriend, S. and Meienhofer, J. ed.) Vol. 9, pp. 1-38, Academic Press, New York.

Table II shows some properties of CRF analogs.

The activity of the new CRF analogs has been tested on a human Y79 retinoblastoma cell line (ATCC HTB 18) in which a functional CRF receptor which stimulates adenylate cyclase activity after occupation with CRF has been observed (Olianas, M.C., Lampis, G., and Onali, P., J. Neurochem. 64, No. 1 (1995), 402-407).

For the determination of the binding affinity of the peptides, CRF analogs were tested in competitive binding assays with [¹²⁵I-Tyr⁰]oCRF in membrane homogenates of Y79 cells. The binding constants for the high-affinity (K_d1) and low-affinity site (K_d2) of the peptides at the receptor were calculated with the nonlinear curve fitting program LIGAND.

The ability of peptides to stimulate (agonistic activity) or inhibit oCRF-stimulated (antagonistic activity) cAMP production in Y79 cells was assayed as described and data analysis to determine the half-maximal response (EC50) or the apparent inhibitory constant (K_i) of the CRF analogs was obtained with the sigmoidal dose-response curve fitting program ALLFIT. The results are shwon in Table ill and in Fig. 1 and 2.

(Ala20,22,25,32,33) oCRF(1-41) is an agonist of CRF which shows a higher potency and affinity to the CRF receptor than oCRF (see Fig. 1 and Table III). It exhibits approximately two to three times higher binding affinity to the CRF receptor and stimulates cAMP production with an EC₅₀ value which was five times lower than that found for oCRF (1-41).

(D-Phe12, Ala20,22,25,32,33) oCRF (12-41) is an antagonist of CRF. It is similar to the known CRF analog (D-Phe12, Nle12,38) h/rCRF (12-41) when binding to the receptor

(K_d = 33 nM) and shows similar biopotency (K_i = 34 nM) in suppressing cAMP stimulation by oCRF (1-41) in a concentration dependent manner (see Fig. 2 and Table III). The Kd-value was determined according to Olianas et al., supra. The activity was determined as described in Olianas, M.C., Lampis G. and Onali, P., J. Neurochem. 64, No. 1

(1995), 394-401.

Ala32-Astressin is an antagonist of CRF. Table III shows that it has a better or at least about the same CRF antagonist activity when compared with astressin.

The CRF analogs of the present invention show a significantly raised lipophilicity compared with native CRF. This high lipophilicity facilitates the passage through the blood-brain barrier and makes the compounds of the invention useful as tools in investigations of peptidergic systems or as therapeutic or diagnostic agents, e.g. in prevention and treatment of stress, depression and anxiety and other psychosomatic diseases whereby the compounds of the present invention can be used alone or in combination with other agents and/or treatments. Example 1

Synthesis of Peptides

The CRF peptides were synthesized with Fmoc chemistry on TentaGel S RAM resin (0.1 mmole scale, Rapp, Tubingen, F.R.G.) with a model ABI 433A peptide synthesizer (Applied Biosystems). After cleavage of the peptides from the resin, the crude peptides were purified by preparative reverse-phase HPLC (RPHPLC) performed on a Waters Prep Nova-Pak HR C₁₈ silica gel column (5 × 30 cm, 6 μm-particle size, 6-nm pore size) with a mixture of aqueous 0.1% trifluoroacetic acid (TFA) and MeCN. The mass spectra of the purified peptides were measured with ESI MS on a Micromass AutoSpec-T tandem mass spectrometer.

For the synthesis of the cyclized CRF analogs, amino acid derivatives Fmoc-Glu(OA1)-OH and Fmoc-Lys(Aloe)-OH (PerSeptive Biosystems GmbH, Hamburg, F.R.G.) were used. The side-chain protected peptides were reacted with Pd⁰[PPh₃]₄ in HOAc/N-methylaniline/dichloromethane (v/v; 2:1:40) for three hours and then cyclized with HOBt/HBTU in DMF and DIEA in NMP for eight hours. After removal of the N-terminal Fmoc group with piperidine in NMP, the peptide resins were treated as mentioned above.

Example 2

Characterization of Peptides

For further characterization of the physicochemical properties of the pure peptides, all CRF peptides were subjected to analytical RPHPLC on a Vydac C₁₈ silica gel column (0.46 × 25 cm, 5 μm-particle size, 30-nm pore size) with solvents A (0.1% TFA in water) and B (80% MeCN in 0.1% TFA in water) at a flow rate of 1 ml/min. The samples were eluted with 5% B for 5 min and then with a linear gradient of 5-95% B in 30 min.

Circular dichroism (CD spectra were obtained with a computer-controlled J720 spectropolarimeter (Jasco, Groβ Umstadt, F.R.G.). Spectra were collected at 1.0 nm intervals in the range of 190-250 nm in one run using a 8.0 s response time and a spectral band width of 1.0 nm in 1.0 mm cuvettes thermostated at 20°C. Peptides were dissolved in either sodium phosphate buffer (20 mM, pH 6.5) or a mixture of trifluoroethanol (TFE) and sodium phosphate buffer (SPB) (v/v; 50:50). Concentrations were based on the calculated molecular weight of the TFA salt of the purified lyophilized peptide. Data analysis waε achieved with the Provencher computer program.

Claims

CLAIMS :

1. Lipophilic CRF analog.

2. CRF analog of claim 1 wherein several amino acids of the native CRF have been replaced by a lipophilic amino acid.

3. CRF analog of claim 1 or 2 wherein the lipophilic amino acid is Ala.

4. CRF analog of any one of claims 1 to 3 which is (Ala20,22,25,32,33) oCRF (1-41).

5. CRF analog of any one of claims 1 to 3 which is (D- Phe12, Ala20,22,25,32,33) oCRF (12-41).

6. CRF analog of any one of claims 1 to 3 which is cyclo (30-33) [ D-Phe¹², Nle^21,38, Glu³⁰, Ala³², Lys³³] h/rCRF- (12-41).

7. CRF analog of claim 4 for use as an agonist of CRF.

8. CRF analog of claim 5 or 6 for use as an antagonist of CRF.

9. Pharmaceutical composition comprising a CRF analog of any one of claims 1 to 6 and, optionally, a pharmaceutically acceptable carrier.

10. Diagnostic composition comprising a CRF analog of any one of claims 1 to 6 and, optionally, a suitable carrier.