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FORSCHUNG 145 ClIIMtA ~5 (t~~ll Nr.'

Chimia45 (1991) /45-/53 © Schwei:. Chemiker-Verhand; /SSN 0009-4293

Peptides - From Research Tools to 'Soft' Drugs? [1]

Alex N. Eberle*

Alex N. Eherle: Born 1945 in St.Gallen, Switzer- Ahsrracr. Over the last 30 years, regulatory have played an ever increasing role as land, studied chemistry, biochemistry. and molecular research tools but as therapeutics they have been applied in only a very limited number of biology at the Swiss Federal Institute of Technology (ETH) in ZUrich. There he also wrote his Ph.D. indications. The recent call by the public for 'soft' pharmaceuticals and 'soft' chemistry, the thesis on synthesis and biology. He thcn increasing knowledge on peptide receptors and intracellular signalling, the design of more worked as a research fellow at the MRC Laboratory potent and specific peptide ligands as well as the availability of sufficient quantities oflarge of Molecular Biology in Cambridge U.K. together peptides by genetic engineering may be the starting point for a new era of peptides as with F. Slinger. i.E. Walkl'1'. C. Milstein. and R.C. 'natural' therapeutic agents in human and animal medicine. In the first part, this review Sheppard. In 1982 he joined the Dcpartmcnt of Researchofthe University Hospitals in Basel where focusses on new achievements in peptide chemistry as well as on the role of peptides as he became its deputy chairman in 1990. His research research tools, i.e. their pleiotropic nature and their mechanisms of action. The second part focusses on the mechanism of action of peptides, summarizes new concepts for the appl ication of peptides in medical diagnosis and therapy. i.e. peptide receptors and signallransduction path- ways, as well as on the application of peptides to medical diagnosis and therapy. His particular intcr- 1. Introduction comparatively small although a few of them est at present are the effects of the mdanotropins and various growth factors onmclanoma. In 1988 have become very important therapeutics. he received the Leonidas Zen'us A wurdfor Pcp tide The peptides comprise a great variety of The availability of large peptide and Chemistry and in 1991 the Rohcrt-Wennl'1'-Pri:e of biologically active linear and cyclic bio- factors by genetic engineering together with the Swiss Cancer League. polymers with diverse functions. They can be new therapeutic concepts will considerably divided into different classes, such as signal- widen the list of peptide medicals in the ling or regulatory factors (i.e. and future. Only a few original publications and re- growth regulators), antibiotics, alkaloids, The scope of this review is to give a short views will be cited. The reader will find toxins, and sweeteners (Tahle J). While account of the present role of peptides as more detailed information in the proceed- antibiotics became very important therapeu- research tools and pharmaceuticals, the em- ings of the last three peptide symposia [21 tics as soon as they could be produced in phasis being put on some of the major and in the relevant journals [3]. large enough quantities after world war II, chemical and biological topics of interest. A there has been a continued argument on the short historical retrospective will be follow- potential for peptide hormones in medical ed by a few remarks on modem methods 2. A Short Historical Retrospective therapy, except perhaps for . The high for the preparation of peptides and by a biological potency of peptides and their summary on the mechanisms of action of The beginning of peptide chemistry is complete metabolism by the organism into regulatory peptides and their interaction with usually traced back to the early 20th century the natural subunits, the amino acids, would membrane receptors. The second part of the when Emil Fischer in Berlin coined the term suggest that they are ideal therapeutic agents. review will concentrate on the use of pep- 'peptide' after he and E. FOllmeall had However, the susceptibility of peptides to tides as diagnostics and 'soft' therapeutics. synthesized glycylglycin, the simplest di- enzymatic attack in the gut and the circula- peptide. Fischer, the nobel laureate of tion with a resulting short half-life as well as Table 1. The Different Classes of Peptides chemistry of 1902, attracted many scientists the need to administer them by the route of and Some Selected Examples to his Institute, among them famous names injection were the principal arguments such as Max Bergmann, and with his team against their application as drugs. Antihiotics made important contributions to the field of 0, Since the 1950's, when chemists began Actinomycin bacitracin. penicillin, peptides during its first epoch until world gramicidin to develop methods for efficient peptide war I. The real roots of peptide chemistry, synthesis, signalling peptides became more Eny:me inhihitors and sl/hstrates however, are to be found much earlier in and more important as research tools, result- ACE inhibitors, inhibitors, Munich where Theodor Cllrtills in 1881 ing in an ever increasing demand for these HIV protease inhibitors; synthetic accidentally obtained benzoyldiglycine by oligopeptides for enzyme diagnostics substances by biologists and pharmacolo- condensing hippuric acid with . The gists. The great interest in peptides is also Hormones and regulatory factors second epoch of peptide chemistry between reflected by the continuously growing num- Pituitary hormones, , the two world wars was characterized by a ber of participants at peptide symposia as insulin, growth factors, cytokines number of important discoveries of peptidic well as of specialized companies that pro- factors, such as insulin by Banring and Best, Peptide alkaloids duce and sell peptides. However, the number Ergotamine penicillin by Fleming, Chain, and Florey, of peptide drugs on the market has remained glutathione by Harrington andMeadand the Toxins peptide alkaloid ergotamin by Sroll. In this Amanitine. phalloidine, a-, period, the first useful protecting group still abrin, ricin used in peptide chemistry today, the benzyl- *Correspondence: PO Dr. A.N. Eberle oxycarbonyl or Z group, was introduced by Departement Forschung (ZLF) Sweeteners Kantonsspital, Hebelstrasse 20 Aspartam Bergmann and Zervas. The third epoch after CH-4031 Basel world war II was very producti ve and brought FORSCHUNG 146 CHtMJA 45 (t991) Nr. 5 (MlIi)

Table 2. Regulatory Peptides Arranged According to the Year of Structural Analysis of choice in any modern peptide research laboratory [5]. Originally, the Merrifield 1951 1953 Insulin technique was based on the attachment of 1954 Adrenocorticotropin, the first Nil-protected amino-acid residue to 1956 j3-Melanotropin, gelatinous chloromethylated polystyrene 1957 a-Melanotropin, beads, obtained by copolymerization of sty- 1960 1962 rene with 1% divinylbenzene. The Nil-pro- 1964 , tecting group was usually a t-butoxycar- 1965 j3- bonyl (Boc) group which is cleaved under 1966 , somatotropin (growth ) moderately acidic conditions, leaving the 1968 , , C-peptide benzyl-type side-chain protecting groups as 1969 Thyrotropin-releasing hormone, , 1971 Choriongonadotropin, vasointestinal peptide, gastric inhibitory peptide, lutropin, lutropin well as the Ts groups (-7 protection of ar- releasing hormone, , , neurophysin ginine) unaffected. As the polystyrene resin 1972 Follitropin, requires apolar solvents for swelling, each 1973 , , tuftsin synthesis cycle consisted of a complex wash 1975 , , 1976 Delta-sleep inducing peptide, /3-endorphin, insulin-like growth factor scheme with various solvents in order to 1977 , granuloliberin meet the criteria for optimal solvation of 1978 Fibroblast growth factor both the gel resin and the growing peptide 1979 Gastrin relcasing peptide, glicentin, a-lj3-neoendorphin,kyotorphin, chain. The whole procedure offered itself to 1980 Interferons- aI /3 automation, which resulted in a number of 1981 Corticotropin-releasing factor, , , peptide histidine (PHI), calcitonin -related peptide, , commercially available instruments [6]. The 1982 releasing factor, Y, peptide YY, rimorphin, katacalcin, uroten- cleavage of the peptide from the resin and sin, interferon-y the side-chain deprotection, however, still 1983 , neuromedin K, , melanin-concentrating hormone, atriopeptin, platelet- remains a manual step and is carried out in derived growth factor, transforming growth factor aI/3, interleukin-2 1984 Tumour necrosis factor ex and ~, cere bellin, interleukin-3 strong acid, such as liquid HF. As these 1985 Valosin, granulocyte-macrophage colony stimulating factor, interleukin-I a and {3, conditions frequently caused a number of neuropeptide K, neutrophil peptide side-reactions, new resins were developed 1986 Cyclolinopeptide A, leucopyrokinin, leucokinin, leukomyosuppressin, pancreastatin, with which novel strategies of synthesis galanin-llssociated peptide, interleukin-6 1987 Follicular -releasing peptide, cardioactive peptide, interleukin-4, interleukin-5, could be applied, in particular simplified interleukin-8 coupling reaction cycles and relatively mild 1988 Corticostatin, interleukin-7, neuropeptide-y, cleavage conditions. The most noteworthy method was developed by Sheppard and The list covers the first 100 regulatory peptides discovered between 1951 and mid 1988, i.e. from oxytocin to Atherton [7] who introduced a physically endothelin. supported polyamide gel resin consisting of

the field of peptide chemistry much at- tention [4]: after the structure of oxytocin had been published by DuVigneau in 1951, more than fifty new peptide hormones and 'regulatory factors were structurally ana- lyzed and chemically synthesized up to 1980 (Tahle 2). Important contributions were made by the peptide groups in Basel which synthe- sized, amongst others, adrenocorticotropin, calcitonin, parathormone, and insulin. Among the new synthetic methods intro- duced during this period, the solid-phase peptide chemistry technique invented by B. Merrifield (the Nohellaureate of chemistry of 1984) in the early sixties made it possible Sample to produce thousands of peptide analogues Loop at reasonable costs and in a short time. Duri ng the fourth epoch of the last ten years the number of known peptides has doubled (Table 2) and the field of peptide and protein DMF Piperidine chemistry has been opened up to molecular Sample biology and genetic engineering. The latter has resulted in the str,uctural analysis of an increasing number of prohormones and Non-retum receptors as well as the preparation of large valve peptides which are not accessible through conventional chemical synthesis. Waste

Fig. I. Scheme of a (semi- }automatic equipment for continuous-flow peptide synthesis designed hy Atherton and 3. Modern Synthetic Methods Sheppard [7]. The polymer is packed into a flow reactor which is connected to a pump, UV monitor and sample loop. Valve 1switches the system from recirculation (coupling reaction) to flow-through into the waste (washing Peptides for research purposes are usual- steps; deprotection). Valve 2alh introduces loaded sample from the sample loop into the system. Valve 3 reverses ly required in only mg-to-g amounts. This is the flow direction in the reactor. Valve 4 selects the solvent (DMF or piperidine). Vail'e 5 is activated for flushing the bottles with nitrogen. Fully automated instruments contain a sample pipetting system with which not only the the optimal scale for the time-saving solid- amino acids but also additional reagents for the coupling step can be introduced into the recirculation system. An phase peptide synthesis which is the method increased number of valves allows the selection between more than two different solvents. FORSCHUNG 147 CHIMIA45 (1491) Nr. 5(M"il a macroporous Kieselgur carrier filled with a copolymer of N,N-dimethylacrylamide, ethylene bisacrylamide, and acryloylsarco- Niu..prote::on Nr,-H~ Q.... ~ jl'>S H,c+/~ O;',-6;;-O-oc,-1- sine methyl ester. The latter is converted to H,C+CH, 0- -0 JyC I a primary amine by reaction with ethylene 0, TH, T fa, TOO TMBHA diamine, thus forming the C-terminal at- 11 tachment point. This pressure-resistant poly- HOCH, -~0-CH,-C- mer is characterized by excellent swelling Na-protection ~ HMPA properties in N,N-dimethylformamide ~~\\ W)'---- ~ ~ (DMF) and its suitability for the continuous y 0 tBu tBu Aan OlBu Trt Prnc2) Trt Soc r -<>'" I I I I I I I I flow technique: the polymer is packed into a . o-c- -- Fmoc-Ser-Tyr-Cys-Met-Glu-His-Phe-Arg-Trp-Asn-Lys-Pro-Val-Linker-Nle-Polymer column and the reactions are carried out by 2'"- }'H Thr3j ASp4) Gln5) Orn6) I either recirculation of the activated amino Intemal acid residues (4 coupling reactions) or f1ow- Reference through of solvents (4 cleavage of the N°-_ Coupling Activated HOBt Carbodiimide active esters protecting group and washing steps). The BF " >-N=C=N-< I 4 orthogonal protection strategy is based on -N + 0 Ho..~V the base-labile f1uorenylmethoxycarbonyl -fo..~-o 'Nn' I (Fmoc) group for N°--protection and t-Bu and \ N'::'N N·N N°N *FF F other acid-labile groups for ro-protection. TBTU DICIH081 -DHBt -Pip This results in a simple reaction cycle where the solvent is either DMF (for coupling Cleavage 76% CFs·COOH /20% HS..CH2-CH2-SH /4% H20 (....•protects tryptophan efficiently), or TFA with other scavengers such thioanisol, anisol + ethane dlthlol, phenol etc. reactions and washing steps) or 20% piperi- Purification Gel and ion exchange chromatography, partition chromatography, counler-current dine in DMF (for the cleavage of Fmoc). distribution, reversed-phe.e hlgh-performance liquid chromatography (RP.HPLC). Automated instruments based on this con- cept have been developed by R.C. Sheppard Fig. 2. Elements of the colllinuous-fiow solid-phase peptide s)'lIIhesis using cOlltinuous-f/ol'l' methodology. The and E. Atherton (Fig. 1) and have also be- example shows an analogue of a-melanotropin with 13 different amino acids containing all those residues for come commercially available [8]. which specific side-chain protection is required. The orthogonal protection strategy uses Fmoc for Nn-protection The current methodology used by many and acid-labile groups for side-chain protection. The linkers are also labile to diluted TFA (TMBHA) or more concentrated TFA (HMPA). Both types oflinkerexist in several variants with different acid-Iabil ity, forming either investigators, including the author, is sum- C-terminal free acid functions upon cleavage (~ HMPA) or carboxamide groups (~ TMBHA). The physically marized in Fig. 2. The resin is either the supported polymers are either Kieselgur/polyamide, Polyhipe or TentaGe!. The coupling reaction is carried out macroporous Kieselgur (4 Pepsyn K) or a by using coupling reagents such as TBTU and related compounds, carbodiimides with HOBt, active esters of the macroporous polystyrene containing a poly- Fmoc-amino acids or preformed symmetrical anhydrides, The cleavage and deprotection is perfol111ed in TFA using scavengers to protect residues such as tryptophan, histidine, , and others. The dimethylacrylamide gel (4 Polyhipe) or purification and analysis employs established methodology. polyoxyethylene grafted onto polystyrene Abbreviations: Acm, acetamidomethyl; Boc, l-butoxycarbonyl; DHBI. dihydrobenzotriazinyl; Ole, diiso- (4 TentaGel). All these resins have excel- propylcarbodiimide; HOBt, I-hydroxybenzotriazol; HMPA, 4-hydromethylphenylacetic acid; Pfp, pentafluoro- lent performance in DMF as the sole solvent. phenyl; Pmc, JVG-(2,2,5,7,8-pentamethylchroman-6-sulfonyl); TBTU, 2-( I H-benzotriazol-I-yl)-l, I,3.3-tetra- They are usually derivatized with an internal methyl uronium letrafluoroborale; TFA, trifllloroacetic acid; TMBHA, trimethoxybenzhydrylamine; Trt, trityl. ') Trityl is cleaved by acid treatment; if disulfide fomlation is required, this must be done before cleavage from amino-acid residue (4 reference) and an the resin. 2) may alternatively be protected by Mtr (methoxytrimethylphenylslllfonyl) or be applied in acid-labile linker which, after cleavage, ei- unprotected, protonated form. ') Thr is protected as Ser. 4) Gin is protected as Asn. 5) Om is protected as Lys, ther produces a C-terminal free acid or car- boxamide. The protection by Fmoc of N°-_ groups is combined with t-Bu-type pro- tecting groups for side chain -NH2, -OH and ting from cleaved protecting groups. The al. [II]. While peptides synthesized in this -COOH. Arginine is frequently protected purification scheme usually combines ion way may be useful in the search for antigenic by NG-(2,2,5,7 ,8-pentamethylchroman-6- exchange and gel chromatography with re- determinants etc., the quality of the products sulfonyl) (Pmc) and histidine, versed-phase chromatography on CIS col- are often insufficient for pharmacological and by trityl (Trt) whereas the umns and, less frequently, partition chroma- studies, as the peptides are frequently used protection of cysteine may consist of tography, counter-current distribution, and without further purification. acetamidomethyl (Acm), trityl or combina- ion pair chromatography. Modem analysis The classical peptide synthesis by frag- tions thereof (Fig. 2). In addition, a number no longer relies solely on HPLC runs, TLC ment condensation still plays an important of other valuable protecting groups have and amino-acid. analysis and sequencing, role for those sequences which cannot be been introduced in the past few years [5][7]. but includes high performance capillary obtained in good yield or quality by the The coupling reaction is carried outa) by the electrophoresis, fast atom bombardment solid-phase technology. The synthesis in active ester method using pentafluorometh- (FAB) mass spectroscopy, HPLC com bined homogeneous solution is also the most fre- yl (Pfp) or dihydrobenzotriazine (DHBt) with ion spray mass spectroscopy or laser- quently used method for the production of esters of Fmoc-amino acids, b) by applying desorption ionization (LDI) mass spectros- large quantities of peptides (although an a condensation reagent forming an interme- copy and other specialized analytical meth- increasing number of 'peptide products is diary uronium ester with the Fmoc-amino ods (CD, NMR spectroscopy, IR spectros- prepared by solid-phase technology). The acid (e.g. TBTU; see Fig. 2), c) by the copy, polarimetry etc.). special features of peptide production were 'classical' carbodiimide method using the Synthesis with the continuous-flow reviewed by Feurer [12] many years ago reagent pair diisopropy\carbodiimide and technology is very rapid, particularly with and are still valid. Like all chemical process- hydroxybenzotriazole (DICIHOBt), or d) by the TentaGel resin where a synthesis cycle is es, both solid-phase and classical peptide symmetrical anhydrides of Fmoc-amino complete in less than 30 min. For more rapid chemistry require energy and considerable acids. Cleavage from the resin with triflu- syntheses of a large number of peptides, amounts of solvents, particularly when me- oroacetic acid (TFA) requires the presence multiple synthesis methods have been de- dium-sized or long peptides are produced, of scavengers such as 20% dithioethane, veloped, such as the so-called tea-bag meth- such as the 32-residue peptide calcitonin or thioanisol, or others, particularly when the od by Houghton [9], the pin-synthesis by the 39-residue peptide corticotropin which peptide contains tryptophan, in order to sup- Geysen et al. [10] and a robotic work-station require almost a hundred steps or more. press side-reactions of the cations origina- with a 48-column reactor by Gausepohl et However, as practically all reactions take FORSCHUNG 148 CHIMIA 45 (1991) Nr. ~ (Mail

Table 3. Peptide Hormolles alld Cell Growth Factor.l· Produced by Genetic Engineering which Are Either Commercially Amilable or Being Del'e1oped for Medical Application

Ami>AlDS therapeutics CD-4 Soluble receptor acts as a decoy for the AIDS virus AIDS vaccine Viral or peptide fragments are being tested as vaccinc candidates

Cardiovascular and clotting factors Atrial Vasodilator/diuretic peptide; potential use in the prophylaxis and treatment of acute renal failure Applaggin Peptide from snake , inhibits platelet aggregation, potential use in prevention and treatment of arterial clots Hirudin Thrombin inhibitor; potential use in prevention and treatment of venous blood clots Hirugen Peptide derivative of hirudin; same application Plasminogen activator Helps dissolve blood clots in attack and cardiovascular disorders Factor VIII Blood clotting factor protein deficient in the major forms of hemophilia

Colony stimulating factors (CSF) and erythropoietin Erythropoietin Regulates production of red blood cells; used to treat am,mia in dialysis patiellls, AIDS and cancer G-CSF Granulocytc-CSF stimulates production of white blood cells; used to trcat cancer patients following chcmotherapy and infectious diseascs GM-CSF Granulocyte-macrophage-CSF stimulates production and functional activities of white blood cells; use similar as for G-CSF M-CSF Macrophage-CSF stimulates production and functional activities of monocytes and macrophages; potential use in cancer therapy

Growthfactor.I' (GFs) and metabolic regulators BDNF Brain-derived neurotrophic factor affects survival and maintenance of central nervous system EGF Epidermal growth factor stimulatcs growth of skin cells; potential use in skin grafting, eye surgcry, bUll1s FGF Fibroblast growth factor promotes growth of fibroblasts, keratinocytes and f0ll11ation of new blood vessels Growth hormone Growth regulator acting I'ia IGF; promotes growth in children IGF Insulin-like growth factor is a metabolic reguJator; potential use in wasting syndromes, severe burns and wound healing Insulin Regulates blood sucrose levels; important therapeutic for the treatment of type I diabetes lnsulinotropin that modulates release of insulin by the ; potential use in type II diabetes NGF Nerve growth factor affects growth and differentiation Alzheimer's disease PDGF Platelet-derived growth factor has a potelllial similar to FGF TGF-{:I Transforming growth factor-{:I stimulates growth of numerous. cell types; potential application in wound hcaling and burns

Illterferolls (INFs) a-Interferon Immune stimulant for cancer therapy {:I-Interferon Immune stimulant for treatment of viral diseases and multiple sclerosis )'-lnterferon Immune stimulant for treatment of infectious diseases, cancer and rheumatoid arthritis

Imerleukins (ILs) Interleukin-I aI{:I Multiple direct and indirect effects on blood cells and various tissues; potential use in cancer therapy and inflammatinn IL-I-RA Interleukin-I ; potential use as antiinflammatory agent Interleukin-2 Stimulates production and function of T cells; application in cancer therapy and infectious diseases Interleukin-3 Stimulates differentiation of white blood Cells. May be used in combination with GM-CSF Interleukin-4 Promotes growth of activated T and B cells to kill tumours; treatment of immune deficiency Interlcukin-5 Acts on specific white blood cells (eosinophils, B cells) Illlerleukin-6 Stimulates T and B cells, early blood cells and platelet production; potential application in cancer therapy Interleukin-7 Stimulates T and B cells; potential use to speed up recovery following chemotherapy Illlerleukin-8 Chemotactic factor for neutrophils Interlcukin-9 Regulator of the hematopoietic and immune systems; stimulates growth of T cells Interleukin-IO Inhibits cytokine synthesis by T helper cells Interlcukin-Il Synergistic action with IL-3 in stimulating white blood cells. May be used in combination with lL-3 and GM-CSF IL receptors Soluble versions of natural cytokine receptors; potential use to regulate autoimmune and inflammatory reactions

Other peptide faclOrs Collagenase inhibitor Protein inhibitor of collagenase; potential application in rheumatoid arthritis and other inflammatory conditions Elastase inhibitor Protein inhibitor of elastase; potential usc in cystic fibrosis and emphysema Relaxin Hormone that softens birth canal TNF-a Tumour necrosis factor-a is toxic for many cells and displays a wide range of immunoregulatory activities, similar to IL-I. A potential use in tumour killing is by transplantation of its gene into TILs (see text) TNF-,B Tumour necrosis factor-{:I (see TNF-a) TNF inhibitor Tumour necrosis factor inhibitors may limit the systemic damage in certain immune-nlediated conditions. c.R. septic shock, cachexia Trypsin inhibitor Protein inhibitor of trypsin for treatment of TSH stimulating hormone; for use in thyroid imaging procedures

Vaccines, a/1tibodie.I', immu/1otoxi/1s Antibodies Monoclonal antibodies are used for diagnostic and imaging purposes of various diseases Immunotoxins Monoclonal antibodies linked to toxin molecules for potential use against cancer cells Vaccines Recombinant viral and bacterial proteins used to prepare subunit vaccines (safer and more effective than killed whole organisms) place at ambient temperature and since the be accounted for by peptide antibiotics, the ease, osteoporosis and pain (mainly of pa- amounts produced are very much lower than immunoregulator ciclosporin (cyclosporin tients with bone cancer). The 'softness' of those of other chemicals (because of the A) and the sweetener aspartam. The hor- these peptides is illustrated by the fact that high biological potency of most regulatory mones and regulatory factors play only a e.g. I kg of salmon calcitonin yields ca. 30 peptides), the risks involved with peptide minor role. However, in terms of value million single therapeutic doses. production are very much smaller. There- added, they are very much more important. fore, it is not unreasonable to regard peptides Some of these peptides, e.g. insulin, reach a as substances that are produced by so-called turnover of more than one billion dollars per 4. Recombinant Peptide Medicals 'low-risk' or 'soft' chemistry. year. Slightly lower is the share for calcito- The weight share of peptides of the world- nin, a peptide hormone which in the past few An alternative and even 'softer' method wide annual chemical production is only years has become an important drug for the for the preparation of medium-sized pep- about 0.002-0.003%, and most of this can treatment of hypercalcemia, Paget's dis- tides such as calcitonin and corticotropin is FORSCHUNG 149 CIIiMIA ~511441) Nr.:; (Ma;l the biotechnological production in bacteria, Precursor yeast, or cultured mammalian cells. This -.j L ..t v' method is also the only economic way to produce peptides of more than 50 amino I acids in length and peptides with complicat- ed glycosyl or other functional groups. ~l Attempts are even being made to apply this method to the production of short peptides such as aspartam, e.g. by using DNA clones . ~r-l------1l~ containing up to 150 repeats of the [13]. The principle of the expression of a 'for- -c;J--G}- eign' gene in host cells is relatively simple: in order to be expressed and controlled l l through the normal biosynthetic and genetic E 1 E 2 E3 E4 E5 E6 E7 machinery of the host cell, the cloned gene has to be linked to the genetic control ele- Fig. 3. Pleiotropic (/c(ion oj pep/ide hormones [171. A biologically inactive precursor is processed illlo active ments that are characteristic of the host peptides which themselves may be further cleaved into smaller fragments with a different function. Certain parts species. This is accomplished through the of the information within these peptides - although contained in the larger sequence - are scarcely transferred at all when the respective message sequences are obscured. Homlone H stimulates receptor R which transduces the use of specialized extra-chromosomal ele- signal, eventually leading to the effect E. (U ) = Message (stimulating portion of the hormone); ('1J ) = buried ments called expression vectors (e.g. plas- (obscured) message; (I'l) = address (binding portion of the honnone). H I interacts well with R I (-) and weakly mids) that are specifically designed for over- with R2 ( ). H2 contains two message sequences, both of which are necessary for full stimulation of R2 (it is expression of the relevant in the host nOl known whether R2 transduces the signal via one or two pathways). H3 stimulates with one of its messagcs R3 and R4 (receptors of different tissues having the same topography) and with another message R5 (a differcnt kind cells. The different expression vectors avail- of receptor); the full effect E4 may depend on simultaneous stimulation of R4 and R5. H6 and its fragmcnt H7 able to date are specific for the different interact well only with their own respective receptors R6 and R7, despite an identical message; truncation at the types of host cell (prokaryotic and eukaryo- C-terminus of H6 changes the topography of the address region. tic cells) and there are a number of different host cell systems available for the produc- troduced as therapeutics, including human cortins, comprIsing adrenocorticotropin tion of polypeptides [14]. The relevant prob- insulin, human growth hormone, erythro~ (ACTH), the , the melanotropins lems with which the biotechnologists are poietin, interferons, interleukin-2, colony (MSHs), and the , each of which confronted include 1) the selection of the stimulating factors and several growth and has a major target (ACTH: adrenal cortex; appropriate expression strategy, 2) the se- other regulatory factors [IS]. For research MSH: melanocytes and melanoma cells; lection of the host cell system, 3) the se- purposes an increasing number of mutants endorphins: certain neurons) but also inter- lection of a plasmid that displays a high thereof are prepared via site-directed muta- act with a number of additional tissues. The enough copy number and good stability (~ genesis of the gene in order to analyze the precursor ofthese peptides, proopiomelano- stability of DNA), 4) the control of tran- contribution of a specific structure of a pro- cortin (POMC), occurs in many tissues, script stabi Iity (~ stabi Iity of mRNA), 5) the tein to its function. This is an important part mainly the pituitary gland and the brain, and control of stability ofthe translated polypep- of modern protein engineering: potentially is processed differently according to the tide, and 6) the isolation and purification of important side chains are identified in the gland cells or neurons in which it is pro- the protein product. structural model of a protein and the corre- duced [17]. Fig. 3 is a schematic represen- The expression strategy is either based sponding residues are then removed or tation of a hypothetical prohormone similar on direct expression or on secretion. In the changed by mutagenesis. This technique is to POMC showing the different possible first, promoter sequences are linked directly not only applied to the structure-function forms of interrelations. to the ATG translation initiation codon fol- analysis of peptide and protein ligands but The pleiotropy of peptides is a disadvan- lowed by the coding region of the 'foreign' also to the study of structure-function rela- tage when they are used in therapy because gene. The protein product is usually highly tionships in receptors [16]. they may induce side effects owing to the expressed but accumulates in the cytoplasm redundancy of signalling information. A of the cells and has to be isolated from cell well-known example is the problem of Iysates, purified and (if it contains disulfide 5. Peptides Are Pleiotropic Molecules ACTH treatment of young children affected bonds) refolded and cyclized. In addition, by the West syndrome, one of the most fre- the N-terminal extra methionine is generally Peptides are known to elicit a variety of quent forms of epilepsy in the very young. not part of the desired sequence and has to be effects and to interact with different tissues Although the severe side effects induced by cleaved. Secretion vectors contain as addi- and cells. There are two reasons for this prolonged administration of ACTH (mainly tional element the gene of a multiple or 'pleiotropic' action of most pep- metabolic and psychic disorders) disappear which directs the translated protein into the tide hormones and regulatory factors: i) after ternlination of therapy, they could per- secretion pathway of the host cell. The sig- receptors for almost all peptides are ex- haps be avoided if a derivative was at hand nal peptide is removed during secretion by a pressed by different target cells, linking the that contained precisely the essential signal- signal peptidase, generating the desired hormonal signal to slightly differing biolo- ling infonmuion but had a reduced adreno- amino terminus. Disulfide bond formation, gical effects; ii) many peptides contain a cortical activity. An ACTH/MSH compound and when eukaryotic cells are being used as multiplicity of signalling information, re- with a very much reduced pleiotropic char- hosts, glycosylation or other modifications sulting in the interaction with different types acter has been developed by D. DeWied and also occur during the secretory process. Thus, of receptors, thus again linking the hormo- WH. Gispen and their colleagues in Holland the secretion strategy is the method of choice nal signal to several biological responses for the treatment of nerve regeneration and if posttranslational modifications of the prod- (Fig. 3). Another important aspect is that the protection from nerve damage by cis- uct are required. peptide hormones are produced via large platin which is uscd to treat cancer patients. There are at present more than 50 regu- prohormones which frequently serve as Thiscompound, Org 2766, was derived from latory peptide factors produced by genetic precursor molecules for several biologically the ACTH410 fragment which still contains engineering that are being developed for active peptides with different functions. The very weak adrenocortical activity; oxida- medical application (Tahle 3). Some of these various forms of pleiotropy of peptides is tion of Met, replacement of Arg by D-Lys and recombinant products have already been in- best illustrated by the group of opiomelano- of Trp-Gly by Phe yielded a compound FORSCHUNG 150 CHIMIA 45 (1991) NT. 5 (Mail

time course of action or ability to inhibit the A Acetyl - Met - Glu - His - Phe - Arg - Trp - Gly - amide natural hormone (antagonistic properties). Such studies have also proved to be very useful for the elucidation of both the mecha- nism of hormone-receptor interactions and the organization of information within the peptide. It was found, e.g., that in many lin- ear (short) peptides the hormonal messages are formed by discrete sequences of ad- jacent amino-acid residues and that a pep- tide may contain several different messages which may even partly overlap. Typical examples are the POMC peptides ACTH, MSH, and endorphin [17].

B Acetyl - Nle - Asp - His - D-Phe - Arg - Trp - Lys - amide

HN NH 6. Peptides for Receptor Analysis y 2 1 NH2 When Paul Ehrlich, the father of che- motherapy and Nobel laureate in medicine of 1908, performed his studies on toxins and antitox ins, he discovered the principle of the receptor: 'corpora non agunt nisifixata'. The dual function of a receptor as discriminator I "..... NH for the ligand on the cell surface and as ~ \,.A transducer of the hormonal signal into the cell was established in the nineteen fifties and sixties but our knowledge on the struc- tural features of peptide hormone receptors C H - Met(O~ - Glu - His - Phe - Lys - Phe - OH is very young. To know more about the discrimination function of a receptor and in NH2 I q-NH particular to develop more specific ligands ( and antagonists), acomplete three- dimensional structure of a receptor is of greathelp. One of the major problems, how- ever, is the low number of receptor mole- H 0 H 0 H 0 cules that are expressed per cell. In most cases the identification of receptor mole- Fig.4. Modified st/"UCIlI- eOOH ,"- ,"- res of the MSHIACTH4./ll - - cules requires elaborate protein chemical fragment with selective techniques, such as photoaffinity labelling. acitivi(y on the pigmen- There are only a few fortunate examples tary or central nervous systems. A) Structure of where a partial protein structure could be o H - Met(O~ - Glu - His - Phe - Lys - Phe - (CH~8 - NH2 the MSH4 10 fragment. B) obtained. When the modem DNA cloning Structure of an analogue strategies were applied to receptor cDNA with 1-2000fold higher and genes, it became possible to obtain com- pigmentary activity than A. C) Structure of the Org plete primary sequences of receptors. Such 2766 analogue with a sequences are now known for about two IOOOfoldhigherCNS ac- dozen peptide receptors but there is still only tivity than A and unde- one crystallographic diffraction analysis, tectable pigmentary ac- namely that of the G protein-coupled type of tivity. 0) Structure of the receptor molecule bacteriorhodopsin. Hoe427 analogue with a eOOH , "- f "- IOfold higher eNS acti- - - Most analyses of receptors would be vity than C. impossible without a set of labelled peptide ligands containing tritium, iodine-125, a fluorescence marker, biotin or another affin- which was IOOOfoidmore potent in certain tween structure Band C of Fig. 4 in the ity or photoaffinity group (Fig. 5). As for neuronal assays. Additional modifications pigment cell system is more than 107 which structure-activity studies (see above), it is to this molecule by R. Geiger and collabo- demonstrates that it is possible to produce important to apply different types of peptide rators in Frankfurt yielded a compound (Hoe very selective agonists. ligands that are labelled with different types 427) suitable for treating mild mental dis- Structure-activity studies during which of marker groups suitable for, e.g., hor- orders without side effects (Fig. 4). It is in- a large number of peptide analogues and mone-receptor binding studies, chemical or

teresting to note that the original ACTH4_IO fragments are synthesized and tested in dif- photochemical cross-linking of the hormone fragment is also a weak for ferent biological assays have become the with its receptor or quantification of recep- melanocytes and melanoma cells (with an most popular tool for the development of tors in tissues by receptor autoradiography. acetylated N-terminus and amidated C-ter- new peptide drugs during the past 20 years. Labelled peptides are also indispensable for minus ca. 1-2000 times less potent than (J.- The principal aim of such studies is the their measurement in biological fluids (see MSH). If Met is exchanged by Nle, Glu by design of hormone analogues with features below). Asp, Phe by D-Phe and Gly by Lys (Fig. 4), of practical utility for medical application, The study of peptide-receptor interactions the biological activity is increased over such as increased potency, greater metabol- and signal transduction as well as the 1000fold. Thus, the potency difference be- ic stability, increased selectivity, prolonged structural analysis by cloning of receptor FORSCHUNG 151 CHIMIA 45 \1991 \ Nr. 5 (Mai\

(bioluminescence, chemiluminescence, Affinity and photo- Fluorescent groups fluorescence etc.). An important aspect is affinity groups the use of peptides as antigens for the gener- ation of specific antibodies: a partial peptide "~~ sequence is selected as optimal antigenic determinant and attached specifically and vy uniformly to the carrier protein. This usually S02 results in more specific antibodies than '--y------.J I random attachment to the caJTier. Alterna- Br<:H.co- tively, the peptides may be linked in small B~(CH2kCO- Amino side-chains.--- (Lys) sets to can-ier templates thus stabilizing sec- o ------ondary structures in these peptides. An ex- - N-Terminus Peptide C-Terminus ample for this kind of concept are the tem- GN-(CH2ls-CO- plate-assembled synthetic proteins (TASP) o Tyr Trp \ developed by Mutter and Vuilleumier [18], t which can be applied to the generation of N3-o-CO- y ,---A-----., specific antibodies or to the study of peptide- N~ I I I protein interactions.J ung and coworkers [19] Icq~cO- ,,-COOH co introduced tripalmitoyl-S -glycerylcysteiny I N CF3 - N'-"""'N~ peptides as immunoadjuvants in combination N -Q-A s- (Cll •.••.C ln3+-OOc> with or covalently linked to the antigens. 3 N A H~ 12S)y 3 ¥3H \\ II co.::::..; (9.9C These are potent stimulators of OH OH N02 B lymphocytes, similar to the lipoproteins Radioactive markers (indium-111, iodine-125 and tritium) from the outer cell membrane of bacteria, and thus appear to be very promising as Fig. 5. Radioactive, fluorescent, and chemica/ marker groups for peptides. The fluorescent groups (e.g. rho- synthetic vaccines. damine-glycyl; dansyl) and the affinity and photoaffinity groups (e.g. biotinyl; bromoacetyl; 5-bromovaleryl; NC_ Another important application of syn- maleimidocaproyl; 4-azidobenzoyl; 4-(3-trifluoromethyldiazirino )benzoyl) are introduced into the amino terminus thetic pep tides is their use as substrates or a side-chain amino group at a late stage during synthesis. The same applies to the 2-nitro-4-azidophenylsulfenyl in enzymology. At present hundreds of chloride reagent (bottom, left) which can be attached specifically to the 2'-position of tryptophan. Photoreactive amino acids (e.g. p-azidophenylalanine, not displayed) can be introduced at any time during the synthesis. For enzyme substrates containing suitable radioactive labelling, a suitable precursor is preparcd, e.g. a peptide containing L-propargylglycine, which in- chromophores are on the market for the corporates4 tritium atoms (~3H4-L-norvaline), or L-3',5'-diiodotyrosine which incorporates 2 tritium atoms (~3H,_ quantification of the activity of the various I.-tyrosine). Radioiodination with Na'L'[ requires a tyrosine (or a histidine) residue in the peptide chain. The DTPA enzymes. chelator for "'In (see text) is usually attached to the N-terminus of the peptide. More recently tumour-specific synthetic pep tides carrying a suitable radioactive marker have proved to be valuable tools for molecules has led to a fairly precise idea 7. Pep tides in Medical Diagnosis tumour diagnosis. They are either applied in how receptors are arranged in a cell mem- vitro on tumour sections foi' the determina- brane and coupled to signal transducing ele- Endocrinological diagnosis has long been tion of the 'receptor status' of the cells (Fig. ments. An example for the arrangement of based on the measurement of peptides in 7) or injected into the body for tumour lo- two G protein-coupled receptors linked to biological samples. The radioimmunologi- calization. For the i/1l'itro application, 1251is each other is given in Fig. 6: the MSH cal techniques required have become very the most frequently used radioisotope where- receptor, which induces dispersion of mela- important in the clinic sinceS. Berson and R. as for in vivo administration, the peptides are nin granules in melanophores and melanin Ya/ow introduced the method for insulin labelled with the short-lived 1231 or with IllIn (pigment) production in melanocytes and about thirty years ago. Numerous radio- or 99Tcboth of which require peptides carry-

melanoma cells, and the a2-adrenergic immunoassays have been developed for re- ing a suitable chelator. Diethylenetriamine- receptor, which reverses the action of the search purposes as well as for clinical appli- pentaacetic acid (DTPA) as chelator for IllIn MSH receptor. The MSH receptor stimu- cation, and there is an increasing number of has been studied extensively for labelling lates adenylate cyclase by activation of the immunometric assays available that do no tumour-specific antibodies [20] and several G, regulatory protein whereas the a.z-ad- longer require radioisotopes as markers peptides, e.g. the somatostatin-octapeptide renergic receptor inhibits adenylate cyclase

by activation of the Gi regulatory protein. Peptide receptors frequently interact with

Gp regulatory proteins that activate phos- pholipase C, resulting in inositol-triphos- Fig. 6. Schematic repre- phate as intracellular signal (-? releases sentation of the stnlctUI'e calcium ions from intracellular calcium of /11'0 associated G pro- stores) and diacylglycerol which stimulates tein-coup/ed receptors. protein kinase C. Another group of recep- BOlh the MSH receptor (hypothetical) and the a,- tors, those for growth factors, contain tyro- adrenoceptor are present sine kinase activity and regulate cellular on pigment cells and both processes directly by phosphorylation of the are linked I'ia stimula- relevant proteins. Yet another group of re- tory G proteins (MSH) and inhibitory G proteins ceptors are ligand-induced ion channels (a,) to the same adenyla- transporting either calcium, sodium or po- te cyclase (e). The G tassium ions. It is interesting that one and the proteins are composed of same peptide may interact with more than three different subunits (a, {3.YJ and requircGTP. one receptor. Thus, the understanding of ATP cAMP +PPi MSH receptor (hypoth.) (X2-adrenoceptor The intracellular signal intracellular signalling is crucial for the de- ofMSH iscAMPformed velopment of new and specific therapeutics. by C from ATP. FORSCHUNG 152 C1HMIA ~5 (19911 Nr. 51Mail aspartyl proteases developed by Roche: A) an orally active inhibitor of renin that is specif- ic for the primate enzyme and does hardly affect the activity of other aspartyl proteases (e.g. , cathepsin D or angiotensin- converting enzmye), and B) a HIY protease inhibitor which contains a pseudo-peptide group and displays a high specificity and potency [22]. The latter is currently being A) 8) tested in AIDS patients. Another important area are non-peptide Fig. 7. Sillily of Ihe MSH receptor 'slalus' of me/alloma lissue hy qualllilali\'e receplor aUloradiography. receptor antagonists which block the effect Cryosections of melanoma were incubated with MSH radioligand in the absence A) and presence 8) of excess of excessively produced endogenous pep- unlabelled homl0ne. Quantitative anlaysis of a series of such autoradiograms reveals the receptor numberpercell. tide [23]. Well known examples are antagonists such as naloxone or nor- (Sandostatin) and a fragment of a-melano- properties of the parent hormone but exhibit binaltorphimine, and CCK and gastrin antag- tropin. Such peptides appear to be very a very much prolonged duration of the effect onists such as and loxiglumide. promising not only for tumour diagnosis but (Fig. 8). Similarly, truncated or conforma- Naloxone is an important pharnlacological also for therapeutic application. tionally restricted analogues of a- MSH were agent for experimental studies but is also found to be over tenfold more stable in the used to treat opiate intoxications. Loxi- circulation than the natural hormone and to glum ide is a very specific blocker of the 8. Peptides in Therapy bind at least as well to the receptor or even CCK receptor in the gut and may be used to better (see Fig. 4 and [17]). Such com- treat pancreatitis. One of the principal problems in the pounds are potential drugs for cancer-diag- general use of peptides as 'natural' thera- nosis and therapy,.e.g. as carriers of radioi- peutic agents is less the availability of suit- sotopes or cytotoxins. 10. New Therapeutic Concepts able compounds but rather their route of ap- plication, which should circumvent the sy- An area of great interest at present are the ringe. Several alternatives have already been 9. Pseudopeptides and Peptidomimetics cytokines, a group of large polypeptides that developed and are being used in the clinic, regulate a variety of cells, mainly of the such as the nasal sprays for calcitonin, lutein- Increasingly important are peptidomi- hematopoietic and immune systems (see also izing hornlOne-releasing hormone (LHRH; metics, compounds that are derived from Table 3). As the cytokines communicate Buserelin), oxytocin, and vasopressin. Rec- peptide structures but are no longer true between the neural, endocrine, and immune tal suppositories for calcitonin will soon be peptides. In particular, protease inhibitors systems, they affect a number of tissues and on the market, and ointments are being tested are currently developed for the treatment of participate in the regulation of various pro- for application of peptides through the skin. cardiovascular diseases, such as renin inhib- cesses, including tumour growth and While the oral administration of the vaso- itors and angiotensin-converting enzyme suppression, activation of T cells and con- pressin analogue desmopressin is well estab- inhibitors, as well as for the control of in- trol of inflammation. Genetic engineering Iished, a first series of tests has been carried fections (e.g. inhibitors of HIY protease). has made it possible to clone and express the out on orally administered insulin: a micro- The aim of these efforts is to find molecules genes of most of these regu latory factors and emulsion of peptide, fatty acids, lecithin, that can be administered orally and that to produce them in quantities large enough and cholesterol was formed and absorbed on exhibit an equally high specifiCity as the for medical application (see above). The cellulose and then embedded in gelatine corresponding peptides. Fig. 9 displays the cytokines are regarded as potentially im- capsules. In this way, it was possible to treat structure of two transition state inhibitors of portant therapeutics for the treatment of diabetic patients for a short period of time almost as effectively as with injections [21]. However, long-term administration of hor- m-ones such as insulin requires a well con- trolled dosage adapted to each individual Ala- Gly-cr -Lys- Asn-Phe -Phe - Trp) patient and, therefore, will probably prove SRIF Cys-Ser-Thr-Phe-Thr- Lys unsuitable for oral application. Although a number of excellent peptide * compounds have become available for ther- cys-Phe-Phe-Trp) apeutic application over the last ten years, OctapeptideSalk there is always a need for more specific cts-Phe-Thr- Lys substances (see e.g. the problem with the ACTH therapy of the West syndrome men- * tioned above). Theoretical modell ing of ASU-Phe-Phe-Trp) bioactive conformations and synthesis of OctapeptideCiba-Geigy stabilized molecular forms of peptides are Fig 8. SII'/IClilreofminiJorms GA~A-- Phe - Thr - Lys getting more and more important in the ofsomaloslalin (SR/F). These structurally reduced cyclic search of new therapeutics. The aim is to * find peptides with reduced structures exhib- peptides retainpharmacologi- pro-Phe-Trp) cal properties similar to the HexapeptideMSD iting a higher potency and longer duration of parent honnone and display a pte - Thr - Lys action than the natural compounds. An in- higher potency and/or a pro- creased stabil ity of the peptides to biodegra- longeddurationofaction.E.g., dation frequently turns out to be the most the octapeptide Sando: (San- dostatin) exhibits a 3fold hig- Phe*-Cys-Phe- TrP) Octapeptide Sandoz decisive factor. E.g., it was possible to syn- her potency in viIro and a thesize fonl1S of somatostatin with reduced (Sandostatin) 70fold increased activity in Thr(Oll-cts-Thr - Lys structures that retain the pharmacological vim as compared to SRIF. FORSCHUNG 153 CHIMIA 4511491) :-Ir. -' \Mail

cals also for the patient, new peptide ana- logues and possiby orally active and meta- A Transition-state inhibitor of renin bolically more stable peptidomimetics will have to be designed which display a higher specificity and less side reactions than the natural compounds. Another challenge will be to develop 'softer' routes of application for the new peptide mcdicals.

Received: March 21,1991

[I J Modified version of the inaugural lecture ("Antritlsvorlesung») held at the University of Basel on June 19, 1990. B HIV protease inhibitor [2J Symposia proceedings: a) G. Jung, E. Bayer. Eds., Peptides 1988, Proc. 20th EUl'llpean Pep- tide Symposium, DeGruyter, Berlin, 1989; b) Fig. 9. Structure of two orally actil'e J. Rivier, E. Marshal, Eds., Peptides, Proe. peptidomimetic inhibitors of' aspartyl 10th American Peptide Symposium, Eseom, proteases. A) A new transition state renin Leiden, 1990; c) E. Giralt, D. Andreu, Eds., I .# N.... ~~V-.~O ,~ H. "H inhibitor with high specificity for renin of Peptides 1990, Proc. 21 st European Peptide , N, N primates (Ro 42-5892; F. Hoffmann-La CX\ , H#s?H , Symposium, Escom, Leiden, 1991. Roche AG, Basel). B) A transition stale [3J Specialized journals: Peptides, Peptide Re- 0:"'" CONH OH 'H+ 2 o N inhibitor of HIV protease with excellent search, Regulatory Peptides, International H selectivity and potency; the in vitro anti- Journal of Peptide and Protein Research, Neuro- viral activity is in the nanomolar range peptides, Journal of Receptor Research. (Roche U.K. [22]), [41 T. Wieland, M. Bodanszky, 'The World of Peptides; a Brief History of Peptide Chemi- stry', Springer. Berlin-Heidelberg, 1991. hematopoietic deficiencies (e.g. by inactivated toxin is, e.g., the abrin or ricin A [5J R. Epton, Ed., 'Innovations and Perspectives in Solid Phase Synthesis', SPCC, Binning- erythropoietin, colony stimulating factors), chain which is toxic to cells only in the ham, 1990. viral infections (by interferons), cancer (by presence of the B chain (the B chain of the l6J Automated Merrifield peptide synthesizers interleukins, tumour necrosis factor), in- native toxin binds to the cell surface and have been developed, amongst others, by flammatory processes (by interleukins) and induces uptake of the toxic A chain) [25]. Beckman, Applied Biosystems. Blichem, Vegll in wound healing (by tissue growth factors Thus, in the peptide-toxin conjugates the Instruments. O'l)cgeJl. and Zinssl'/' (~ mul- tiple peptide synthesizer). such as TGF-/3). Some of them have already toxic part becomes only activated upon bind- [7J E. Atherton. R.C. Sheppard, 'Solid-phase been administered successfully to large ing of the hormone to its receptor. The peptide synthesis', IRL Press, Oxford, 1989. groups of patients, e.g. erythropoietin which second concept, the boron-activation, is [8] Continuous-flow solid-phase peptide synthe- sizers are available from MilligenIBio.\'('lIrch, is used to treat anemia of hemodialyzed based on the ability of the lOB isotope to Plwrmllcill-LKB, and NO\'llhiochl'/11 (UK). patients with kidney disease. capture slow neutrons, thus producing ex and [9J R.A. Houghton, Proc. Natl. Acad. Sci. U. S. A. The general application of certain cyto- 7Liparticles. This process is very efficient in 1985, 82, 5131. kines in vivo may however produce severe cell killing [26] and seems to be less damag- [10J H.M. Geysen, S.J. Rodda, T.J. Mason, G. side effects owing to their multiple biologi- ing for the surrounding tissue than y-irra- Tribbick, P.G. School's, ./.I/II/IIIIJlol. Methods diation. The application of boron-contain- 1987.102,259. cal activity (~ pleiotropic character). There IIIJ H.Gausepohl. M. Kraft,C. Boulin. R.W. Frank. are two possibilities to transform such mol- ing peptides to target tumours may also have in [5], p. 487. ecules into 'soft' medicals, i) by designing the advantage that side effects can be mini- [12J M. Feurer, in 'Peptides', Eds. M. Goodman analogues with a much reduced pleiotropy mized as compared to peptide-toxin conju- and J. Meienhofer, Wiley & Sons, New York, thus displaying only the desired effect, and gates. The principal problem is, however, . 1977, p. 448. [13J M.T. Doel, M. Eaton, E.A. Cook, H. Lewis, T. ii) by delivery of the cytokine directly into the specific accumulation of large enough Patel, N.H. Carey, Nlle/eic Acids Rl's.1980, 8, the site of action, e.g. into a tumour. This quantities of boron in the tumour. 4575. could be achieved either by coupling the [14J G.K. Jacobson, S.O. Jolly, Eds., 'Gene Tech- cytokine to a tumour-specific antibody or by nology'. 'Biotechnology', Eds. H.J. Rehm and loca[ production at the site of action. This 11. Conclusion G. Reed, VCH, Weinheim, 1989, Vol. 7b. [15J D.K. Stone, Biotechnology Industry 1990, latter approach is currently being investigat- September 14, p.1. ed by research teams in the United States Peptides have become very widely used [16J W.H.J. Ward, D. Timms, A.R. Fersht, Trends where, e.g., the tumour necrosis factor (TNF- tools for research and are part of almost any P1wrmacol. Sci. ]990, II, 280. ex) gene is cloned into tumour-infiltrating modem project in the field of immunology, [17] A.N. Eberle, 'The mclanotropins; chemistry, physiology and mechanisms of action', Kar- lymphocytes (TILs) isolated from a patient's oncology, infectiology, endocrinology, car- ger, Basel, 1988. tumour. These genetically altered TTLs are diovascular diseases, or neurobiology. In [18J M. Mutter,S. Vuilieumier,Angel1'.Chelll. 1989, reinjected into the patient and should find medical therapy too, peptides play an increas- /01,551; ibid. Int. Ed. 1989,28,535. their way back into the tumour where TNF- ingly important role: apart from the estab- [19] K.H. WiesmUller, J. Metzger, R. Schaude, A. lished trealment of certain infectious disea- Beck, G. Hess, B. Haas, W.G. Bessler, G. ex is locally produced and released, thus Jung, in [5], p. 601. killing the tumour [24]. ses with peptide antibiotics and of hormone [20J D.J. Hnatowich,Nllci. Med. Bioi. 1990,17,49. Other concepts for the treatment of tu- deficiencies with natural or synthetic pep- [21] y.w. ClIO. M. Flynn, Llincet1989, 2,1518. mours include the construction of peptide- tide hormones, new peptide factors such as [22] N.A. Roberts, J.A. Marlin, D. Kinchington e( toxin (or antibody-toxin) conjugates or the cyclosporin and several cytokines are being al .. Science 1990, 248,358. [23] R.M. Freidinger, Trends Phllrnlllcol.Sci. ]989, application of boron-containing peptides used in modem therapeutic concepts. Most 10,270. through which radioactivity is generated in of the larger peptide factors are produced by [24J S.A. Rosenberg, Scient. Am. 1990, May, p. 34, situ by activation with thermal neutrons. biotechnological methods and, thus, from I.M. Vernla, ihid. 1990, November, p. 34. The first approach targets peptide receptors the point of view of production, can be [25J A.E. Frankel, Ed., 'Immunllloxins', Kluwer regarded as 'soft' medicals. However, many Academic Publishers, Boston, 1988. on tumour cells whereby the peptide acts as [26J H. Hatanaka, Ed., 'Boron-neutron capturc ther- carrier for a 'partially inactivated toxin and of them still induce severe side effects dur- apy of tumours', Nishimura, Niigata/Japan, directs it to the site of action. The partially ing treatment. In order to become soft medi- 1986.