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ISSN 1068 1620, Russian Journal of Bioorganic Chemistry, 2010, Vol. 36, No. ... Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of ...
ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2010, Vol. 36, No. 6, pp. 690–695. © Pleiades Publishing, Ltd., 2010. Original Russian Text © M.G. Akimov, I.V. Nazimov, N.M. Gretskaya, V.I. Deigin, V.V. Bezuglov, 2010, published in Bioorganicheskaya Khimiya, 2010, Vol. 36, No. 6, pp. 753– 759.

Investigation of Peptide Stability upon Hydrolysis by of Fragments of the Organs of the Gastrointestinal Tract of Rats M. G. Akimov1, I. V. Nazimov, N. M. Gretskaya, V. I. Deigin, and V. V. Bezuglov Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. MiklukhoMaklaya 16/10, Moscow, 117997 Russia Received March 1, 2010; in final form, March 24, 2010

Abstract—The hydrolytic stability of a range of cyclic tripeptides, including the therapeutically important dalargin and stemokin, as well as peptides modified by ibuprofen and aspirin, has been studied. The first two experimental systems used utilized purified enzymes (pepsin, trypsin, and chymotrypsin), while the second one utilized fragments of the stomach and small intestine of rats. The linear peptides containing only Lamino acid residues were shown to be hydrolyzed by stomach and intestine fragments, although some of these peptides were resistant to hydrolysis by individual enzymes. The peptides containing Damino acid res idues and cyclic peptides were stable under all of the conditions used, but the peptides modified by aspirin lost the acetyl group of the aspirin moiety in acidic media, this process being accelerated in the presence of pepsin. Keywords: hydrolysis, peptides, stomach, intestine, dalargin, stemokin, hemopressin DOI: 10.1134/S1068162010060038 1INTRODUCTION

Peptides are promising drug candidates. First, these compounds are composed of natural compo nents connected by bonds easily degraded by the organism and, therefore, do not cause problems for its 2

detoxification and excretion systems. Second, the peptide pools of living organisms contain peptides capable of performing almost any therapeutic action [1, 2] and, therefore, it is often sufficient to find a suit able natural peptide in order to develop a drug. It should also be noted that the methods of peptide syn thesis are well developed and the peptides obtained can be relatively easily modified, for example, with molecules of other drugs, in order to change or extend the range of their action [3–5]. Drug administration per os is one of the most attractive methods, because the patient does not feel almost any physiological or psychological discomfort in this case. However, this route of drug administration poses additional requirements to the preparation, which must be stable enough to pass through the gas trointestinal tract, get into the bloodstream, and remain intact until the contact with the target. The stomach is the main barrier for proteins because the cleavage of food peptides and proteins occurs there: this organ is characterized by extremely low pH values (approximately 1.5–2) and high concentrations of 1 Corresponding

author; phone: (495) 3306592; email: aki [email protected]. 2 Abbreviations: GIT, gastrointestinal tract; SSM, artificial stom ach salt mixture; ISM, artificial intestine salt mixture.

hydrochloric acid (up to 40 mM) and pepsin (13.3 g/l) [6]. The absorption of most peptides also occurs in the stomach. Trypsin and chymotrypsin are the main hydrolytic enzymes of the intestine. Therefore, it is necessary to confirm the stability of the peptide in the organs of the gastrointestinal tract (GIT) in order to make a decision about the route of its administration. The aim of the present work was to study the stabil ity of several peptides in the rat GIT and compare it to the resistance of the peptides to hydrolysis by purified enzymes. RESULTS AND DISCUSSION Studies of Peptide Cleavage by Individual Enzymes The conventional approach to the determination of peptide and protein resistance to hydrolysis in the gas trointestinal tract (GIT) is the modeling of the com position of the medium in a specific part of the GIT by adjusting the salt concentrations and the concentra tions of the main enzymes, namely, pepsin, trypsin, and chymotrypsin [7, 8]. Pepsin (EC 3.4.23.1), trypsin (EC 3.4.21.4), and chymotrypsin (EC 3.4.21.1) are the principal proteinhydrolyzing enzymes of rat GIT [9, 6], and the first test system for the analysis of peptide resistance to hydrolysis was based on these enzymes. The salt mixtures used as incubation media were iden tical to stomach (SSM) and intestine (ISM) media, respectively, and allowed for the maintenance of the correct pH values (2.6 and 7.0, respectively) [10]. Ste mokin, dalargin, linear peptide (VI), cyclopeptide (Ia), and its analogs modified with aspirin (Ib) and

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ibuprofen (Ic) (see Table 1 for the structures of the substrates) were incubated in these reaction media. The research performed allowed for the estimation of the stability of peptides (Ia), (Ic), and (VI) in artifi cial salt mixtures modeling the internal media of the stomach and the intestine, as well as in these salt mix tures with a pepsin or trypsin–chymotrypsin mixture added. The behavior of peptide (Ib) modified with aspirin was more complex. The incubation of this pep tide with the artificial salt mixture of the stomach in the presence of pepsin caused a complete loss of the acetyl group of the aspirin moiety, while in the absence of the enzyme the extent of the reaction was less than 50%. Thus, the peptide itself and its bond with aspirin are stable in gastric juice but the bound drug molecule is degraded. The conjugate (Ib) was stable in the artifi cial intestinal medium. Peptide (Ia) is not hydrolyzed by pepsin; therefore, it was used to investigate the effector action of the pep tides studied on the hydrolysis of specific substrates by this enzyme. The peptide ProValAsnPheLysVal ValSerHis, which is 50% hydrolyzable by pepsin, was used as the substrate. Equal amounts of this sub strate and the cyclopeptide were incubated with pep sin. Surprisingly, peptide (Ia) was found to accelerate the hydrolysis 1.3fold (the degree of hydrolysis equaled 55 ± 3% in the absence of the cyclopeptide and 68 ± 3% in its presence, respectively; the difference is statistically significant, р = 0.01 in the Holm–Sidak test). The phenomenon observed can be explained by the optimization of the interaction between the enzyme and the hydrolyzed substrate by the cyclopep tide, but the details of this interaction require further investigation. The peptides dalargin and stemokin were not hydrolyzed by pepsin under the conditions described above. The optimum pH of pepsin activity towards dif ferent substrates is known to vary from 1.8 to 4.2; therefore, at a specific pH some substrates can be hydrolyzed while others remain intact [11]. Therefore, additional experiments were performed to check whether the stability of dalargin and stemokin was due to suboptimal pH values of the incubation medium. It was shown that the use of an acetate buffer instead of SSM, as well as incubation at pH 3.0 and 4.0, did not result in dalargin and stemokin hydrolysis by pepsin. Investigation of Peptide Hydrolysis by GIT Fragments A detailed analysis of the GIT model based on indi vidual GIT enzymes shows that the results obtained using this model are only partially relevant for the esti mation of the stability of the substances in the organ ism. This model does not include the enzymes located on the cell surface, as well as the possibility of intrac ellular peptide transport with subsequent hydrolysis. Accordingly, some authors suggest using the enzymes of the intestinal walls, along with the secreted protein RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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Degree of hydrolysis 1.2 1 1.0

2

0.8 0.6 0.4 0.2 0 2

6

4

8

10

12 pH

Fig. 1. The pH dependence of dalargin hydrolysis in intes tinal (1) and stomach (2) enzymes.

ases, for modeling, or implanting a catheter into the GIT of the animal for subsequent studies of the hydrolysis in vivo [12, 13]. The present work reports the use of a new test system including fragments of rat GIT tissues instead of the purified enzymes; the hydrolysis of dalargin and stemokin was detected in this test system. A range of experiments was performed to charac terize the model of stomach and intestinal hydrolytic systems based on GIT fragments. First, the depen dence of the degree of dalargin hydrolysis on the pH of the incubation medium in the whole pH range was studied. The mixture of sodium citrate and sodium phosphate with the pH ranging from 2.6 to 7.2, as well as sodium carbonate (pH 9.0–10.6), were used as buff ers in these experiments. The pH optimum for the hydrolysis of this peptide by stomach fragments was shown to be near 5.9, while for the fragments of the intestine it was near 7.2 (Fig. 1). The influence of several buffers capable of main taining the optimal pH value (sodium phosphate and a mixture of sodium citrate and phosphate for the frag ments of intestine, and sodium acetate and a mixture of sodium citrate and phosphate for stomach frag ments) during dalargin hydrolysis was studied in the subsequent experiments. SSM and ISM with pH val ues adjusted to 5.9 and 7.2, respectively, were also studied. At the end of the incubation period, the pH of the media was checked to rule out any changes. There were no statistically significant differences in the hydrolysis efficiency between the buffers studied, and, therefore, further research was performed with ISM and SSM in order to compare the results with those obtained with individual enzymes. The time dependence of dalargin and stemokin hydrolysis by enzymes from fragments of the stomach Vol. 36

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Table 1. Structures of the substrates used Indication

Sequence or formula

H N

H N

(I)

H N

O

NH

R

N H

O

OH

O O

a:R=H

c:R=

O O

O O

b:R=

O (II)

H2N

NH

HN

HN

O

NH

R

O O (III)

NH

HN

HN

H2N

R

O O

N H

O

HN

OH

O (V)

O

HN

OH

N H

NH2

NH

(VI)

H2N

NH2 H N O

Dalargin Stemokin

NH2

a: R=H b: R=OH c: R=OCH3

NH2

O

H N

R

NH2

a: R=H b: R=OH c: R=OCH3

O

H N

R

a: R=H b: R=OH c: R=OCH3

O

NH

(IV)

a: R=H b: R=OH c: R=OCH3

O

O N H OH

OH O

TyrDAlaGlyPheIleArg IleGluTrp RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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0.8

Degree of hydrolysis 1.2 1 2 1.0 3 0.8

0.6

0.6

1.0

693

0.4

0.4 1 2 3 4

0.2 0

0.2 0 –0.2

–0.2 0

10

20

30

40

50

0

60 70 Time, min

Fig. 2. Time dependence of dalargin (1, 2) and stemokin (3,4) hydrolysis in intestinal (1, 3) and stomach (2, 4) enzymes.

and intestine was studied under the chosen conditions (Fig. 2). According to the data obtained, the efficiency values of dalargin and stemokin hydrolysis by enzymes of rat GIT fragments are in the following order: ste mokin in the stomach < stemokin in the intestine ≤ dalargin in the stomach < dalargin in the intestine. The mass spectrometric analysis of the products of stemokin hydrolysis in the intestine and in the stom ach under the conditions described above showed that the Сterminal tryptophan residue of the peptide is cleaved off and the dipeptide LeuGlu (ion [М]2+, m/zcalc 130.0686, m/zexp 130.455 (intestine) and 130.456 (stomach)) is the product of the reaction. The mass spectrum of the stemokin standard contained the peak of the [M + H]+ (m/zcalc 447.2243, m/zexp 447.029), while the mass spectrum of the dalargin standard contained the peaks of the [М]+ ion (m/zcalc 725.386, m/zexp 725.425) and the [M + 2H]2+ ion (m/zcalc 363.7008, m/zexp 363.703). Dalargin hydrolysis by enzymes from stomach fragments also yields a product with the Cterminal arginine cleaved off—the pentapeptide TyrAlaGly PheIle (ion [М]+, m/zcalc 569.2850, m/zexp 569.757). Dalargin hydrolysis by the enzymes from the frag ments of the intestine is more complicated: namely, the tripeptide TyrAlaGly (ion [M + 2H]+, m/zcalc 310.1403, m/zexp 310.263) and the tetrapeptide Tyr AlaGlyPhe (ion [M + 2H]+, m/zcalc 457.2087, m/zexp 457.023) are formed. The dependence of the changes of the content of these products in the incubation medium on time allows for the conclusion that they are products of competing reactions, and not of the stepwise degradation of the substrate (Fig. 3). The results of the experiments investigating the time RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

10

20

30

40

50

60 70 Time, min

Fig. 3. Time dependence of the accumulation of the products of dalargin hydrolysis by fragments of rat small intestine. The percentage content of each component (plot ted on the ordinate axis) was calculated as the ratio of the product peak area to the sum of all peak areas. (1) all prod ucts; (2) TyrDAlaGly; and (3) TyrDAlaGlyPhe.

dependence of dalargin hydrolysis allow for the assumption that this peptide contains two sites of hydrolysis recognized either by two independent endopeptidases or by a single enzyme. The Cterminal fragment of dalargin is probably cleaved into individ ual amino acids immediately after it is formed, since it could not be detected chromatographically. The possibility of the hydrolysis of a range of pep tides modified by tryptamine, 5hydroxytryptamine, and 5methoxytryptamine (Table 1, structures (Ia) and (II)–(IV)) under optimized conditions was stud ied. All the cyclic peptides ((IIa)–(IIc), (IIIa)– (IIIc), and (Ia)), as well as the linear peptides contain ing Damino acids (Va)–(Vc), were resistant to hydrol ysis by the enzyme systems of the fragments of rat stomach and small intestine. Linear peptides (IVa) and (IVc) were resistant to hydrolysis by the enzymes from the stomach fragments, but the enzymes from the frag ments of the intestine hydrolyzed these peptides com pletely. Linear peptide (IVb) was also hydrolyzed by the enzymes from the fragments of the intestine, while the percentage of the peptide cleaved after incubation with the stomach fragments was less than 50%. Thus, the use of models based on the purified prin cipal hydrolases of the stomach and small intestine (pepsin, trypsin, and chymotrypsin) is insufficient for a reliable estimation of peptide resistance to hydrolysis in the GIT. Models involving fragments of these GIT parts seem more adequate, but the limitations on their use include the incomplete standardization of the amount of enzymes in each incubation mixture, as well as the limited possibility of taking the partial uptake and immobilization of the substrates and prod ucts in tissue fragments into account. In addition, Vol. 36

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since the incubation mixture contains various compo nents of the GIT wall in addition to the layer of cells that are in immediate contact with food, it is difficult to make an unambiguous conclusion concerning the participation of digestive or intracellular enzymes in hydrolysis in this model. The results of the research performed in the two experimental systems allow for the conclusion that cyclic peptides (Ia), (II), and (III), as well as the pep tide (Ia) modified with aspirin (Ib) and ibuprofen (Ic), and the branched peptide (VI) are stable in the intesti nal medium, while the unmodified peptides are stable both in the stomach and in the intestine, and the linear peptides containing Damino acid residues are resis tant to hydrolysis in both parts of the GIT. The loss of the acetate group by the peptides covalently modified by aspirin (Ib) can be unambiguously postulated, as well as the fact that linear peptides consisting only of Lamino acids will be hydrolyzed in the intestine and, in some cases, also in the stomach. Thus, it has been shown that the use of artificial gastric or intestinal juice containing a limited number of enzymes can lead to an overestimation of the stabil ity of therapeutic peptide preparations in some cases. We have developed test systems for the investigation of peptide resistance to hydrolysis in rat GIT, which are more adequate than the systems based on purified GIT enzymes. This procedure can be applied to other ani mal species, including humans. EXPERIMENTAL Bovine trypsin (AO Samson, Russia), porcine pep sin (Olaine Chemical Plant Biolar, Latvia), bovine chymotrypsin (SigmaAldrich, United States), phos phatebuffered saline (Helicon, Russia), NaCl, KH2PO4, NaH2PO4, KCl, CaCl2, NaHCO3, Na2CO3, sodium acetate, and sodium citrate with a purity of not less than 99% (SigmaAldrich, United States) were used in the present work. The composition of the internal media of the stom ach (2.05 g/l NaCl, 0.6 g/l KH2PO4, 0.11 g/l CaCl2, 0.37 g/l KCl, pH 2.6 or 5.9) and the intestine (6.14 g/l NaCl, 0.68 g/l KH2PO4, 0.3 g/l NaH2PO4, 0.68 g/l NaHCO3, pH 7.0 or 7.2) was reproduced according to the data of Beumer [10]. The peptides were synthesized in the Laboratory of Peptide Chemistry of the Institute of Bioorganic Chemistry, Russian Academy of Sciences; the purity of the peptides was not lower than 95% according to the results of HPLC (the conditions of HPLC are described below) Preparation of rat GIT fragments. Young Wistar rats with a body mass of 150–200 g were used in the present work. The organs were extracted on ice and the buffers used were cooled to 4°С. The stomach and small intestine were extracted, separated, cleaned of food debris, joined in a 2ml syringe, washed by 6 ml

of cold phosphatebuffered saline (pH 7.4), cut into pieces, and frozen in liquid nitrogen. Before the beginning of the experiment, the tissues were stored at –52°С. Prior to the experiment, the tissue pieces were defrosted, weighed, and cut into 20mg fragments. Investigation of peptide resistance to hydrolysis. The incubation mixture contained the peptide sub strate (1 mg/ml), 99 μl of SSM (pH 5.9) or ISM (pH 7.2), and a fragment of the stomach or the intes tine; the total volume of the mixture equaled 100 μl. The influence of pH on peptide hydrolysis was tested using 100mM sodium citrate phosphate buffers (pH 2.6–7.2) and a sodium carbonate buffer (pH 9.0– 10.6). The influence of the buffer composition on pep tide hydrolysis was tested using a 100mM sodium cit rate phosphate buffer, sodium acetate–CH3COOH buffer, or sodium phosphate buffer (pH 5.9 for stom ach fragments and pH 7.2 for small intestine frag ments). Incubation mixtures lacking a tissue fragment or containing a tissue fragment previously incubated at 80–100°С for 5 min were used as negative controls. The positive controls for the experiments devoted to studying the resistance of synthetic peptides (except dalargin) to hydrolysis by the enzymes of GIT frag ments contained the incubation mixture with dalargin as the substrate. The incubation mixture for modeling the stomach medium upon the investigation of peptide hydrolysis by purified enzymes contained 1 μM pepsin or 1 μM acidinpepsin (commercially available preparation), 100μM substrate and SSM, or a 100mM sodium acetate buffer, pH 2.6, 3.0, or 4.0; the mixture for the modeling of the intestinal medium contained 1 μM trypsin, 1 μM chymotrypsin, 100μM substrate, and SSM, pH 7.0. Human insulin and hemopressin were used for the control of enzyme activity; the reactions were performed with individual enzymes and with a trypsin–chymotrypsin mixture. The reaction was conducted at 37°С with intensive stirring in a thermostated Biosan TS100 mixer (Latvia) for 60 min and stopped by heating at 100°С for 3 min. In order to confirm the resistance of the substrates not hydrolyzed after 1 h to the hydrolysis by the purified enzymes, the incubation was performed for 4 h. The incubation mixture used for the investigation of the effector action of the peptides on the pepsin activity contained 1 μM pepsin, 100 μM substrate (ProValAsnPheLysValValSerHis), 100 μM of the peptide studied, and SSM; the pH of the mixture equaled 2.6, and its volume equaled 100 μl. The incu bation was performed at 37°С with intensive stirring in a thermostated mixer Biosan TS100 (Latvia) for 1 h and stopped by heating at 100°С for 3 min. The prod ucts were subsequently separated using HPLC (see below), and the results were compared using the Holm–Sidak test [14].

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After the incubation, 10 μl of glacial acetic acid was added to each sample, the mixtures were centrifuged (10 min, 18000 g) at room temperature in an Elmi CM50 centrifuge (Latvia), and aliquots of the super natants were analyzed by HPLC. HPLC analysis of the products of peptide hydrolysis. The supernatant obtained was loaded onto a ProntoSil 1205C18 AQ HPLC column (Ekonova, Russia) installed on a Milihrom A02 device (Ekonova, Rus sia) and equilibrated by 0.1% TFA. The substances were eluted by a 0 to 100% gradient of an acetonitrile concentration in 0.1% TFA at a flow rate of 0.15 ml/min with the simultaneous optical detection at 220 and 280 nM. The degree of hydrolysis was cal culated as the ratio of the sum of the areas of the prod uct peaks to the sum of the areas of the substrate and product peaks; the areas of the peaks recorded at the wavelength of 220 nm were used. Mass spectrometric analysis of the products of pep tide hydrolysis. The products of the hydrolysis of a 40μg substrate obtained after the HPLC separation of the incubation mixtures under the abovedescribed conditions were lyophilized in a SpeedVac device (Savant Instruments, United States), redissolved in 20 μl of water, and analyzed on an MX 5310 timeof flight mass spectrometer (Institute of Analytical Devices, Russian Academy of Sciences) using electro spray ionization in the positively charged ion mode. ACKNOWLEDGMENTS The authors are grateful to T.V. V’yunova, PhD (Chem.) from the Institute of Molecular Genetics, Russian Academy of Sciences, for kindly providing rat GIT organs. The present work was partially funded by grants from the Federal Target Program “National Techno logical Base,” for 2007–2011 (State Contract

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no. GP/07/599/NTB/k of 23. 10. 2007) and the Fed eral Target Program “Research and Development of the Priority Directions of the Development of Science and Technological Complex of Russia, for 2007– 2012” (State Contract no. 02.512.11.2005 of 23.06.2008). REFERENCES 1. Loffet, A., J. Peptide. Sci., 2001, vol. 8, pp. 1⎯7. 2. Latham, P.W., Nat. Biotechnol., 1999, vol. 17, pp. 755⎯ 758. 3. Craik, D.J. and Adams, D.J., ACS. Chem. Biol., 2007, vol. 2, pp. 457⎯468. 4. Chapter, M.C., White, C.M., Deridder, A., Chadwick, W., Martin, B., and Maudsley, S., Pharmacol. Ther., 2009, vol. 125, pp. 35⎯54. 5. Lundblad, R.L., Chemical Reagents for Protein Modifi cation, Boca Raton, FL: CRC, 2005. 6. Khmelevskii, Yu.V. and Usatenko, O.K., Osnovnye biokhimicheskie konstanty cheloveka v norme i patologii (The Main Biochemical Constants of Humans in Nor mal State and in Pathology), Moscow: Zdorov’e, 1987. 7. Gauthier, S.J., Vachon, C., Jones, J.D., and Savoie, L., J. Nutr., 1982, vol. 112, pp. 1718⎯1725. 8. Gargallo, S., Calsamiglia, S., and Ferret, A., J. Anim. Sci., 2006, vol. 84, pp. 2163⎯2167. 9. Zebrowska, T., Low, A.G., and Zebrowska, H., Br. J. Nutr., 1983, vol. 49, pp. 401⎯410. 10. Beumer, R.R., de Vries, J., and Rombouts, F.M., Int. J. Food. Microbiol., 1992, vol. 15, pp. 153⎯163. 11. Fox, P.F. and Whitaker, J.R., Biochem. J., 1977, vol. 161, pp. 389⎯398. 12. Wickham, M., Faulks, R., and Mills, C., Mol. Nutr. Food. Res., 2009, vol. 53, pp. 952⎯958. 13. Thresher, W.C., Swaisgood, H.E., and Catignani, G.L., Plant. Foods. Hum. Nutr., 1989, vol. 39, pp. 59⎯65. 14. Glantz, S.A., Primer of Biostatistics, 3rd ed., New York: Mc GrawHill, 1992.

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