Conjugates of lipophilic moieties and fragments of vasoactive intestinal peptide (VIP)

The invention concerns novel conjugates of peptide fragments of vasoactive intestinal peptide (VIP) or analogues thereof having 3-12 amino acid residues, and lipophilic moieties, which may be present at the N- or C-terminus. The invention further concerns pharmaceutical compositions containing these novel conjugates which may be used for treatment of male impotence or for the treatment of neurodegenerative diseases.

FIELD OF THE INVENTION
 The present invention concerns novel conjugates of a lipophilic moiety and
 a peptide of 3-12 amino acids. The present invention further concerns
 pharmaceutical compositions comprising as an active ingredient said novel
 conjugates. The pharmaceutical compositions of the invention are
 preferably used for the treatment of male impotence or for the treatment
 of neurodegenerative diseases.
 BACKGROUND OF THE INVENTION
 Vasoactive intestinal peptide (VIP), a 28 amino acid neuropeptide widely
 distributed in the mammalian nervous system, has potent neurotrophic
 actions that influence nerve cell function. In the central nervous system,
 this role of VIP is translated into developmental effects, display of
 growth factor activities and maintenance of neuronal survival and
 function. Neurons, which are capable of releasing VIP, innervate blood
 vessels throughout the body, as well as the trachea in the lung, and the
 released VIP serves as a potent vasodilator, inducing smooth muscle
 relaxation. Radioligand binding assays, pharmacological experiments,
 molecular cloning and development of superactive novel derivatives have
 indicated several classes of VIP receptor sites and several potential
 therapeutical uses.
 Two possible therapeutical uses of VIP, modified VIP or lipophilic VIP
 derivatives were reported in our previous Patents IL 87055, EP 0354992 and
 U.S. Pat. No. 5,147,855 and published patent applications EP 0540969 and
 EP 0620008 which are directed to the treatment of male impotence by
 transdermal administration and to the treatment of neurodegenerative
 diseases, respectively.
 VIP is a hydrophilic peptide of a very short half life in the serum (Said,
 S. I., Editor, Vasoactive intestinal peptide in: Advances in Peptide
 Hormone Research Series, Raven Press, New York, 1-512 (1982)) having the
 following sequence:
 (SEQ ID NO:1)
 1 2 3 4 5 6 7 8 9 10 11 12
 His-Ser-Asp-Ala-Val-Phe-Tyr-Asp-Asn-Tyr-Thr-Arg-
 13 14 15 16 17 18 19 20 21 22 23 24
 Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-
 25 25 27 28
 Ser-Ile-Leu-Asn-NH.sub.2
 To enhance its biological availability and increase its stability the
 present inventors have resorted to two chemical modifications reported in
 said patents and applications. The first was lipophilization, namely, the
 addition of a fatty acid moiety, designed to augment VIP's ability to
 penetrate biological membranes without loss of activity; thus,
 stearoyl-VIP, a molecule combining VIP with a stearic acid moiety at its
 N-terminal was designed (EP 0354992). The second modification was the
 replacement of native amino acids with unnatural amino acids, namely, a
 substitution of methionine (amino acid 17 of VIP) by norleucine, aimed at
 stabilizing the molecule against oxidation as well as at increasing
 lipophilicity; thus, stearoyl-Nle-VIP was designed (Gozes et al.,
 Endocrinology, 134:2121-2125 (1994); Fauchere et al., Int. J. Peptide
 Protein Res., 32:269-278 (1988); EP 0540969). Unmodified VIP fragments
 derived from the 17-24 positions of the VIP sequence are described in EP
 0225020 as ulcer inhibitors.
 A major obstacle in the use of any substance as a medicament is its
 distribution in the body. The modified VIP or lipophilic VIP used for
 transdermal treatment of male impotence reported in the abovementioned EP
 0354992 and EP 0540969 have to penetrate through the dermis and reach the
 erectile tissues in a short a time span as possible.
 VIP, modified VIP or lipophilic VIP used to treat neurodegenerative
 diseases described in EP 0620008 have to pass the blood brain barrier in
 order to exert their therapeutic effect on brain cells.
 It would have been desirable, both for the purpose of treatment of male
 impotence and for the purpose of administration to the CNS for the
 treatment of neurodegenerative diseases, to use molecules that, while
 having the physiological activity of the full VIP peptide, are smaller in
 size and thus are able to improve the bioavailability of the therapeutic
 compound at the target tissue. Furthermore, smaller molecules are at times
 more stable to degradation than larger molecules since, as a rule, they
 have less sites available to degradation.
 SUMMARY OF THE INVENTION
 The present invention is based on the surprising finding that short
 fragments of VIP or modified VIP conjugated to a lipophilic moiety, which
 are 3-12 amino acids long, are physiologically active in the treatment of
 impotence and/or neurodegenerative diseases. The advantage of using short
 physiologically active peptides conjugated to a lipophilic moiety versus
 the usage of the full VIP molecule is better biodistribution and
 bioavailability in the body, as well as ease of preparation. Furthermore,
 the invention concerns short cyclic peptides containing said short
 fragments of VIP or of modified VIP conjugated to a lipophilic moiety
 which in addition to the above advantages feature the advantage of being
 relatively degradation resistant.
 The present invention is concerned with a conjugate of a peptide coupled to
 a lipophilic moiety, wherein the peptide has at least 3 and at most 12
 amino acid residues, said conjugate being selected from the formulae:
EQU (i) R.sub.1 -X.sub.1 -X.sub.1 '-X.sub.1 "-X.sub.2 -NH--R.sub.2 (SEQ ID
 NO:2);
EQU (ii) R.sub.1 -X.sub.3 -Ser-X.sub.4 -Leu-Asn-NH--R.sub.2 (SEQ ID NO:3);
 ##STR1##
 wherein
 R.sub.1 is H or a lipophilic moiety;
 R.sub.2 is H, a lipophililc moiety, a lipophilic moiety substituted by
 X.sub.3 -Ser-X.sub.4 -Leu-Asn-NHR.sub.1 (SEQ ID NO:79) or a spacer
 consisting of 1-3 residues of a non-charged amino acid coupled to X.sub.1
 -X.sub.1 '-X.sub.1 "-X.sub.2 NHR.sub.1 (SEQ ID NO:80),
 with the proviso that at least one of R.sub.1 and R.sub.2 is a lipohilic
 moiety;
 X.sub.1 is a covalent bond, Ala, Val, Ala-Val, Val-Ala, L-Lys, D-Lys,
 Ala-Lys, Val-Lys, Ala-Val-Lys; Val-Ala-Lys or Orn;
 X.sub.1 is L-Lys, D-Lys or Orn;
 X.sub.1 " is L-Tyr, D-Tyr, Phe, Trp or the residue of p-amino
 phenylalanine;
 X.sub.4 is Ile or Tyr;
 X.sub.5 is a residue of a hydrophobic aliphatic amino acid;
 X.sub.2 is X.sub.5, X.sub.5 -Asn, X.sub.5 -Ser, X.sub.5 -Ile, X.sub.5 -Tyr,
 X.sub.5 Leu, X.sub.5 -Nle, X.sub.5 -D-Ala, X.sub.5 -Asn-Ser, X.sub.5
 -Asn-Ser-Ile (residue 1-4 of SEQ ID NO:75), X.sub.5 -Asn-Ser-Tyr (residues
 1-4 of SEQ ID NO:76); X.sub.5 -Asn-Ser-Ile-Leu (residues 1-5 of SEQ ID
 NO:75), X.sub.5 -Asn-Ser-Tyr-Leu (residues 1.5 of SEQ ID NO:76), X.sub.5
 -Asn-Ser-Ile-Leu-Asn (SEQ ID NO:75) or X.sub.5 -Asn-Ser-Tyr-Leu-Asn (SEQ
 ID NO:76);
 X.sub.3 is a covalent bond, Asn, X.sub.5, X.sub.5 -Asn, Tyr-X.sub.5,
 Tyr-X.sub.5 -Asn, Lys-X.sub.5, Lys-X.sub.5 -Asn, Lys-Tyr-X.sub.5,
 Lys-Tyr-X.sub.5 -Asn (residues 4-7 of SEQ ID NO:77), Lys-Lys-Tyr-X.sub.5
 ((residues 3-6 of SEQ ID NO:77), Lys-Lys-Tyr-X.sub.5 -Asn (residues 3-7 of
 SEQ ID NO:77), Val-Lys-Lys-Tyr-X.sub.5 (residues 2-6 SEQ ID NO:78),
 Val-Ala-Lys-Lys-Tyr-X.sub.5 -Asn (SEQ ID NO:77), or
 Ala-Val-Lys-Lys-tyr-X.sub.5 -Asn (SEQ ID NO:78);
 X.sub.6 is a covalent bond or Asn, Ser, Ile, Tyr, Leu, Asn-Ser,
 Asn-Ser-Ile, Asn-Ser-Tyr, Asn-Ser-Ile-Leu (residues 2-5 of SEQ ID NO:75),
 Asn-Ser-Tyr-Leu (residues 2-5 of SEQ ID NO:76), Asn-Ser-Ile-Leu-Asn
 (residues 2-6 of SEQ ID NO:75) or Asn-Ser-Tyr-Leu-Asn (residues 2-6 of SEQ
 ID NO:76);
 X.sub.7 is a covalent bond or Asn;
 X.sub.8 is a covalent bond, X.sub.5, Tyr, Lys, Tyr-X.sub.5, Lys-X.sub.5,
 Lys-Tyr-X.sub.5, Lys-Lys-Tyr-X.sub.5 (residues 3-6 of SEQ ID NO:77),
 Val-Lys-Lys-Tyr-X.sub.5 (residues 2-6 of SEQ ID NO:78),
 Ala-Lys-Lys-Tyr-X.sub.5 (residues 2-6 of SEQ ID NO:77), or
 Ala-Val-Lys-Lys-Tyr-X.sub.5 (residues 1-6 of SEQ ID NO:78);
 Z is --CCNH--, --NHCO--, --S--S--, --S(CH.sub.2).sub.t CO--NH-- or
 --NH--CO(CH.sub.2).sub.t S--;
 m is 1 or 2 when Z is --CONH--, --S--S-- or --S(CH.sub.2).sub.t CO--NH--,
 or m is 2, 3 or 4 when Z is --NH--CO-- or --NH--CO(CH.sub.2).sub.t S--;
 n is 1 or 2 when Z is --NH--CO--, --S--S-- or --NH--CO(CH.sub.2).sub.t S--,
 or n is 2, 3 or 4 when Z is --CONH-- or --S(CH.sub.2).sub.t CO--CO--NH--,
 and
 t is 1 or 2,
 with the proviso that the conjugate
 stearoyl-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID
 NO:24) is excluded.
 The hyrdophobic aliphatic amino acid represented above by X.sub.5 may be a
 residue of D- or L- amino acid selected from Ala, Ile, Leu, Met, Val, Nva
 and Nle.
 The term "lipophilic moiety of the conjugates of the invention" will refer
 in the following description and claims to: a saturated or unsaturated
 hydrocarbyl or carboxylic acyl radical having at least 3 carbon atoms such
 as propionyl, caproyl, laurly, palmitoyl, stearoyl, oleyl, eicosanoyl,
 docsanoyl and the respective hydrocarbyl radicals propyl, hexyl, dodecyl,
 hexadecyl, octadecyl, eicosanyl and docosanyl. Preferably the hydrocarbyl
 or acyl radical is saturated, and has 3-22 carbon atoms.
 The term "spacer" refers to residue of a non-charged natural or non-natural
 amino acid such as alanine, proline and aminocaproic acid.
 Examples of the conjugates of the invention are conjungates of a lipophilic
 moiety and peptides of the sequence Lys-Lys-Tyr-Leu derived from position
 20-23 of the VIP sequence (SEQ ID NO:1) and/or peptides of the sequence
 Asn-Ser-Ile-Leu-Asn, derived from positions 24-28 of the VIP sequence (SEQ
 ID NO:1). modified peptides thereof in which amino acid residues have been
 replaced, added, deleted or chemically modified or combinations of these
 two sequences such as:
 St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6);
 St-Lys-Lys-Tyr-D-Ala-NH.sub.2 ;
 St-Ala-Val-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:7);
 St-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:8);
 St-Lys-Lys-Tyr-Val-NH.sub.2 (SEQ ID NO:9);
 St-Ser-Ile-Lau-Asn-NH.sub.2 ;
 St-Lys-Lys-Tyr-Leu-Nle-NH.sub.2 (SEQ ID NO:10);
 St-Asn-Ser-Tyr-Leu-Asn-NH.sub.2 (SEQ ID NO:11);
 St-Asn-Ser-Ile-Tyr-Asn-NH.sub.2 (SEQ ID NO:12);
 St-Lys-Lys-Tyr-Leu-Pro-Pro-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:13;
 Lau-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:14);
 Cap-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:15);
 St-Lys-Tyr-Leu-NH.sub.2 ;
 St-Lys-Lys-Tyr-Nle-NH.sub.2 (SEQ ID NO:16);
 St-Val-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:17);
 St-Leu-Asn-Ser-Ile-Leu-Asu-NH.sub.2 (SEQ ID NO:18;
 St-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:19);
 St-Lys-Lys-Tyr-Leu-Asn-NH.sub.2 (SEQ ID NO:20);
 St-Lys-Lys-Tyr-Leu-Asn-Ser-NH.sub.2 (SEQ ID NO:21);
 St-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-NH.sub.2 (SEQ ID NO:22); and
 St-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-NH.sub.2 (SEQ ID NO:23).
 In the following, the symbol "St" stands for stearoyl, "Lau" stands for
 lauroyl and "Cap" stands for caproyl.
 By another aspect the present invention concerns pharmaceutical
 compositions comprising as an active ingredient, active conjugates of the
 invention together with a pharmaceutically acceptable carrier.
 The pharmaceutical composition of the invention comprising a conjugate of
 the invention or the conjugate
 St-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:24)
 (hereinafter "Peptide") described in EP 0620008 may be used for the
 treatment of sexual disfunctions such as male impotence, preferably by
 transdermal or urinary tract application. Preferred conjugates used for
 this purpose are:
 St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6);
 St-Lys-Lys-Tyr-D-Ala-NH.sub.2 ;
 St-Ala-Val-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:7);
 St-Asn-Ser-Ile-Leu-Asn-NH.sub.2 ;
 St-Lys-Lys-Tyr-Val-NH.sub.2 (SEQ ID NO:9);
 St-Ser-Ile-Lau-Asn-NH.sub.2 ;
 St-Asn-Ser-Tyr-Leu-Asn-NH.sub.2 (SEQ ID NO:11);
 St-Asn-Ser-Ile-Tyr-Asn-NH.sub.2 ;
 St-Lys-Lys-Tyr-Leu-Nle-NH.sub.2 (SEQ ID NO:10); and
 St-Lys-Lys-Tyr-Leu-Pro-Pro-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:13).
 The pharmaceutical compositions of the invention may also be used for the
 treatment of neurodegenerative diseases, such as Alzheimer, Down Syndrome,
 hypoxia, decline in motor or cognitive function due to ischemia, stroke,
 hereditary diseases of the central and peripheral nervous system, decline
 in motor or cognitive function due to injury of the central or peripheral
 nervous system, decline in cognitive functions due to old age and
 neurological disorders associated with blood circulation and neuronal
 survival. The term "treatment" should be understood in the context of the
 present invention as alleviation, improvement or abolishment of the
 abnormal conditions manifested in those diseases and more particularly to
 improvement in cognitive functions damaged by those diseases. Preferred
 conjugates used for this purpose are:
 St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6);
 St-Lys-Lys-Tyr-D-Ala-NH.sub.2 ;
 St-Ala-Val-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:7);
 St-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:8);
 St-Lys-Lys-Tyr-Val-NH.sub.2 (SEQ ID NO:9);
 St-Ser-Ile-Lau-Asn-NH.sub.2 ;
 Lau-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:14);
 Cap-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:15);
 St-Lys-Tyr-Leu-NH.sub.2 ;
 St-Lys-Tyr-Leu-NH.sub.2 ;
 St-Lys-Lys-Tyr-Nle-NH.sub.2 (SEQ ID NO:16);
 St-Val-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:17);
 St-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:18);
 St-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:19);
 St-Lys-Lys-Tyr-Leu-Nle-NH.sub.2 (SEQ ID NO:10);
 St-Lys-Lys-Tyr-Leu-Asn-NH.sub.2 (SEQ ID NO:20);
 St-Lys-Lys-Tyr-Leu-Asn-Ser-NH.sub.2 (SEQ ID NO:21);
 St-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-NH.sub.2 (SEQ ID NO:22); and
 St-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-NH.sub.2 (SEQ ID NO:23).
 Optional modes of administration of pharmaceutical compositions of the
 invention are subcutaneous, intravenous, oral, nasal, ocular, by an
 intracerebroventricular pump, through the urinary tract or transdermal
 administration.
 Where the pharmaceutical compositions of the invention are used to treat
 impotence, urinary tract or transdermal administration are preferable. For
 transdermal application the carrier is preferably selected from amongst
 those which enhance the tissue penetration of the active ingredient.
 Examples of suitable carriers are olive oil, glycerine, lubricants,
 nitroglycerin and Sefso.TM., and mixtures thereof. Sefsol is a trademark
 (Nikko Chemicals, Tokyo) for, 1-glyceryl monocaprylate, propylene glycol
 didecanoate, propylene glycol dicaprylate, glyceryl tricaprylate and
 sorbitan monocaprylate and they are the preferred carriers in compositions
 according to the invention. Of these, 1-glyceryl monocaprylate and olive
 oil are particularly preferred. For urinary tract application a gel is
 preferably used as a carrier.
 The present invention further provides, for the sustained release of a
 conjugate of the invention, a transdermal dispenser comprising an
 applicator loaded with said conjugate and adapted for application to the
 skin.
 If desired, the conjugate in the applicator may be formulated into a
 pharmaceutical composition of the kind specified above.
 Treating male impotence by transdermal administration exhibits several
 advantages over modes of parenteral, such as subcutaneous, administration.
 For one, it is non-surgical and does not entail tissue destruction.
 Moreover, it does not cause priapism or the burning pain associated with
 other modes of administration. Furthermore, the transdermal application is
 a much more discreet and convenient mode of application as compared to an
 intracavernosal injection. Transdermal administration enables the use of a
 continuous slow release device which may enable spontaneous sexual
 activity without the need for a lengthy preparation, thus sparing an
 inflicted individual much of the usual embarrassment.
 Where the pharmaceutical compositions of the invention are to be used as
 drugs acting on the central nervous system, it is preferable to administer
 them through the nose, which enables the penetration of the aerosol
 composition to the CNS through the olfactory nerve (WO 91/07947), via the
 ocular route (Chiou, G. C. Y., (1991) An. Rev. PharmacoL Toxical.,
 31:457-67) or by any other suitable method of administration as described
 in W. M. Pardridge, Peptide Drug Delivery, Raven Press, N.Y. 1991.
 The pharmaceutical compositions of the invention may be also directly
 targeted to the brain by an intracerebroventricular pump.
 The present invention further concerns a method of treatment of
 neurodegenerative diseases or male impotence by administering to a host in
 need of such treatment a therapeutically effective amount of the conjugate
 of the invention.
 The present invention still further provides use of the conjugate of the
 invention for the preparation of a pharmaceutical composition.
 As will be appreciated by any person versed in the art, the conjugates as
 defined above in formulae I above include a large number of possible
 conjugates. Those which fall under the scope of the invention and those
 defined as "active conjugates" in the pharmraceutical composition of the
 invention are the conjugates which are active in at least one of the
 following assays:
 (1) conjugates which are able to induce erection in an animal model of
 impotence (normal and castrated animals);
 (2) conjugates which have the activity of protecting electrically blocked
 neurons from death;
 (3) conjugates which are able to protect untreated neurons in culture from
 naturally occurring death;
 (4) conjugates which are able to protect cultured neurons from death caused
 by a 25-35 fragment of .beta.-amyloid peptide;
 (5) conjugates which are able to avoid deterioration of learning and memory
 acquisition of either old animals or animals treated with a dementia
 causing agent as tested in an acceptable learning of memory acquisition
 assay, as well as conjugates which are able to improve recollection of a
 previously acquired task in animals treated with a dementia causing agent,
 for example, as described in example.
 (6) conjugates which are able to avoid or ameliorate decline of motor and
 cognitive functions in an animal model of ischemia and/or models of
 stroke;
 (7) conjugates which are able to protect neurons from damage caused due to
 lack of oxygen;
 (8) conjugates which are able to improve motor and cognitive functions in
 models for hereditary neurodegenerative diseases of the central and
 peripheral nervous system such as models of mice with a knock out of ApoE
 (Cell, 71:343 (1992)); transgenic models of amyloid over-expression
 (Nature, 373:523 (1995)); models for ALS which are mutant super oxide
 dismutase expression (Science, 264, 1772 (1994)) and a model for Down
 Syndrome which is trisomy of chromosome 16; and
 (9) conjugates which are able to improve motor and cognitive functions in
 models of injury of the central and peripheral nervous system such as
 lesions of the nucleus basalis in rats (PNAS, 85:9481 (1988));
 scopolamine-induced acetylcholine release in ventral hippocampus (PNAS,
 90:11287 (1993)); NMDA induced convulsions (Brain Res. 448:115 (1988)).
 In the following, the invention will be further illustrated with reference
 to some non-limiting drawings and examples.

DETAILED DESCRIPTION OF THE INVENTION
 A. Synthesis of Linear Peptides
 To obtain a large battery of small peptides an automatic peptide
 synthesizer was utilized. Syntheses of the peptides of the invention were
 achieved by automatic procedure employing an ABIMED AMS 422 synthesizer
 (ABIMED, Langenfeld, Germany) using the commercially available protocols
 via the Fmoc strategy. All protected amino acid derivatives were as
 recommended by the company. Thus, the following side-chain protection was
 utilized: Lys, N-epsilon-t-butyloxycarbonyl (Boc), Tyr, Thr, Ser,
 O-t-butyl; Arg; 2,2,5,7,8-pentamethylchroman-6-sulfonyl (PMC); Trp,
 N.sup.int -Boc; Cys, S-trityl, Asn, beta-trityl, PyBOP, i.e.
 benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate,
 was used as a coupling agent. Peptide chains were assembled on a
 4-([2",4"-dimethoxyphenyl] Fmoc aminoethyl) phenoxy resin (Rink amide
 Resin, Nova, Switzerland).
 Final cleavage of the peptide chain from the resin along with the side
 chain deprotection was achieved as follows: cleavage mixture: 90% TFA, 5%
 water, 5% triethylsilane. The resin, 100 mg, loaded with peptide was
 incubated for 30 min with a 3 ml cleavage mixture inside the reaction
 column used for solid phase synthesis. After 30 min, the reaction was
 separated from the cleaved resin and cleavage continued for an additional
 3 hrs. The cleaved peptide was precipitated with ice cold tert-butylmethyl
 ether and centrifuged (4.degree. C., 2000 rpm). To ensure optimal
 precipitation, petroleum ether (b.p. 40-60.degree. C., 1:1 v/v) was
 occasionally added. The solution was decanted and the pellet was dissolved
 in water and frozen for lipophilization to yield a white powder.
 Purification of the crude peptides was performed by semi-preparative HPLC
 on an RP-8 column (Merck 7 .mu.M; 250.times.10 mm) employing linear
 gradient established between 35% acetonitrile in water containing 0.1%
 TFA, and 0.1% TFA in 75% acetonitrile in water at a flow rate of 10 m/min.
 Elution was monitored at 220 nm. Yields were 30-45%. Purity of the
 products was ascertained by analytical HPLC on an RP-18 column (Merck;
 250.times.4 mm) and amino acid analysis following exhaustive acid
 hydrolysis gave the expected values of each constituent amino acid.
 Examples of the conjugates comprising linear peptides that were synthesized
 by this method are those listed on pages 5-9 hereinbefore and the
 following conjugates are: