NO176022B - Analogous Procedure for Preparation of Small Peptides That Prevent Binding to T4 Receptors and Act as Immune Organs - Google Patents

Description

The invention relates to an analogue method for the preparation of a therapeutically active peptide with the formula

R<1> - R2 - R3 - R4 - R5 - R6 - R7 - Tyr - R<8> - R9 (I) where R<1> is an amino terminal residue Cys or is absent,

R<2> is Ala or D-Ala or an amino terminal residue Ala or

D-Ala or absent,

R3 is Ser or absent,

R<4> is Thr or an amino terminal residue Cys or absent, R<5> is Thr, Ser, Asn or amino terminal residue Thr, Ser, Asn, R<6> is Thr or Ser,

R7 is Thr, Ser, Arg or Asn,

R<8> is a Thr, Arg, Gly or Ser or carboxy terminal residue

Thr, Arg, Gly, Ser or Thr amide or absent,

R<9> is a carboxy terminal residue Cys or absent, or a physiologically acceptable salt thereof.

Synthetically produced short peptide sequences prevent binding of HTLV-III/LAV (hereafter referred to as HIV) to human cells by blocking the receptor sites on the cell surface, thus preventing viral infectivity of human T cells. The peptides, although they prevent infectivity, also induce antibody production against the envelope protein of the HIV virus. Consequently, these peptides also have application as vaccines to prevent the development of Acquired Immune Deficiency Syndrome (AIDS). Monoclonal antibodies for the peptides can also be used as diagnostic agents to identify the HIV virus. Accordingly, peptides and antibodies to the peptides have applications in the manufacture of test kits for the identification of HIV carriers or persons suffering from AIDS.

The complete nucleotide sequence of the AIDS (HIV) virus has been reported by several researchers. (See Lee Råtner et al., Nature 313, p. 277, January 1985; Muesing et al., Nature, 313, p. 450, February 1985; and Wain-Habson et al., Cell 40, pp. 9-17 , January 1985). The capsule gene has been particularly associated with antigenicity and infectivity. However, it is known that the capsule part also has areas that are very different. HIV virus capsular glycoprotein has been shown to attach covalently to the brain membranes of humans, rats and monkeys and to cells of the immune system.

The understanding that viruses can exert cell and tissue tropism by attaching to very specific sites on cell membrane receptors has encouraged researchers to look for agents that bind to the receptor sites for the virus in cell membranes and thus prevent binding of the particular virus to these cells. A demonstration that specific receptor-mediated infectivity of cowpox virus is blocked by synthetic peptides has previously been given (Epstein et al., Nature 318, pp. 663-667).

The HIV virus has been shown to bind to a surface molecule known as the CD4 or T4 region present on various cells susceptible to HIV infection, including T lymphocytes and macrophages. (See Shaw et al., Science 226, pp. 1165-1171 for a discussion of tropism of HTLV-III).

In addition to the symptoms arising from immunodeficiency, patients with AIDS exhibit neurophysiological defects. The central nervous and immune systems share a large number of specific cell surface recognition molecules that serve as receptors for neuropeptide-mediated intercellular communication. The neuropeptides and their receptors exhibit extensive evolutionary stability, being well conserved in largely unchanged form in unicellular organisms as well as in higher animals. Furthermore, the central nervous and immune systems share common DC4 (T4) cell surface recognition molecules that serve as receptors for the binding of HIV capsular glycoprotein (gp 120). Since the same well-conserved neuropeptide information substances integrate immune and brain function through receptors remarkably similar to those of HIV, the inventors have postulated that a very similar amino acid sequence between the HIV glycoprotein gp 120 and a short peptide previously identified in a different context from capsular region of the Epstein-Barr virus may indicate the core peptide essential for viral receptor binding. It was postulated that such a peptide could be useful in preventing infection of cells with HIV by binding to receptor cells and blocking the binding of HIV gp 120, that such peptides binding to the receptor sites would give rise to the production of antibodies directed at the peptide sequence, and that these peptides can be used to provide an immunological basis for contraception

AIDS.

It is a purpose of the present invention to provide an analogue method for the production of peptides which will serve to alleviate symptoms of AIDS by preventing the binding of HIV (AIDS virus) to receptor sites in cells of brain membranes and the immune system.

It is also a purpose of the invention to obtain, by the analogical method, peptides for use as vaccines which can be used to give rise to antibodies which will protect against the development of AIDS in persons who may be exposed to HIV (AIDS virus).

It is a further object of the invention to provide, in the same way, diagnostic means for identifying the presence of antibodies to HIV or HIV capsular protein.

These purposes are achieved by an analogous method as stated in the patent claim.

Detailed description of the invention.

An octapeptide in the HIV capsular glycoprotein (gp 120) was identified by computer-assisted analysis. This peptide, called "peptide T" because of its high threonine content, has been shown to prevent binding of gp 120 to the brain membranes. The peptide has the sequence Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr. Later analysis revealed a class of related pentapeptides with similar binding properties.

According to a first aspect of the present invention, a peptide of the formula (I) has been obtained: where Ra is an amino-terminal residue Ala- or D-Ala and R<b> is a carboxy-terminal residue -Thr or -Thr-amide or a derivative thereof with an additional Cys residue at one or both of the amino and carboxy termini, or a peptide of the formula (II):

where R"<1> is an amino terminal residue Thr-, Ser- or Asn-,

R 2 is Thr or Ser,

R 3 is Thr, Ser, Asn or Arg,

R<4> is Taurus,

and R<5> is preferably a carboxy terminal residue -Thr, -Arg or -Gly or a Thr amide. Although the preferred amino acids at R5 • are both indicated, it is known that the amino acid in this position can vary greatly. It is actually possible to terminate the peptide with R<4 >(tyrosine) as the carboxy-terminal amino acid, R<5> being absent. Such peptides retain the binding properties of the groups discussed here. Serine and threonine seem to be interchangeable for the biological purposes discussed here. The active compounds according to the invention can exist as physiologically acceptable salts of the peptides.

The most preferred peptides, as well as peptide T above, are the following octapeptides of formula (I):

and and the following pentapeptides of the formula (II):

optionally with an amide derivative at the carboxy terminus.

The compounds produced by the analogue method according to the invention can advantageously be modified by the methods known to promote the passage of molecules through the blood/brain barrier. Acetylation has been shown to be particularly useful in promoting the binding activity of the peptide. The terminal amino and carboxyl sites are particularly preferred sites for modification.

The peptides can also be modified in a limiting structure that provides increased stability and oral availability.

The following abbreviations are used hereafter:

Unless otherwise stated, the amino acids naturally exist in the natural form of L-stereoisomers.

A comparison of amino acid sequences for 12 pentapeptides is shown in Table 1. Although the original computer investigation historically showed that peptide T (contained in the ARV isolate) was the relevant molecular moiety, as additional virus sequences became available it became clear that the relevant bio-active sequence could be a shorter pentapeptide comprising, nominally, peptide T t4-8] or the sequence TTNYT. In isolates compared (Table 1), significant homologies were detected only in this shorter region. Most of the changes are interconversions of serine (S) and threonine (T), two closely related amino acids. The tyrosine at position 7 of peptide T is an invariant feature in all these engineered fragments indicating that it may be mandatory for bioactivity. Substitutions occurring at position 5 include T, G, R or S. Positions 4 and 6 were initially restricted (with one exception) to S, T and N, all of which are amino acids containing uncharged polar groups with closely related steric properties. An assessment of general sequence agreement among 5 different AIDS virus isolates shows that the region surrounding and including the peptide T sequence is a highly variable area. Such variability may suggest specialization through strong selective variation of the function(s) that can be defined at this locus. Like the opiate peptides, these peptide T analogs appear to exist in multiple forms reminiscent of met- and leu-enkephalin. These pentapeptide sequences represented in these different AIDS virus isolates are biologically active and capable of inter-reacting as agonists of the CD4 receptor - previously known largely as a surface "marker" of T helper cells.

The peptide of seven amino acids Cys-Thr-Thr-Asn-Tyr-Thr-Cys is also active. Addition of cysteines to a core does not adversely affect activity.

The peptides were custom synthesized by

Peninsula Laboratories under a confidentiality agreement between the inventors and the manufacturer. Merrifield's method for solid phase peptide synthesis was used. (See US-PS 3,531,258 herein incorporated by reference.) The synthesized peptides are particularly preferred. Although peptide T and the pentapeptide which is part of it could be isolated from the virus, the peptides produced according to Merrifield's method were free of virus and cell debris. Consequently, no adverse reactions with polluting materials take place when the synthesized peptides are used.

The peptides according to the invention can be prepared by usual methods for peptide synthesis. Both solid-phase and liquid-phase methods can be used. Merrifield's solid phase method has been found to be particularly suitable. In this method, the peptide is synthesized in a stepwise manner while the carboxy end of the chain is covalently attached to the insoluble support. During the intermediate synthetic steps, the peptide remains in the solid phase and can therefore be easily manipulated. The solid support is a chloromethylated styrenedivinylbenzene copolymer.

An N-protected form of the carboxy-terminal amino acid, e.g. a t-butoxycarbonyl protected (Boe) amino acid is reacted with the chloromethyl residue of the chloromethylated styrenedivinylbenzene copolymer resin to produce a protected aminoacyl derivative of the resin, where the amino acid is linked to the resin benzyl ester. This is deprotected and reacted with a protected form of the next desired amino acid, thus producing a protected dipeptide which is attached to the resin. The amino acid will generally be used in activated form, e.g. using a carbodiimide or active ester. This sequence is repeated and the peptide chain grows one residue at a time by condensation at the amino end with the desired N-protected amino acids until the desired peptide has been built up on the resin. The peptide resin is then treated with an anhydrous hydrofluoric acid to cleave the ester that binds the assembled peptide to the resin to release the desired peptide. Functional side chain groups of amino acids that must be blocked during the synthetic process using conventional methods can also be removed at the same time. Synthesis of a peptide with an amino group at its carboxy terminus can be carried out in a conventional manner

using a 4-methylbenzhydrylamine resin.

The compounds of the invention were found to effectively block cell receptor sites and to prevent cell infectivity with HIV (AIDS virus) in monkey, rat and human brain membranes and cells of the immune system.

The compounds produced by the analog method according to the invention can e.g. is used in a pharmaceutical mixture comprising such a compound in addition to a pharmaceutically acceptable carrier or excipient adapted for use in human or veterinary medicine. Such mixtures can be presented for use in the usual way in admixture with one or more physiologically acceptable carriers or excipients. The mixtures can optionally further contain one or more other therapeutic agents which, if desired, can be a different antiviral agent.

The peptides according to the invention can thus be formulated for oral administration, administration via the cheek or cavity (buccal), parenteral, topical or rectal administration.

More specifically, the peptides produced by the analog method according to the invention can be formulated for injection or infusion and can be given in unit dose form in ampoules or in multi-dose containers with an added preservative. The mixtures can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulating agents such as suspending agents, stabilizing agents and/or dispersing agents. Alternatively, the active ingredient can be in powder form for composition with a suitable vehicle, e.g. sterile, pyrogen-free water before use.

Such pharmaceutical mixtures may also contain other active ingredients such as antimicrobial agents or preservatives.

The mixtures can contain 0.001-99% of the active material.

For administration by injection or infusion, the daily dose used to treat an adult person of approx. 70 kg body weight be in the range from 0.2 mg to 10 mg, preferably from 0.5 to 5 mg, which can be given in 1-4 doses, for example, depending on the route of administration and the condition of the patient.

It was postulated that the affinity constants are similar to those for morphine. On the basis of this affinity, dosages of 0.33-0.0003 mg/kg per day suggested. This has proven to be effective. A blood concentration of 10~*> - 10"^ molar blood concentration has been proposed. In monkeys, at 3 mg/kg per day, a serum concentration of 150 x 10"^ M is achieved. This concentration is 15 times greater than necessary to achieve a concentration of 10-<8> M. Primates generally require 10 times the dose used in humans.

Vaccine preparations containing a peptide according to the invention provide protection against infection by the AIDS virus. The vaccine will contain an effective immunogenic amount of peptide, e.g. lug-20 mg/kg based on the host's weight, optionally conjugated to a protein such as human serum albumin, in a suitable vehicle, e.g. sterile water, saline or buffered saline. Aids can be used, e.g. aluminum hydroxide gel. The supply can be by injection, e.g. intramuscularly, intraperitoneally, subcutaneously or intravenously. The supply can take place once or at a number of different times, e.g. at 1-4 week intervals.

Antigen sequences from crab as well as proteins from other invertebrates can also be added to the peptides produced by the analog method according to the invention to promote antigenicity.

Another application of the peptide concerns test kits for detecting the AIDS virus and antibodies for the AIDS virus containing a peptide produced according to the invention as a source of antigen, or a monoclonal antibody produced by a peptide produced according to the invention. E.g. can such a peptide be used in a test kit to detect AIDS infection and diagnose AIDS and pre-AIDS conditions by using it as the test reagent in an enzyme-linked immunosorbent assay (ELISA), or an enzyme-immunospot assay (enzyme immunodot assay). Such test packs may include an insoluble porous surface

or solid substrate, to which the antigenic peptide or monoclonal antibody has been preabsorbed or covalently bound, such a surface or a substrate preferably being in the form of microtiter plates or wells, test sera, heteroantisera which specifically bind to and saturate the antigen or antibody absorbed on the surface or support, various diluents and buffers, labeled conjugates for the detection of specifically bound antibodies or other signal-generating reagents such as enzyme substrates, cofactors and chromogens.

The peptide can be used as an immunogen to produce monoclonal antibodies that specifically bind to the relevant part of the capsule sequence in the AIDS virus using common techniques.

EXPERIMENTAL METHODS AND DATA

Radioactive labeling of gp 120, preparation of brain membranes, binding and cross-linking of gp 120 to receptor and immunoprecipitation of T4 antigen

HTLV-IIIb of HIV was propagated in H9 cells, and gp 120 was isolated by immunoaffinity chromatography and preparative NaDodSO^PAGE.

125

Purified gp 120 was labeled with I by the chloramine-T method.

Fresh hippocampus from humans, monkeys and rats was quickly homogenized (Polytron, Brinkmann Instruments) in 100 volumes of ice-cold 50 mM Hepes (pH 7.4). The membranes collected by centrifugation (15,000 x g) were washed in the original buffer volume and used fresh or stored at -70°C. Before use, the brain membranes and highly purified T cells (ref. 16; gift from Larry Wahl) were preincubated for 15-30 minutes in phosphate-buffered saline (PBS). Membranes obtained from 20 mg (initial wet weight) brain (approximately 100 µg protein) were incubated with 28,000 cpm of <125>I-gp 120 for 1 h at 37°C in 200 L (final volume) of 50 mM Hepes containing 0.1% bovine serum albumin and the peptidase inhibitors bacitracin (0.005%), aprotinin (0.005%), leupeptin (0.001%) and chymostatin (0.001%). The incubations were rapidly vacuum filtered and counted to determine the receptor bound material.

Immunoprecipitation

Immunoprecipitates were prepared by incubation (overnight at 4°C) of 0.5% Triton X-100/PBS solubilized, lactoperoxidase/glucose oxidase/I-iodinated brain membranes or intact T cells indicating mAbs at 10 µg per reaction. A solid-phase immunoabsorbent (immunobeads, Bio-Rad) was used to precipitate immune complexes prior to their reresolution by NaDodSO 4 /PAGE. Control incubations contained no primary mAb or a subclass control mAb (OKT8).

Chemical neuroanatomy and computer-assisted densitometry Cryostat-cut 25-μm sections of fresh-frozen human, monkey, and rat brain were thaw-mounted and dried on gelatin-coated slides, and receptors were visualized as described (18). Incubations with or without antibodies (10 µg/ml) against T4, T4A, T8 and T11 were performed overnight at 0°C in RPMI medium, cross-linked on o their antigens and visualized, with <125>I-labeled goat anti-mouse -antibody. Incubations of slide-mounted tissue sections to o label the antigen receptor with <125>I-gp 120 were performed in 5 ml slide carriers with (1 *iM) or without unlabeled gp 120 or mAb OKT4A (10 µg/ml) (Ortho Diagnostics).

Separation of T lymphocyte subsets

Subsets of T cells were obtained by treating Percoll density-purified peripheral blood T cells with specific monoclonal antibodies (T4 or T8) at 10 µg/ml. The treated cells were then plated (21) onto a plastic Petri dish coated with goat anti-mouse immunoglobulin [F(ab') 2 ^ (Sero Lab, Eastbury, MA) for 30 minutes at 4°C. The non-adherent cells were then removed, washed and analyzed for reactivity by flow cytometry. The separated T4 and T8 cell populations have <5% contamination by other T cell subsets. Cells were then cultured with phytohemagglutinin (1 µg/ml) for 72 hours and exposed to HIV as described below. Infected cells were phenotypically characterized when cytotoxicity assays were performed.

Viral infection

The HTLV-lll virus used for infection was isolated from an interleukin 2 (IL-2)-dependent cultured T-cell line established from fresh material from AIDS patients and introduced into HuT 78, a permissive IL- 2-independent cell line.

Description of the drawing

1 25

Fig. 1A shows a cross-linking of I-gp 120 to brain membranes and T cells (a) <125>I-gp 120 only; (b) monkey; (c) rat; (d) human brain and (e) human T cells. Figures 1B and 1C show immunoprecipitation of 1 ^Si^e^e^e monkey brain membranes and human T cells, respectively; (f,i) no primary antibody control; (g,j) OCT4 Mab; (h,k) 0KT8 Mab. Fig. 2a shows a displacement of specific <125>j_gp 120 binding to healthy rat hippocampal membranes. Each determination was performed in triplicate; the results of an experiment performed three times with similar results are shown. Specific binding displaceable by 10 µg/ml OKT4 and 4A was in the range 27-85% of the total binding which was 2201 ± 74 cpm in the experiment shown. Fig. 2b shows that the viral infectivity is blocked by peptide T and its synthetic analogues. Each determination was performed in duplicate. The results represent a single experiment that was repeated three times with similar results.

Example 1

A single radioactively labeled cross-linked product of approx. 180 Kd is obtained after specific binding of <125>j_gp 120 to membranes from either squirrel monkey, rat or human brain membranes which is indistinguishable from that of human T cells (Fig. 1A). This result suggests that gp 120 can be linked to a protein of approx. 60 Kd; unreacted ^<25>j_gp 120 runs alongside the non-membrane control (lane a).

Immunoprecipitation of radioiodinated human brain membranes with 0KT4 and OKT8 (10 µg/ml) (Fig. 1B) shows that brain membranes contain a T4 antigen of approx. 60 Kd indistinguishable from that identified on human T lymphocytes (Fig. 1C); in contrast, 0KT8 immunoprecipitates a low molecular weight protein (about 30 Kd) from T lymphocytes (Fig. 1C) that is absent from brain membranes (Fig. 1B), suggesting that brain T4 is not derived from lymphocytes present On-site. Similar results are observed in monkey and rat (not shown) hippocampal membranes. These results show that the T4 antigen serves as the virus receptor and is a well-conserved 60 Kd molecule that is shared with the immune and central nervous systems.

The discovery that Epstein-Barr and HTLV-III/LAV share an almost identical octapeptide sequence led to the synthesis and investigation of "peptide T". Fig. 2 shows that the high affinity (0.1 nM range) and saturability (Fig. 2a) of <125>I-gp 120 binding to freshly prepared rat brain membranes. Specificity (Fig. 2b) is shown by blockade with OKT4 and OKT4A, but not 0KT3 (0.1 )Ag/ml). Peptide T and two of its synthetic analogs (but not the irrelevant octapeptide substance P [1-8]) significantly inhibited <1>2<5>I-gp 120 binding in the 0.1 nM range (Fig. 2c). Substitution of a D-threoninamide in position 8 led to at least a 100-fold greater loss of receptor binding activity. The classical [D-Ala] substitution for [L-Ala] results in a consistently more potent, apparently more peptidase-resistant analog than peptide T; amidation of the C-terminal threonine also consistently gives a somewhat greater efficiency (Fig. 3).

When the synthetic peptides were tested for their ability to block viral infection of human T cells, the experimenters were unaware of the results of the binding assays. At 10"<7>M, the three peptides active in the binding assay can reduce detectable levels of reverse transcriptase activity by nearly 9-fold. The less active binding displacer [D-Thr]-peptide T similarly showed greatly reduced blocking of virus infection and required concentrations 100-fold higher to achieve significant inhibition.Thus, not only is the ranking of efficacy of the four peptides (D- [Ala] j-peptide T-amide > D-[Ala] j-peptide T > peptide T > D-[Thr] 8-peptide T-amide), but also their absolute concentrations with regard to inhibiting receptor binding and virus infectivity closely correlated (fig. 3).

Example 2

A protein of approximately 60 Kd that is similar if not identical to human T-cell T4 antigen was present in apparently conserved molecular form on membranes prepared from human brain; furthermore, the radioactively labeled HIV capsular glycoprotein (^<2>^I-gp 120) may be covalently cross-linked to a molecule present in three mammalian brains, whose size and immunoprecipitation properties could not be distinguished from those of the T4 antigen. Using a method for visualizing antibody-bound receptors on brain slices, the neuroanatomical distribution pattern of brain T4, which is densest over the cortical neuropil and analogously organized in all three mammalian brains, was presented. Furthermore, radiolabeled HIV viral capsular glycoprotein bound in an identical pattern on adjacent brain sections again suggested that T4 was the HIV receptor.

Example 3

Chemical neuroanatomy, computer-assisted densitometry. Cryostat-cut 25 micrometer sections of freshly frozen human, monkey and rat brain were thaw-mounted and dried on gel-coated slides and receptors visualized as described by Herkenham and Pert, J. Neurosci., 2, pp. 1129-1149 (1982) . Incubations with or without antibodies (10 µg/ml) against T4, T4Ak T8 and T11 were performed overnight at 0°C in RPMI, cross-linked to their antigens and visualized with <125>j_goat anti-mouse antibody. Incubations of slide-mounted tissue sections to label the antigen/receptor with 12^I_9P 120 were carried out in 5 ml slide containers with (10"<*> M) or without unlabeled gp 120 or Mab OKT4A (10 ug/ml) (Ortho Diagnostics ) as described above for membranes.

Computer-assisted transfer of autoradiographic film opacity to quantitative color images was performed. Co-exposure of standard known increases of radioactivity with monkey brain sections generated a linear plot ( 4 = > 0.99) of log O.D. as a function of cpm, from which the relative concentration of radioactivity can be meaningfully extrapolated. Cell-staining of brain sections with thionin was carried out by classical methods and visualization of receptors that lay on top of stained tissue.

Example 4

Experiments were performed to determine the distribution of T4 antigen on a rostral to caudal series or coronal sections of squirrel monkey brain. These experiments show that there are detectable levels of T4 monoclonal antibody binding to cytoarchitectonically meaningful areas of the brainstem (eg, substantia nigra), but the striking pattern of cortical enrichment is evident at every level of the neuroaxis. 0KT8, a T-lymphocyte-directed monoclonal antibody from the same subclass as 0KT4, shows no observable pattern. In general, the superficial layers within the cerebral cortex contain the densest concentrations of the T4 antigen; the frontal and perilimbic cerebral cortex that lies on top of the amydala is particularly rich in receptors through all the deep layers. The hippocampal formation has the densest concentration of receptors in the brains of monkeys, rats and humans. Dark-field microscopy of squirrel monkey sections dipped in photographic emulsion revealed that the band with the densest receptor labeling lies within the molecular layers of the dentate gyrus and hippocampus proper (which contains very few neurons). Thus, the receptors appear to be properly distributed across the neuropil (the neuronal extensions of dendrites and axons) or they may be localized to a specific subset of unstained astroglial cells.

Evidence for the specificity of the chemical neuroanatomy and results showing that T4 and the viral capsid recognition molecule are indistinguishable from each other have been determined. Coronal sections of rat brain showed a very similar cerebral cortex/hippocampus-rich pattern of receptor distribution, whether 0KT4 or <125>j_gp 120 was used for visualization. Furthermore, this pattern was not evident when incubation took place in the presence of unlabeled gp 120 (1 m), OKT4A (10 µg/ml) or OKT4 (10 µg/ml). Other mouse Mabs directed against other human T-cell surface antigens including OKT8 and 0KT11 produced no detectable pattern on rat brain when visualized by <125>i goat anti-mouse IgG secondary antibody, just as there was no reproducible detectable antigen/receptor with secondary antibody alone.

Claims (1)

Analogous method for the preparation of a therapeutically active peptide of the formula where R<1> is an amino terminal residue Cys or is absent, R<2> is Ala or D-Ala or an amino terminal residue Ala or D- Alas or absent, R<3> is Ser or absent, R<4> is Thr or an amino terminal residue Cys or absent, R<5> is Thr, Ser, Asn or an amino terminal residue Thr, Ser, Asn, R<6> is Thr or Ser, R<7> is Thr, Ser, Arg or Asn, R<8> is a Thr, Arg, Gly or Ser or carboxy terminal residue Thr, Arg, Gly, Ser or Thr amide or absent, R<9> is a carboxy terminal residue Cys or absent, or a physiologically acceptable salt thereof, characterized in that a) the N-protected form of the carboxy-terminal amino acid is attached to a resin on the surface of polystyrene beads; b) the amino group is deprotected; c) addition of the next amino acid in protected form and dicyclohexylcarbodiimide; d) repeating steps b) and c) for each added amino acid; e) washing the beads to remove excess reagent and unwanted products after each step; and f) releasing the completed peptide from the resin.