Peptide Factors as Pharmaceuticals: Criteria for
`Application
`
`E. WUNSCH, Max-Planck-lnstitut fur Biochemie, Abteilung
`Peptidchemie, 8033 Martinsried, Federal Republic of Germany
`
`Synopsis
`
`The increasing interest in the pharmaceutical use of peptide factors in human medicine
`presents formidable challenges for peptide chemistry. Fully reproducible multistage syn(cid:173)
`theses with a definite assessment of the degree of purity represent the crucial premise for
`the introduction of peptide factors as pharmaceuticals. Extensive studies of the stability
`of peptidic material on storage allows identification of the most suitable form of adminis(cid:173)
`tration. Nevertheless, the high clearance rate of peptides as pharmaceuticals presents new
`challenges for improvement by structural modification of resistance to enzyme degradation
`without creating new problems related to metabolites.
`
`INTRODUCTION
`
`After years of stagnation, peptide factors are regaining great importance
`as powerful pharmaceutical agents both in diagnosis and in therapy. From
`structure-function studies of a series of biologically active peptides, it is
`known that even slight structural changes can provoke drastic modifications
`both in the potency and in the activity profile. Therefore, the pharma(cid:173)
`ceutical use of peptides strongly implies an unequivocal control of their
`state of purity and extensive studies of their stability on storage and of their
`form of application.
`One main difficulty in the control of purity in the case of peptide factors
`arises from the fact that no reference substance characterized by 100%
`purity is available. Additionally, depending on the production method,
`i.e., by isolation from natural sources, chemical synthesis, semisynthesis
`or gentechnological procedures, the resulting peptide preparations may
`contain side products or contaminants of a completely different nature.
`Thus, to qualify such peptide preparations as of high purity-a quality
`indispensable for their pharmaceutical use-a large spectrum of analytical
`tests of differentiated specificity has to be performed. Nevertheless, a final
`judgment will only be possible on the basis of the greatest probability and
`this only until a new analytical method may demonstrate the opposite.
`However, standardized procedures, both in the isolation of the natural
`products and in the different synthesis strategies, may simplify analytical
`control of peptide preparations once the most efficient and significant as(cid:173)
`says have been specifically elaborated.
`
`Biopolymers, Vol. 22, 493-505 (1983)
`© 1983 John Wiley & Sons, Inc.
`
`CCC 0006-3525/83/010493-13$02.30
`
`-493-
`
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`MAIA Exhibit 1020
`MAIA V. BRACCO
`IPR PETITION
`
`

`

`494
`
`WUNSCH
`
`CRITERIA OF PURITY
`
`This analytical process and the related critical aspects are exemplified
`on synthetic somatostatin-14, a peptide factor 1 that in a short time has
`become an important pharmaceutical with several different therapeutical
`applications. 2
`As already traditional, the material obtained by chemical synthesis3 in
`the first instance was analyzed by thin-layer chromatography (TLC) and
`high-performance liquid chromatography (HPLC). This was followed by
`amino acid analyses, both qualitative and quantitative, of the acid and
`aminopeptidase-M hydrolysates with concomitant determination of the
`peptide contents and of the recoveries from the enzymatic digests. In this
`context, it is noteworthy that different conditions of hydrolysis may lead
`to different results. Thus, the most indicative conditions of hydrolysis have
`to be experimentally determined by comparative studies for any new
`In the case of somatostatin-14, the amino acid
`peptide preparation.
`analysis of the aminopeptidase-M digest was found to produce more sig(cid:173)
`nificant values than the analysis of the acid hydrolysate, even if performed
`under different standardized conditions.4 Consequently, for an analytical
`comparison of different somatostatin-14 samples, the acid hydrolysis was
`performed in the presence of 2.5% thioglycolic acid5 to recover tryptophan
`and in the absence of this additive for reliable evaluation of the serine
`(Table I); the values for tryptophan and cystine, however, were more pre(cid:173)
`cisely determined by analyses of the enzymatic digests, as shown in Table
`ll. Unfortunately, asparagine is partly hydrolyzed in the incubation media,
`preventing an exact determination of possibly partially desamidated so(cid:173)
`matostatin-14. Valuable analytical information results from comparison
`of the values related to peptide content and to recovery from enzymatic
`digestion (Table III). For most of the somatostatin-14 samples analyzed
`these values coincide fairly well within the limits of error of this analytical
`procedure. The limits of error of the quantitative amino acid analysis are
`at least 2-3%.
`
`TABLE I
`Amino Acid Analyses of Acid Hydrolysatesa of Different Somatostatin-14 Samples
`
`Diamalt-
`Serono
`A
`
`B
`
`CuraMed
`B
`A
`
`Clin Midy Cenentech
`B
`A
`B
`A
`
`UCB
`B
`A
`
`Bachem
`B
`A
`
`1.01
`1.98
`0.96
`1.00
`1.00
`2.92
`1.96
`
`Asp
`Thr
`Ser
`Gly
`Ala
`Phe
`Lys
`Trp
`
`1.02
`1.01
`2.02
`1.98
`0.97
`0.88
`1.01
`1.00
`1.00 0.99
`2.91
`2.99
`2.06
`1.92
`0.92
`
`1.07
`1.99
`0.85
`1.00
`1.00
`3.10
`1.96
`1.00
`
`1.00
`1.98
`0.94
`1.00
`1.00
`2.90
`1.99
`
`1.19
`1.92
`0.85
`0.98
`1.02
`2.82
`1.90
`1.03
`
`0.97
`1.93
`0.93
`1.00
`1.01
`2.92
`2.02
`
`1.01
`1.94
`0.97
`1.00
`1.00
`2.94
`1.91
`
`1.08
`1.93
`0.83
`1.00
`1.00
`2.91
`1.98
`0.94
`
`1.00
`1.93
`0.88
`1.00
`1.00
`2.94
`1.93
`0.96
`
`1.14
`LOI
`1.98 1.96
`0.95 0.87
`1.00 1.01
`1.00 1.01
`2.96 2.90
`2.05 1.94
`1.04
`
`a Acid hydrolysis: A, 6M HCI, 24 h, 110°C; B, 6M HCI, 24 h, 110°C; 2.5% thioglycolic
`acid.
`
`-494-
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`PEPTIDE FACTORS AS PHARMACEUTICALS
`
`495
`
`TABLE II
`Amino Acid Analyses of Aminopeptidase-M Digests• of Different Somatostatin-14
`Samples
`
`Diamalt-Serono CuraMed Clin Midy Genentech UCB Bachem
`
`Asn + Asph
`Thr
`Ser
`Gly
`Ala
`(Cysh
`Phe
`Lys
`Trp
`
`0.99
`1.99
`1.02
`0.97
`0.96
`0.98
`2.93
`2.11
`l.02
`
`1.04
`1.99
`1.01
`1.00
`1.00
`0.97
`3.09
`2.01
`0.92
`
`l.10
`2.01
`1.03
`0.99
`1.01
`0.92
`3.09
`2.04
`1.01
`
`0.97
`1.99
`0.99
`1.03
`1.03
`0.96
`3.07
`1.98
`0.97
`
`0.90
`1.96
`0.96
`0.99
`1.01
`0.96
`2.97
`2.00
`0.97
`
`1.07
`1.98
`1.02
`0.97
`1.03
`1.02
`3.09
`2.04
`1.00
`
`a Aminopeptidase-M digestion: Sigma AP-M, 37°C; 24 h.
`b Aspartic acid is formed by desamidation in the incubation media; the observed values
`correspond to those obtained with asparagine.
`
`The peptide content determined by amino acid analysis constitutes basic
`information that is then used for most of the physicochemical, biological,
`and immunological assays. Therefore, the results of these analyses, even
`if the methods applied a priori possess a higher degree of precision, are
`characterized by a limit of error that can not be lower than 2- 3%.
`Additionally, the various analytical methods exhibit different quanti(cid:173)
`tative specificity versus the possibly accompanying substances, impurities
`etc. An example in this context is given by the differentiated sensitivity
`of the HPTLC and HPLC of a series of somatostatin-14 samples (Figs. 1
`and 2). The Stilamin ampules contain a large amount of mannitol, which
`is weakly visualized by means of the chlorine test and hardly detected in
`HPLC because of the low absorption of this alcohol at 280 nm. On the other
`side, the high degree of purity of the Genentech material, as judged by
`HPTLC, is not confirmed by the HPLC as shown in Fig. 2. In fact, several
`well-separated uv-absorbing contaminants are revealed in the elution
`profile. A quantitative determination of the contaminants by integrating
`the peak areas gives only approximate values, since their absorption coef(cid:173)
`ficients are unknown. Thus, for our somatostatin-14 preparation the
`pooled side fractions presenting visible impurities on HPLC and HPTLG
`
`TABLE lII
`Peptide Contents and Digestion Rates of Different Somatostatin-14 Samples0
`
`Oiamalt-
`Serono
`
`CuraMed Clin Midy Genentech
`
`UCB Bachem
`87.5
`78.6
`
`84.7
`
`73.0
`
`77.4
`
`77.8
`
`79.9
`
`76.4
`
`Peptide content
`(±3%)
`Digestion rate
`(±3%)
`• Determined by quantitative amino acid analysis (M, = 1637.94).
`
`87.6
`
`79.2
`
`59.7
`
`61.2
`
`-495-
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`496
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`WUNSCH
`
`front - -
`
`start __..
`
`2
`
`3
`
`4
`
`5
`
`6
`
`Fig. 1. HPTLC of somatostatin-14 samples on precoated silica gel-60 plates (Merck
`AG, Darmstadt). Solvent system: 1-butanol/acetic acid/water/pyridine (45:10:20:15); 40
`µg/sample. Columns: 1, Stilamin (Serono); 2, Diamalt; 3, Curamed; 4, UCB; 5, Clin Midy;
`6, Genentecb.
`
`CLIN ~IOY 8133
`
`8150 UCB
`
`GENENTE01 LOTQ1-III
`
`j
`T
`
`12
`
`16
`
`20mm
`
`12'0 OIAMA,Lf
`
`S,£111:Qt,fO SflLAMIN
`
`11
`
`16
`
`:mm,n
`
`J
`1
`
`12
`
`'16
`
`20rnin
`
`,,
`J
`
`Fig. 2. HPLC of somatostatin-14 samples on µ-Bondapak Cl8 (30 X 0.4 cm); eluent: ac(cid:173)
`etonitrile/0.2M ammonium acetate (pH 4.0); 29:71 (v/v); flow rate, 1.3 mL/min.
`
`-496-
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`PEPTIDE FACTORS AS PHARMACEUTICALS
`
`497
`
`were chromatographed on a Lobar RP-8 column; the impurities were sep(cid:173)
`arated, pooled again, and then found to represent less than 1 % on a weight
`basis.6 Consequently, the main somatostatin fraction, wherein these im(cid:173)
`purities could not be detected by the above-mentioned methods, should
`exhibit a correspondingly higher degree of purity.
`To summarize our experiences in the case of somatostatin-14, prior to
`its use in human medicine, a whole set of analyses has to be performed as
`follows:
`Chromatographic Methods
`1. Amino acid analysis of acid and enzymatic hydrolysates;
`2. Gas-chromatographic analysis of the derivatized acid hydrolysate
`on Chirasil-V al glass capillary columns for postsynthetic chiral analysis
`(Table IV);
`3. TLC on normal and high-performance precoated plates in different
`solvent systems;
`4. HPLC possibly using different reversed-phase supports and eluent
`systems with qualitative and quantitative runs to confirm the total elution
`of applied material;
`5. Electrophoresis on supports or in the free-flow systems to make
`additional characterization of the homogeneity of the substance on the basis
`of its mobility;
`6. Micropreparative gel filtration to detect polymeric forms in the case
`of somatostatin-14.
`Spectroscopic Methods
`1. Uv measurements;
`2. Nmr to detect, via 1H and 13C resonance, possible synthetic side
`products such as alkylated tryptophans or residual protecting groups,
`etc.;
`Ir to detect possible column material resulting from the chromato(cid:173)
`3.
`graphic steps (repeated Millipore filtrations on 0.2-µm filters were found
`to be useful to avoid such contaminations);
`4. Chemical ionization, field desorption and fast-atom bombardment
`
`TABLE IV
`Gas-Chromatographic Separation of Amino Acid Enantiomers of the Acid Hydrolysates
`of Different Somatostatin-14 Samplesa
`
`D-Amino Acid Diamalt-Serono CuraMed UCB Genentech Clin Midy Bachern
`
`Ala
`Lys
`Asp
`Phe
`Trp
`Thr
`Ser
`
`<0.5
`1.5
`3.3
`1.0
`<0.5
`<0.5
`<0.5
`
`<0.5
`1.8
`2.9
`l.4
`<0.5
`<0.5
`2.7
`
`<0.5
`1.8
`3.1
`1.7
`<0.5
`<0.5
`0.6
`
`<0.5
`1.5
`3.0
`0.7
`<0.5
`<0.5
`<0.5
`
`<0.5
`2.1
`3.4
`1.3
`<0.5
`<0.5
`<0.5
`
`<0.5
`1.8
`3.0
`1.4
`<0.5
`<0.5
`<0.5
`
`"Acid hydrolysis in 6M HCI at 110°C, 20 h. Derivatization to N-pentafluoropropionyl
`amino acid isopropylesters and gas-chromatographic separation on a glass capillary column
`coated with N-propionyl-L-valine lert-butylamide polysiloxane according to Ref. 10.
`
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`498
`
`WUNSCH
`
`TABLE V
`Hydrolysis of the Tyrosine-O-Sulfate Ester on Trifluoroacetic Acid Treatment
`
`H-Arg - Asp(OtBu)-Tyr( SO 1H) • Thr ( tBu) •Cly - T rp - Nle-Asp(OtBu) - Phe- NH l
`
`l 90 \ CF 3COO H / 2-Methyl l nd ole
`
`H- Arg-Asp- Tyr( 50 3H ) - Thr- Cly-Trp- Nle- Asp- Phe - NH l
`
`(crude product)
`
`AM I NO ACID ANALYSIS :
`
`AP· M digest (l7 °C ; zq hrs ): Arg 1. 06 ; Asp 1.97 ; Thr 1. 02 ; Gl y 0.99 ; Nie 0. 99 ; Tyr 0 , 02 ; Ty r( S03H) 1. 00 ;
`Phe 1. 02 : Trp 0. 95 .
`
`H- Tyr( SO 3Bo 112 ) - Thd !Bu ) - Cly-Trp- N le- Asp( OtBu 1- Phe- NH z
`190\ CFfOOH / 2- Methylindole
`H-T y,(S0 1H)-Thr- Gly - Trp - Nlc- Asp - Phe - NH 2 (c,ude product)
`
`AMINO ACID ANA L YSIS :
`
`AP-M dig .. t (17 ° C : 20 h,s) : Asp 1.01 ; Thr 1. 07; Cl y 0 , 99 ; Nie 1.00 ; Tyr 0 . 20; Tyr(S0 3H) 0 . 85 ; Phe 1.03 ; T,p 0. 90 .
`
`mass spectrometry to characterize enzymatic fragments or the entire
`molecule for controlling the correctness of the sequence;
`5. Fluorescence measurements to detect fluorescent contaminants
`resulting from the resins, the solvents, and the ion-exchange water used.
`Enzymatic Methods
`1. Amino peptidase-M digest;
`2. Trypsin digest with the related chromatographic separation;
`3. Dipeptidyl-peptidase digest, again with the related chromatogram,
`representing useful tools for control of purity.
`Biological Methods
`1. Assays in vitro;
`2. Assays in vivo (both necessary to establish the biological activity);
`3. Toxicological properties (which must be determined for use in
`medicine).
`Immunological Methods
`1. Cross-reactivities with different antisera (this allows one to make
`ulterior characterization of the synthetic preparation).
`
`Most of these analyses have been performed on our somatostatin-14
`preparation3A,6 on standardization of the synthetic procedures, which have
`been used commercially to produce this peptide factor in the last three
`years_ This material has since been used in human medication, and no case
`of allergy, toxic effect, or unexpected behavior has been observed. Cer(cid:173)
`tainly other peptide factors such as oxytocin, calcitonin, and ACTH, have
`also been used for years as pharmaceuticals and were admitted for use in
`human medicine at a time when both synthetic methods and analytical
`assays had not reached today's standards. This observation should not,
`however, relieve peptide chemistry from maximum efforts to assure the
`highest quality material for pharmaceutical use.
`
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`PEPTIDE FACTORS AS PHARMACEU'rICALS
`
`499
`
`How the criteria of purity may depend on progress in analytical methods
`is well documented by an example from our laboratory. The hormone
`secretin was studied for years in our laboratory, particularly for its possible
`application as a promising drug in treating ulcers or in the diagnosis of
`diseases of the pancreas in combination with pancreozymin-related pep(cid:173)
`tides. A reproducible standardized synthesis with a simple purification
`procedure by ion-exchange chromatography on DEAE- Sephadex was
`elaborated for possible industrial use.7 In the last fragment-condensation
`step, both N -hydroxysuccinimide (HONSu), as proposed by us, 8 and 1-
`hydroxybenzotriazole (HOBt), proposed by Konig and Geiger,9 were used
`as additives to the dicyclohexylcarbodiimide as coupling reagent. Both
`secretin preparations (HONSu- and HOBt-secretin) exhibited a high degree
`of purity as judged from all the analytical tests performed, and the biological
`activity was found to be slightly superior to that of the best natural secretin
`of Mutt.7
`New developments allowed us to optimize the HPLC conditions for
`natural and synthetic secretin, as well as for secretin analogs. The elution
`profiles (Fig. 3) of the crude HOBt- and HONSu-secretions obtained on
`trifluoroacetic acid treatment of the fully protected heptacosapeptide
`amides indicate a side peak in the HOBt material, with higher tn practically
`absent in the HONSu-preparation. Gas-chromatographic analysis on a
`
`A)
`
`BJ
`
`20
`16
`12
`8
`0
`Fig. 3. HPLC of crude secretin preparations on a Rad P ak C 18 column; eluent: acetoni(cid:173)
`t rile/0.1 3M dibutylammoniumtrifluoroacetate (pH 2.6); 31:69 (v/v); fl ow rate, 2 mL/min.
`(A) crude HOBt-secretin; (B) crude HONSu-secretin.
`
`24
`
`28
`
`32
`
`36
`
`40 Umin
`
`-499-
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`

`500
`
`WUNSCH
`
`Al
`
`Bl
`
`~1c
`
`A.
`
`I
`
`.,.
`
`-
`
`A
`
`'
`
`0
`
`8
`
`12
`
`16
`
`20
`
`24
`
`2B
`
`32
`
`36
`
`40
`
`I.I.min
`
`Fig. 4. HPLC of (A) HOBt-secretin and (8) natural secretin (a generous gift of Prof. Mutt)
`on a Rad Pak Cl8 column; eluent: acetonitrile/0.13M dibutylammoniumtrifluoroacetate
`(pH 2.6); 31:69 (v/v); flow rate, 2 mL/min.
`
`Chirasil-Val glass capillary colurnn10 of the two derivatized acid hydroly(cid:173)
`sates indicates a strong difference in the content of D-phenylalanine. This
`observation, as well as a comparison by HPLC with an authentic sample
`ofo-Phe6-secretin prepared by us as a reference substance, confirmed that
`the impurity eluting with higher tn and present in the "HOBt material"
`corresponds to the o-Phe6 diastereoisomer of secretin. o-Phe6-secretin
`was not separated by the above-mentioned ion-exchange chromatography.
`The HPLC elution profile of both secretin preparations presented an ad(cid:173)
`ditional impurity with lower tR as shown for the HOBt material in Fig. 4
`after ion exchange chromatography. This impurity was then separated
`by chromatography on CM-cellulose (eluent: 0.05M NH40Ac, pH
`6.65).
`By means of a trypsin digest and the related chromatographic identifi(cid:173)
`cation of the fragments (Fig. 5), the modification was located in these(cid:173)
`quence region 15-18. The structural analysis strongly suggests that the
`contaminant may result from the succinimide formation at the level of the
`
`-500-
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`

`PEPTIDE FACTORS AS PHARMACEUTICALS
`
`501
`
`-
`
`start
`
`,.
`
`B
`A
`Fig. 5. HPTLC of the tryptic fragments of (A) secretin and (B) secretin side product (sol(cid:173)
`vent: 1-butanol/acetic acid/pyridine/water, 45:10:15:20). T 1, H-His-Ser-Asp-Gly-Tbr(cid:173)
`Pbe-Thr-Ser-Glu-Leu-Ser-Arg-OH [1- 12]; T 2, H-Leu-Arg-OH [13- 14]; T a, H-Asp-Ser-Ala(cid:173)
`Arg-OH [15-18]; T4, H-Leu-Gln-Arg-OH [19-21]; Ts, H-Leu-Leu-Gln-Gly-Leu-Val-NH2
`[22-27}. Precoated silica gel 60 plates (Merck AG, Darmstadt).
`
`Asp-Ser sequence (positions 15 and 16) with concomitant a -
`peptidation.
`Since our synthetic secretin exhibited a biological activity slightly su(cid:173)
`perior to that from natural sources, a -
`,B-transpeptidation in the central
`region of the hormone molecule seems not to influence the biological ac(cid:173)
`tivity, whereas if the same side-reaction occurs in the N-terminal region
`it is accompanied by a drastic loss of potency.
`
`,B-trans(cid:173)
`
`STABILITY OF PEPTIDE FACTORS
`
`In the 1970s, it was generally believed that secretin is a very unstable
`peptide hormone. Extensive investigations of secretin stability allowed
`us to demonstrate the opposite. 11 By means of TLC following the incu(cid:173)
`bation of secretin solutions at room temperature as a function of pH, an
`unknown substance (X-secretin) was found to appear to an increasing ex(cid:173)
`tent enhancing the proton concentration of the incubation media (Fig. 6).
`Secretin was then kept in dilute acetic acid at pH 3 at room temperature
`for 28 days and the conversion product (X-secretin) was isolated from the
`reaction mixture by gel filtration on Biogel P 6. It was found to be stable
`
`-501-
`
`
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`

`

`502
`
`WUNSCH
`
`front---+
`
`start-+
`
`-
`
`1 2 3 4 5 6 7 8 9 10 11
`.,__ _____ . ... J
`~- ... -
`-
`-
`-
`...J
`
`Fig. 6. TLC of secretin on precoated silica gel 60 plates (Merck AG, Darmstadt); eluent:
`1-butanol/acetic acid/water/pyridine (60:6:24:20). The secretin samples were kept for 4 weeks
`in (1) 10% acetic acid; (2-6) 0.15M ammonium acetate, pH 3.2, 4.0, 5_0, 6.0, 7.0; (7-11) 0.25M
`ammonium acetate, pH 3.2, 4.0, 5.0, 6.0, 7.0.
`
`only in acidic solution and to quantitatively convert into another substance
`or mixture of substances (Y-secretin) at pH 7 within 2 days. The biological
`activities determined in vivo in dogs were 1-2% for X-secretin and 10-20%
`for Y-secretin. The structure determination of the two products was
`achieved by means of chromatographic, enzymatic, and spectroscopic
`methods: X-secretin corresponds to cyclic succinimide derivative, while
`Y-secretin is a mixture of about 80- 90% inactive "3-(3-aspartyl-secretin"
`and 10-20% active "3-a-aspartyl-secretin." To summarize, it has been
`possible to characterize two inactive forms of secretin and to demonstrate
`that the a -
`/3-transpeptidation in the sequence 3-4 postulated earlier by
`Bodanszky et al. 12 in fact represents one of the mechanisms for the inac(cid:173)
`tivation of secretin in solution, whereby this inactivation is catalyzed by
`acids, particularly acetic acid. These results have also been confirmed by
`Beyermann et al.13 A sample of 100% active secretin was kept in neutral
`solution (pH 7) for 30 days at 25°C; on lyophilization it exhibited 90% ac(cid:173)
`tivity. A second sample of secretin was incubated for 28 days in dilute
`acetic acid at pH 3 and then 2 days at pH 7; it showed only about 50% bio(cid:173)
`logical activity. This observation is in contrast to previous findings by other
`laboratories,14•15 which claim that secretin is most stable in slightly acidic
`solutions.
`Of the possible side reactions at the level of the secretin molecule listed
`-y-transpeptidation, the desamidation, and
`by Beyerrnann et al.,13 the a -
`the N - 0 shift, as well as lactone formation, have not been observed. in de(cid:173)
`tectable amounts, whereas transpeptidation both at the level of the Asp-Gly
`(positions 3 and 4) and Asp-Ser bond (positions 15 and 16) is now definitely
`confirmed.
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`PEPTIDE FACTORS AS PHARMACEUTICALS
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`503
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`A peptide hormone, studied for years particularly because of its well(cid:173)
`known instability, is pancreozymin-cholecystokinin (CCK-PZ). The in(cid:173)
`stability of the CCK-PZ-tritriacontapeptide amide, as well as of its C-ter(cid:173)
`minal fully active octa- and decapeptides with concomitant loss of biological
`activity, is mainly due to two factors:
`(1) facile hydrolysis of the tyros(cid:173)
`ine-0-sulfate moiety and (2) the strong tendency of the two methionine
`residues to oxidize.
`In fact, HPLC of ampuled CCK-PZ-octapeptide
`(Scincalide) , as well as of the bulk material (Squibb Laboratories), clearly
`revealed that in the ampule form, most of the active material was de(cid:173)
`stroyed.16 This problem was extensively investigated by us. Finally, using
`tyrosine-0-sulfate as starting material, 17 we succeeded in demonstrating
`that the greatest stability of the sulfate ester moiety is reached at the level
`of the C-term.inal nonapeptide. 18 The relative proximity of the guanido
`function of arginine-25 to the tyrosine-0 -sulfate in position 27 may possibly
`lead to a stabilizing complex formation. This stabilizing effect is so strong
`that even on exposure to trifluoroacetic acid, as needed for the acidolytic
`deprotection step of the synthetic side-chain-protected nonapeptide, no
`hydrolysis of the sulfate ester was observed (Table V) .
`The elongation of the chain to the decapeptide sequence with an aspartic
`acid residue significantly weakens this stabilizing effect, with concomitant
`hydrolysis in identical conditions of about 10%. It may weU be possible
`that similar stabilizing effects can be produced by the complex forming
`cations Ca2+ or Ba2+ added to the octa- and decapeptides.
`Our studies on CCK-related peptides have also shown that biological
`activity is fully retained on substitution of methionine-28 with threonine
`(in analogy with the primary structure of caerulein) and of methionine-31
`with norleucine, but not with leucine. The CCK-nonapeptide analog, as
`lyophilizate, was found to be perfectly stable in the cold for three years; no
`decomposit ion, as monitored by TLC and HPLC, was observed, and bio(cid:173)
`logical activity was fully retained.
`Replacement of methionine by norleucine seems to be of general utility.
`In fact, norleucine-analogs retain the biological potency of the parent
`methionine peptides and, excluding inactivation via oxidation of the me(cid:173)
`thionine thioether function , may represent a useful bypass to stabilize
`peptide factors for their use in medicine. Norleucine should be harmless,
`since it is degraded in biological systems. In this context, it may also be
`noteworthy that even from an immunological point of view, the methio(cid:173)
`nine/norleucine replacement causes negligible effects, as we recently found
`in the case of gastrins.19
`
`FORMS OF ADMINISTRATION OF PEPTIDE FACTORS AS
`PHARMACEUTICALS
`
`Tht CCK-PZ-octa- and decapeptides were long believed to possess a
`higher activity than CCK-PZ-33 or its variant 39. It is now well known that
`this difference is mainly attributable to adsorption on laboratory glassware.
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`504
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`WUNSCH
`
`In the meantime, these adsorption phenomena have been observed for most
`of the peptide factors, e.g., also for secretin,13 and may represent a real
`problem in establishing the right dosages in human medicine. Treatment
`of the glassware, as well as the use of additives, was found to suppress, at
`least partially, this unpredictable loss of material.
`An additional problem derives from the necessity of preparing ampules
`with additives for visual effects, particularly in cases in which the thera(cid:173)
`peutic doses are in the nanogram range. To convince users and physicians
`that peptide factors are really present, mannitol is usually added. This
`addition is made solely for commercial reasons, without taking into account
`the resulting chemical problems. In fact, since mannitol is an alcohol, al(cid:173)
`coholysis of the lyophilized peptide material can occur, particularly if traces
`of free acetic acid are present in the lyophilizates to catalyze these reactions.
`For example, secretin ampuled in the presence of mannitol totally loses
`its biological activity during the lyophilization process; preliminary results
`indicate that somatostatin-14 exhibits a similar tendency on storage. For
`somatostatin-14, which is administered in larger doses, the problem of
`additives may not be so crucial, but for other peptide factors, an alternative
`solution has to be found. Depending on the solubility of the peptide, an
`addition of salts such as Na Cl or of amino acids may resolve this problem.
`In the future, it may also be possible to administer these natural substances
`in the presence of carrier proteins, whereby even the adsorption on glass
`would be prevented and the stability of the peptides against degradation
`enhanced.
`Peptides are usually administered by intravenous, intramuscular, or
`subcutaneous injection. Oral, nasal, or rectal administration would cer(cid:173)
`tainly be much more desirable for patients. Slow resorption with conse(cid:173)
`quently fast degradation makes such forms of application impractical at
`present. Thus, analogs more resistant to degradation are desirable. This
`problem has been intensively investigated recently by synthesis of peptide
`analogs containing D-amino acids, N -methyl amino acids, unnatural amino
`acids, or other building blocks. The design of analogs in some cases has
`been forced to the point where not many of the structural characteristics
`of the parent peptide factors remain; "normal" chemical drugs are the re(cid:173)
`sult, with all the concomitant problems relating to the study of possible
`metabolites.
`
`References
`
`l. Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J. & Guillemin, R. (1973)
`Science 179, 77-79.
`2. Bunte, H. & Hotz, J ., Eds. (1981) Proceedings of Munst ersche Allgemeinchirurgische
`Symposien: Somatostatin , klini.~che und experimentelle Ergebnisse, Systemdruck und
`Verlags-GmbH, Freiburg, FRG.
`3. Moroder, L., Gemeiner, M., Gohring, W., Jaeger, E ., Thamm, P. & Wunsch , E. (1981)
`Biopolymers 20, 17- 37.
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`PEPTIDE FACTORS AS PHARMACEUTICALS
`
`505
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`4. Wunsch, E. (1979) in Hormone Receptors in Digestion and Nutrition, Rosselin, G.,
`Fromageot, P. & Bonfils, S., Eds., Elsevier/North-Holland Biomedical, Amsterdam, pp.
`115-125.
`5. Matsubara, H. & Saraki, R. M. (1969) Biochem. Biophys. Res. Commun. 35, 175-
`181.
`6. Moroder, L., Vaysse, N. & Wunsch, E. (1982) in Proceedings of the 2nd International
`Symposium on Somatostatin, Raptis, S., Ed. (in press).
`7. Wunsch, E. (1972) Natu.rwissenschaften 59, 239- 246.
`8. Wunsch, E. & Drees, F. (1967) Chem. Ber. 100, 816-819.
`9. Konig, W. & Geiger, R. (1970) Chem. Ber. L03, 788-798.
`10. Frank, H., Nicholson, G. J. & Bayer, E. (1977) J. Chromatogr. Sci. 15, 174-176.
`11. Jaeger, E., Knof, S., Scharf, R., Lehnert, P., Schulz, I. & Wunsch, E. (1978) Scand.
`Gastroenterol. (Suppl. 49) 13, 93.
`12. Bodanszky, M., Ondetti, M. A., Levine, S. D. & Williams, N. J. (1967) J. Am. Chem.
`Soc. 89, 6753-6757.
`13. Beyerman, H. C., Grossman, M. I., Solomon, T. E. & Voskamp, D. (1981) Life Sci. 29,
`885-894.
`14. Spignola, L. J. & Grossman, M. I. (1973) in Peptide Hormones, Berson, S. A. & Yalow,
`R. S., Eds., North-Holland, Amsterdam, pp. 1066-1068.
`15. Grossman, M. I. (1969) Gastroenterology 57, 767.
`16. Pradayrol, L., Vaysse, N., Cassigneul, J . & Ribet, A. (1979) in Hormone Receptors in
`Digestion and Nutrition, Rosselin, G., Fromageot, P. & Bonfils, S., Eds., Elsevier/North(cid:173)
`Holland Biomedical, Amsterdam, pp. 95-100.
`17. Moroder, L., Wilschowitz, L., Jaeger, E., Knof, S., Thamm, P. & Wunsch, E. (1979)
`Hoppe Seyler's Z. Physiol. Chem. 360, 787-790.
`18. Moroder, L., Wilschowitz, L., Gemeiner, M., Gohring, W., Knof, S., Scharf, R., Thamm,
`P., Gardner, J. D., Solomon, T. E. & Wunsch, E. (1981) Hoppe Seyler's Z. Physiol. Chem.
`362, 929-942.
`19. Wunsch, E., Moroder, L., Gillesen, D., Soerensen, U. 8. & Bali, J.P. (1982) Hoppe
`Seyler's Z. Physiol. Chem. 363, 665-669.
`
`Received June 20, 1982
`Accepted August 26, 1982
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