The use of inhibitory agents to overcome .

inhibitory agent

Journal of Controlled Release52(1998)1–16

Invited review

The use of inhibitory agents to overcome the enzymatic barrier to perorally administered therapeutic peptides and proteins

*

¨

Andreas Bernkop-Schnurch

Institute of Pharmaceutical Technology,Center of Pharmacy,University of Vienna,Althanstr.14,A-1090Vienna,Austria Abstract

The peroral administration of peptide and protein drugs is a major challenge to pharmaceutical science.In order to provide a suf?cient bioavailability of these therapeutic agents after oral dosing,several barriers encountered with the gastrointestinal (GI)tract have to be overcome by a suitable galenic.One of these barriers is caused by proteolytic enzymes,leading to a severe presystemic degradation in the GI tract.Beside some other strategies to overcome the so-called enzymatic barrier,the use of inhibitory agents has gained considerable scienti?c interest,as various in vivo studies could demonstrate a signi?cantly improved bioavailability of therapeutic peptides and proteins,due to the co-administration of such excipients.In vitro techniques to evaluate the actual potential of inhibitory agents include incubation with pure proteases,freshly collected gastric or intestinal?uids,mucosal homogenates,brush border vesicles and freshly excised mucosa.In situ techniques are based on single-pass perfusion studies cannulating different intestinal segments and determining the amount of undegraded model drug in perfusion solutions or blood.For in vivo studies,insulin is mostly used as a model drug,offering the advantage of a well-established method to evaluate the biological response after oral dosing by determining the decrease in blood glucose level.Generally,inhibitory agents can be divided into:inhibitors which are not based on amino acids(I),such as p-aminobenzamidine,FK-448and camostat mesilate;amino acids and modi?ed amino acids(II),such as a-aminoboronic acid derivatives;peptides and modi?ed peptides(III),e.g.bacitracin,antipain,chymostatin and amastatin;and polypeptide protease inhibitors(IV),e.g.aprotinin,Bowman–Birk inhibitor and soybean trypsin inhibitor.Furthermore,complexing agents and some mucoadhesive polymers also display enzyme inhibitory activity.Drawbacks of inhibitory agents,such as the risk of toxic side effects or high production costs,might be excluded by the development of advanced drug delivery systems.Initial steps in this direction can be seen in the development of delivery systems containing mucoadhesive polymers providing an intimate contact to the mucosa,thereby reducing the drug degradation between delivery system and absorbing membrane,controlled release systems which provide a simultaneous release of drug and inhibitor and in the immobilisation of enzyme inhibitors on delivery systems.©1998Elsevier Science B.V.

Keywords:Enzyme inhibitors;Complexing agents;Proteolytic enzymes;Peroral administration;(Poly)peptide drugs

1.Introduction ing a large number of potential therapeutic peptides

and proteins in commercial quantities.Moreover, Due to the great progress in biotechnology,as well these new techniques also allow the production of as gentechnology,the industry is capable of produc-peptide and protein vaccines based on antigens found

on the surface of various infectious microorganisms *and viruses.The majority of such drugs and vaccines Tel.:1431313368476;fax:143131336779;e-mail:

andreas.bernkop-schnuerch@univie.ac.at is most commonly administered by the parenteral

0168-3659/98/$19.00©1998Elsevier Science B.V.All rights reserved.

PII S0168-3659(97)00204-6

inhibitory agent

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2A.Bernkop-Schnurch/Journal of Controlled Release52(1998)1–16

routes that are often complex,dif?cult and occasion-after oral dosing(e.g.[5–8]).In order to evaluate the ally dangerous.Beside so-called alternative routes of actual potential of enzyme inhibitors,it is the aim of application,i.e.the nasal and transdermal routes[1],this review to document the known information there is no doubt that the peroral route is one of the about their validation and use to overcome the most favored,as it offers the greatest ease of enzymatic barrier to perorally administered therapeu-application.However,in order to gain suf?cient tic peptides and proteins.

blood concentrations after oral dosing,different

barriers encountered with the peroral route of appli-

cation have to be overcome by a suitable galenic. 2.Enzymatic barrier

Besides the barrier function of the mucus covering

gastrointestinal epithelia[2],the absorption barrier The use of inhibitory agents for the peroral [3],degradation during absorption and the hepatic administration of peptide and protein drugs depends ?rst-pass metabolism,the rapid luminal enzymatic on the enzymatic barrier,which has to be considered degradation caused by secreted and membrane-bound both qualitatively and quantitatively.From the quali-proteases can be regarded as a very important factor tative point of view,the enzymatic barrier is pre-for low bioavailability.Attempts to reduce this determined by the structure of the peptide or protein barrier include analogues,prodrugs,formulations drug which should be perorally administered.The such as nanoparticles,microparticles,and liposomes information on the speci?city of proteases is there-that shield therapeutic peptides and proteins from fore essential for the choice of enzyme inhibitor(s)in luminal enzymatic attack,and the design of delivery order to guarantee the stability of the therapeutic systems targeting the colon where the proteolytic agent in the intestine.Table1summarizes the activity is relatively low[4].Moreover,considerable speci?city of luminally secreted as well as mem-interest is shown in the co-administration of enzyme brane-bound proteases.

inhibitors,since due to such excipients various in On the other hand,also the quantity of co-ad-vivo studies could demonstrate a signi?cantly im-ministered inhibitor(s)is essential for the intestinal proved bioavailability of peptide and protein drugs stability of a peptide or protein drug.The potential of

Table1

Substrate speci?city of luminally secreted as well as brush border membrane-bound enzymes[9]

Enzyme Site of cleavage Speci?city

Luminally secreted protease

Trypsin H N h—h—j↓—h—h COOH Arg,Lys

2

Chymotrypsin H N h—h—j↓—h—h COOH Phe,Tyr

2

Elastase H N h—h—j↓—h—h COOH Ala,Gly,Ile,Leu,Val

2

Carboxypeptidase A H N h—h—h—h—↓j COOH Tyr,Phe,Ile,Thr,Glu,His,Ala

2

Carboxypeptidase B H N h—h—h—h—↓j COOH Lys,Arg

2

Membrane-bound protease

Aminopeptidase A H N j↓—h—h—h—h COOH Asp,Glu

2

Aminopeptidase N H N j↓—h—h—h—h COOH Many,but especially Ala,Leu

2

Aminopeptidase P H N j↓—h—h—h—h COOH Pro

2

Aminopeptidase W H N j↓—h—h—h—h COOH Trp,Tyr,Phe

2

g-Glutamyl transpeptidase H N j↓—h—h—h—h COOH g-Glutamic acid

2

Dipeptidyl peptidase IV H N h—j↓—h—h—h COOH Pro,Ala

2

Peptidylpeptidase A H N h—h—h—↓j—j COOH His-Leu

2

Carboxypeptidase M H N h—h—h—h—↓j COOH Lys,Arg

2

Carboxypeptidase P H N h—h—h—h—↓j COOH Pro,Gly,Ala

2

g-Glutamyl carboxypeptidase H N h—h—h—h—↓j COOH g-Glutamic acid

2

Endopeptidase-24.11H N h—h—↓j—h—h COOH Hydrophobic amino acids

2

Endopeptidase-24.18H N h—↓j↓—h—h COOH Aromatic amino acids

2

Enteropeptidase H N h—h—j↓—h—h COOH(Asp)-Lys

24

inhibitory agent

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A.Bernkop-Schnurch/Journal of Controlled Release52(1998)1–163 enzyme inhibitors is generally expressed by the shown in units secreted from the pancreas per enzyme activity in units,which will be inhibited by a minute.The protease activity per ml intestinal certain amount(e.g.1mg)of the corresponding?uid—representing the major parameter for the inhibitor.In order to be able to estimate the quantity chosen quantity of inhibitor(s)in peroral(poly)pep-of an inhibitor which is necessary to guarantee a tide delivery systems—can therefore be estimated suf?cient protective effect of the delivery system,it according to an intestinal?ow rate in the duodenum is essential to be well-informed about the enzymatic of up to several ml per min and 2.16and0.67 activity of intestinal parison between ml/min in the jejunum and ileum,respectively[16]. the enzymatic activity of luminally secreted and For the development of drug delivery systems membrane-bound enzymes demonstrated that the which should protect therapeutic peptides or proteins total peptidase activity present in the lumen of the in a restricted area of drug liberation and absorption, small intestine of rats is16-times greater than the it is also important to be well-versed about the total activity in the brush border membrane(BBM)activity of brush border membrane-bound enzymes

2

of the epithelial cells,using the B chain of insulin as in this area,estimated to be0.5–2cm.However, substrate[10].Similar studies in guinea pigs using data given about the enzymatic activity of brush the same substrate showed in total a40-times higher border membrane-bound proteases are in general activity of luminal enzymes than of the brush border only expressed in units per milligram or gram protein [11].However,although the enzymatic barrier to isolated from the intestinal mucosa.As a matter of perorally administered peptide and protein drugs has fact,it is very dif?cult to obtain detailed information been carefully reviewed in the past by Woodley[9]about the enzymatic activity on a certain surface as well as Langguth et al.[12],no detailed data area—representing the real‘enemy’s strength’. about the proteolytic activity of digestive enzymes in Nevertheless,data about the activity of brush border

2

the small intestine were given.A reason for this membrane-bound enzymes per cm would be very might be seen in the in?uence of various parameters,helpful for the development and evaluation of inhib-e.g.pancreatic stimulation,pH value of the intestinal itor containing dosage forms.Recently,our research ?uid,concentration of activating ions,as well as type group evaluated the enzymatic activity of a de?ned and quantity of nutrients,leading to a highly variable surface area of freshly excised porcine mucosa using enzymatic activity in the intestine.Hence,as shown L-leucine-p-nitroanilide as substrate.Results were of in Table2,data about the enzymatic activity of good reproducibility,taking the surface variability luminally secreted proteases in man can only be due to the elasticity of the mucosa into account,and

Table2

Stimulated enzyme secretion of the human pancreas

Protease Substrate Enzyme activity

(units secreted from

the pancreas per min)

a Trypsin Carbobenzoxy-glycyl-glycyl-arginine-2-naphthylamide23.8IU(7.5–69IU)

b

N a-Benzoyl-L-arginineethylester(BAEE)31U(16.3–61U)

b

p-Toluolsulfonyl-L-arginineethylester136U(55.5–335U)

a Chymotrypsin N-Glutaryl-glycyl-glycyl-L-phenylalanine b-naphthylamide72.8mIU(17.9–260.4mIU)

b

N-Benzoyl-L-tyrosineethylester(BTEE)66U(28.4–154U)

b

L-Tyrosineethylester34U(13.8–86U)

a Elastase Succinyl-L-alanyl-L-alanyl-L-alanine-2-naphtylamide0.356IU(0.189–0.836IU)

b Carboxypeptidase A Hippuryl-L-phenylalanine8.5U(4.9–14.9U)

b

N-Carbo-b-naphtoxy-L-phenylalanine0.21U(0.077–0.57U)

b Carboxypeptidase B Hippuryl-L-arginine61U(23.9–157U)

a Calculated for pancreatic secretion of3.5ml/min;mean(trypsin,chymotrypsin,n515;elastase,n54)plus highest and lowest values [13,14].

b One unit hydrolysis1m mol substrate per minute at258C at optimised assay conditions;mean(n525)plus95%-range[15].

inhibitory agent

inhibitory agent

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A.Bernkop-Schnurch/Journal of Controlled Release52(1998)1–165 based on a direct interaction of the inhibitor with the gastric or intestinal?uids,these tests can also be

enzyme.The reduction or loss in the enzymatic carried out with freshly collected gastric or intestinal activity is caused by the deprivation of essential?uids as described by Takada and Ushirogawa[22] metal ions out of the enzyme structure.This depriva-and Asada et al.[23].Incubation can be performed in tion of cations can be achieved by complexing suitable vessels,e.g.beakers or centrifugation tubes agents.Although they are competitive as far as the under permanent shaking or stirring of the enzyme metal ion is concerned,and can be reversed by containing medium at378C.The protective effect of increasing the concentration of this co-factor,they the inhibitor or dosage form can be estimated by the are non-competitive in the usual sense,since the percentage of undegraded substrate or model drug in inhibition will be independent of the substrate con-the medium and/or in the dosage form[20,21,24]. plexing agents reduce the apparent For formulations delivering not only the drug but maximum enzyme reaction velocity.also the inhibitor,the in?uence of dilution effects of The practical relevance of these theories,however,the auxiliary agent on the protective effect can be is limited by the fact that many inhibitors do not studied using a?ow-through cell[20].

follow exclusively only one of these mechanisms.

For instance,the inhibition of aminopeptidase N by 4.1.2.In vitro test models for brush border complexing agents is,for1,10-phenanthroline and membrane-bound proteases

EGTA,of the competitive type,for sodium sul?de it By analogy with luminally secreted proteases,the is of a mixed-type,and only for EDTA it has protective effect of an inhibitor or a(poly)peptide features of the third mentioned mechanism[19].delivery system,containing such an auxiliary agent,

Furthermore,the Michaelis constant(K)of various towards brush border membrane-bound proteases can

M

intestinal proteases is known for some substrates,but be evaluated by incubating with media containing the in most cases not for peptide and protein drugs.Even pure protease(s)[25].However,such studies are far if the Michaelis constant has already been deter-from the in vivo situation,and can therefore only be mined for a certain peptide or protein of interest,it regarded as preliminary studies.Instead of isolated still depends on the assay conditions used,such as proteases,these tests can also be carried out with pH value,ionic strength,divalent cations and tem-intestinal mucosal homogenates as described by perature.The same variability has to be taken into Asada et al.[23]and Yamamoto et al.[26].How-

account for the inhibition constant(K).ever,the transcellular peptide and protein transport is

i

not the most likely route of absorption for hydro-

philic macromolecules.Hence,therapeutic peptides 4.Methods to evaluate the protective effect of and proteins will be digested to a much higher extent inhibitory agents and dosage forms towards the by such homogenates,containing large amounts of luminal enzymatic attack cytosolic enzymes,than can be expected under in

vivo conditions.Accordingly,more detailed infor-4.1.In vitro test models mation will be obtained from incubation with brush

border membrane vesicles instead of mucosal 4.1.1.In vitro test models for luminally secreted homogenates[12,27].

proteases In vitro evaluations,taking into account the im-The protective effect of an inhibitor or of a portant in?uence of the diffusion barrier caused by (poly)peptide delivery system containing such an the mucus layer,which covers the brush border auxiliary agent towards luminally secreted proteases membrane and its proteases,can be performed with a can be evaluated by incubation with an arti?cial Franz diffusion cell using an arti?cial membrane. gastric or intestinal?uid at378C.In contrast to in This membrane can be prepared by immobilisation vivo evaluations,the method offers the advantage of of the pure protease of interest on a nitrocellulose analysing the protective effect not only towards all membrane,coating the remaining free binding sites luminal proteases,but also towards pure proteases of with a protein,which will not be digested by the a certain enzymatic activity[20,21].Besides arti?cial protease,and covering the membrane with a mucus

inhibitory agent

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6A.Bernkop-Schnurch/Journal of Controlled Release52(1998)1–16

layer.The inhibitor(s)or dosage form containing a port of(poly)peptide drugs by analysing the serosal substrate or model drug is added to the donor?uids and the mucosa after incubation[29]. chamber,and the amount of remaining unhydrolysed

substrate or model drug can be determined in the 4.2.In situ test models

donor and acceptor chamber.The test model also

allows investigations of‘far distance acting’inhib-Perfusion studies,using a single-pass perfusion itory agents—as described below—as well as of the technique can be performed by cannulating different in?uence of diffusion effects of inhibitors on the intestinal segments of anaesthetised animals.Test inhibition of BBM-bound proteases[2,25].solutions containing the model drug and enzyme Instead of an arti?cial membrane,tests can also be inhibitor(s)are delivered continuously through the carried out on the native mucosa,ing only the segment and the amount of undegraded model drug donor chamber of a Franz diffusion cell and de-in the intestinal?uid and blood can be determined in termining the degree of degradation of a substrate or samples taken from perfusion solutions and different model drug in the presence of inhibitor(s)or dosage veins of animals,respectively[6,12,30].

form(Fig.2).The technique offers the advantage of

a de?ned surface area,which provides comparable 4.3.In vivo test models

and reproducible results using the mucosa of similar

intestinal segments[17].Probably the best established in vivo test model, Furthermore,the protective effect of an enzyme for evaluating the in?uence of enzyme inhibitors on inhibitor can also be evaluated with intestinal rings.the bioavailability of perorally administered(poly)-After sacri?ce of test animals,the intestine is peptide drugs,is the insulin test model.It offers the everted,cut into rings and incubated in an appro-advantage that insulin is digested by luminally priate buffer solution purged with a steady stream of secreted as well as membrane-bound proteases oxycarbon.In the medium containing intestinal rings,[11,31],and that an increased bioavailability can be the stability of model drugs in the presence of easily evaluated by determining the decrease in enzyme inhibitors can then be determined[12,28].blood glucose level.Because diabetic animals are Using everted sacs,instead of intestinal rings,for more sensitive to insulin activity,more marked enzyme inhibition studies allows additional inves-results are obtained using such animals for in vivo tigations concerning the absorption and intact trans-studies.Besides some other test models evaluating

an enhanced bioavailability by a biological response,

e.g.the antidiuretic response of vasopressin deriva-

tives or the increase in blood total leucocyte counts

after administration of human granulocyte colony-

stimulating factor[32,33],the concentration–time

pro?le of many perorally administered(poly)pep-

tides in blood can be evaluated by HPLC analysis,

RIA or ELISA[6,12,34–36].However,it is reported

that some inhibitory agents,such as bacitracin,

EDTA and poly(acrylate)derivatives,display pene-

tration-enhancing properties[35,37–39],which

makes the exclusive evaluation of their inhibitory

effect in vivo quite complex and dif?cult.

Fig. 2.Experimental set-up to study peptide degradation by

BBM-bound enzymes.(a)Incubation cell with constant tempera- 5.Enzyme inhibitors

ture of378C,(b)platform,(c)incubation medium containing the

substrate/model drug and inhibitor(s)or dosage form,(d)mucus

In recent years the use of enzyme inhibitors to layer,(e)intestinal mucosa,(f)glass cylinder of certain inside

diameter,(g)cap,[17].overcome the enzymatic barrier to perorally adminis-

inhibitory agent

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A.Bernkop-Schnurch/Journal of Controlled Release52(1998)1–167 tered therapeutic peptides and proteins has gained tion of inhibitory agents based on their chemical considerable interest.However,especially for pep-structure is shown below.

tide and protein drugs which are used in long-term

therapy,the co-administration of enzyme inhibitors 5.1.Inhibitors which are not based on amino remains questionable because of side effects caused acids

by these agents.Even if systemic toxic side effects

and an intestinal mucosal damage can be excluded,With few exceptions,this class of inhibitors is enzyme inhibitors of pancreatic proteases still have a more of theoretical than practical interest,as most of toxic potential caused by the inhibition of these these inhibitors are highly toxic.It seems to make digestive enzymes themselves.Besides a disturbed their use in human therapeutics quite unrealistic. digestion of nutritive proteins,an inhibitor-induced However,they are generally very potent inhibitors stimulation of protease secretion caused by a feed-[55]and the immobilisation of these auxiliary agents back regulation has to be expected[40].Numerous to unabsorbable matrices or the development of studies have investigated this feed-back regulation slightly chemically modi?ed analogues might reduce with inhibitors,such as Bowman–Birk inhibitor,or even eliminate this drawback.

soybean trypsin inhibitor(Kunitz trypsin inhibitor)Organophosphorus inhibitors,such as diisopropyl-and camostat,in rats and mice.They demonstrate?uorophosphate(DFP)and phenylmethylsulfonyl that this feed-back regulation rapidly leads to both?uoride(PMSF),are potent irreversible inhibitors of hypertrophy and hyperplasia of the pancreas.More-serine proteases,such as trypsin and chymotrypsin. over,a prolonged administration of the Bowman–However,the additional inhibition of acetylcho-Birk inhibitor and soybean trypsin inhibitor leads to linesterase makes these compounds highly toxic the development of numerous neoplastic foci,fre-[55].4-(2-Aminoethyl)-benzenesulfonyl?uoride quently progressing to invasive carcinoma[41–44].(AEBSF),has an inhibitory activity comparable to A reduction or even exclusion of this feed-back DFP and PMSF,however,it is markedly less toxic. regulation might be possible by the development of(4-Aminophenyl)-methanesulfonyl?uoride hydro-drug delivery systems which keep inhibitor(s)con-chloride(APMSF)is an other potent inhibitor of centrated on a restricted area of the intestine,where trypsin,but is also toxic.In contrast to these drug liberation and subsequent absorption takes inhibitors,4-(4-isopropylpiperadinocarbonyl)phenyl place.1,2,3,4,-tetrahydro-1-naphthoate methanesulphonate Without taking possible toxic risks into account,(FK-448)is a low toxic substance,representing a various inhibitors of pancreatic and brush border potent and speci?c inhibitor of chymotrypsin.The membrane-bound proteases are listed in Tables3and co-administration of this compound led to an en-4,respectively.Besides this overview,a classi?ca-hanced intestinal absorption of insulin in rats and

Table3

Inhibitors of luminally secreted proteases

Luminally secreted proteases Co-factor Inhibited by

Trypsin(EC3.4.21.4)Calcium Aprotinin[7,31,32,45–48],Bowman–Birk inhibitor[31,40,49],soybean trypsin inhibitor

[7,11,22,31,33,40],chicken ovomucoid[33],chicken ovoinhibitor[50],human pancreatic trypsin inhibitor

[51],camostat mesilate(5FOY-305)[5,7,52],?avonoid inhibitors[53],antipain[21],leupeptin[5],p-aminobenzamidine

[54],AEBSF,TLCK(tosyllysine chloromethlyketone),APMSF,DFP,PMSF[55],poly(acrylate)derivatives[56] Chymotrypsin(EC3.4.21.1)Calcium Aprotinin[7,31,32,45–48],Bowman–Birk inhibitor[31,40,49],soybean trypsin inhibitor

[7,11,40],chymostatin[5,31],benzyloxycarbonyl–Pro–Phe–CHO[57],FK-448[5],chicken ovoinhibitor[50],

sugar biphenylboronic acids complexes[58],DFP,PMSF,b-phenylpropionate[55],poly(acrylate)derivatives[59] Elastase(EC3.4.21.36)Calcium Elastatinal[5],methoxysuccinyl–Ala–Ala–Pro–Val–chloromethylketone(MPCMK)[60],

Bowman–Birk inhibitor[40],soybean trypsin inhibitor[11,40],chicken ovoinhibitor[50],

DFP,PMSF[55]

Carboxypeptidase A(EC3.4.17.1)Zinc EDTA,chitosan–EDTA conjugates[61],poly(acrylate)derivatives[59]

Carboxypeptidase B(EC3.4.17.2)Zinc EDTA,chitosan–EDTA conjugates,poly(acrylate)derivatives[39]

inhibitory agent

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Table4

Inhibitors of membrane bound proteases

Membrane bound proteases Co-factor(s)Inhibitory agents

Aminopeptidase N(EC3.4.11.2)Zinc,cobalt Amino acids[62],di-and tripeptides[27],EDTA,amastatin[11,30,63],bestatin[64–67],puromycin[27,64,65],

bacitracin[7,27,47,65],phosphinic acid dipeptide analogues[68],a-aminoboronic acid derivatives[69],

chitosan–EDTA conjugates[17],Na-glycocholate[7,65]

Aminopeptidase A(EC3.4.11.7)Zinc,calcium Phosphinic acid dipeptide analogues[68],a-aminoboronic acid derivatives[69],

puromycin[70],EDTA[70],1,10-phenantroline[70]

Aminopeptidase P(EC3.4.11.9)Zinc,mangan PMSF,bestatin[65],phosphinic acid dipeptide analogues[68],

a-aminoboronic acid derivatives[69]

Aminopeptidase W(EC3.4.11.16)Zinc Phosphinic acid dipeptide analogues[68],a-aminoboronic acid derivatives[69]

Leucin aminopeptidase(EC3.4.11.1)Zinc,magnesium,mangan Bestatin[65],amastatin[30],phosphinic acid dipeptide analogues[68,71],?avonoid

inhbitors[53],a-aminoboronic acid derivatives[69]

Dipeptidyl peptidase IV(EC3.4.14.5)Zinc N-Peptidyl-O-acylhydroxylamines boronic acid analogues of proline and alanine[72],DFP[73]

g-Glutamyl transpeptidase(EC2.3.2.2)Magnesium Acivicin(amino-(3-chloro-4,5-dihydro-isoxazol-5-yl)-acetic acid)[74],L-serine-borate[75]

Peptidyl dipeptidase A(EC3.4.15.1)Zinc ACE inhibitors(in theory)

Carboxypeptidase M(EC3.4.17.12)Zinc D,L-2-Mercaptomethyl-3-guanidinoethylthiopropanoic acid[76]

Carboxypeptidase P(EC3.4.17.16)Zinc,mangan

Neutral endopeptidase(EC3.4.24.11)Zinc1,10-Phenanthroline[77],thiorphan((2-mercaptomethyl-3-phenyl-propionylamino)-acetic acid)[78],phosphoramidon

(Fig.3)[11,77,79],SQ28,603(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-b-alanine)[80] Endopeptidase-24.18(EC3.4.24.18)Zinc

dogs,resulting in a decrease in blood glucose level.inhibitors of exopeptidases such as aminopeptidase N This increased bioavailability of insulin was found to[62].They are non-toxic and can be produced at be related to the inhibition of digestive enzymes,comparably low cost.However,due to their low especially chymotrypsin[5].Further representatives inhibitory activity,their practical use is quite ques-of this class,also exhibiting low toxic risk,are tionable.In contrast,modi?ed amino acids display a camostat mesilate(N,N9-dimethyl carbamoylmethyl-much stronger inhibitory activity.A special kind of p-(p9-guanidino-benzoyloxy)phenylacetate methane-modi?ed amino acids is represented by‘transition-sulphonate)[7]and Na-glycocholate[7,65].state’inhibitors.Their strong inhibitory activity is

based on the hypothesis that molecules which resem-5.2.Amino acids and modi?ed amino acids ble a substrate in its transition-state geometry,have a

much higher af?nity for the active site of an enzyme Generally,amino acids as well as modi?ed amino than the substrate itself.Transition-state inhibitors acids are low or non-toxic and can be produced at are reversible,competitive inhibitors.Examples for comparably low cost.However,due to their low this type of inhibitors are a-aminoboronic acid molecular size and good solubility,they are exten-derivatives,such as boro-leucine,boro-valine and sively diluted in the intestine as well as quickly boro-alanine.They are potent and reversible in-absorbed.Accordingly,unrealistic high amounts of hibitors of aminopeptidases and it is reported that these inhibitors are necessary to achieve an inhib-boro-leucine is more than100-times more effective itory effect towards luminal proteases in the intes-in enzyme inhibition than bestatin and more than tine.For these reasons,this particular class of1000-times more effective than puromycin[69].As inhibitors has so far gained more interest for other these modi?ed amino acids were so far only tested in transmucosal routes,e.g.nasal,where these effects the nasal cavity,their practical use for peroral are comparably lower.Nevertheless,the use of such administration cannot be predicted.However,prob-inhibitors for peroral(poly)peptide administration lems encountered with their chemical liability,might seems to be feasible,but will mainly depend on have to be taken into account.

delivery systems which guarantee the reduction or Recently,our research group demonstrated a even exclusion of extensive dilution effects.strong inhibitory effect of N-acetylcysteine towards Amino acids can act as reversible,competitive the enzymatic activity of pure aminopeptidase N

inhibitory agent

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[25].As this auxiliary agent displays additional Another example of a transition-state analogue is mucolytic properties which lead also to a reduction the modi?ed pentapeptide pepstatin[84],which is a of the diffusion barrier[2],it is a promising inhibitor very potent inhibitor of pepsin.Structural analysis of with low toxic risk.pepstatin,by testing the inhibitory activity of several

synthetic analogues,demonstrated the major structur-5.3.Peptides and modi?ed peptides al function of the statine components(Fig.3),which

are responsible for the inhibitory activity[84]. An important representative of this class of in-Although an enzymatic attack of perorally adminis-hibitors is the cyclic dodecapeptide,bacitracin,ob-tered peptide and protein drugs in the stomach can be tained from Bacillus licheniformis.Bacitracin A has excluded by a gastric?uid-resistant coating,the a molecular mass of1423Da and shows remarkable inhibition of pepsins is nevertheless of practical resistance against the action of proteolytic enzymes relevance;especially in cases where pepsin-digested like trypsin and pepsin[81].It has several biological therapeutic(poly)peptides should be liberated in the properties inhibiting bacterial peptidoglycan synthe-stomach,e.g.epidermal growth factor in treatment of sis,mammalian transglutaminase activity,and gastric ulcer[85].However,pepstatin can cause proteolytic enzymes such as aminopeptidase N.several side effects by the inhibition of physiologi-Because of its protease inhibitory activity,it has cally essential,but pepstatin-sensitive enzymes[86–been used to inhibit the degradation of various88].

therapeutic(poly)peptides,such as insulin,met-Another special type of modi?ed peptides are kephamid,LH-RH,and buserelin[7,27,82].Besides inhibitors with a terminally located aldehyde func-its inhibitory activity,bacitracin also displays absor-tion in their structure.For example,the sequence ption-enhancing effects without leading to a serious benzyloxycarbonyl–Pro–Phe–CHO,which ful?l the intestinal mucosal damage[37].known primary and secondary speci?city require-Nevertheless,because of its nephrotoxicity[83],ments of chymotrypsin,has been found to be a the use of bacitracin as a suitable adjuvant to potent reversible inhibitor of this target proteinase overcome the enzymatic barrier is quite questionable.[57].The chemical structures of further inhibitors So far,it has almost exclusively been used in with a terminally located aldehyde function, e.g. veterinary medicine and as a topical antibiotic in the antipain,leupeptin,chymostatin and elastatinal,are treatment of infections in man.Recently our research shown in Fig.3.

group demonstrated that bacitracin,which is co-Furthermore,the chemical structures of other valently linked to a mucoadhesive polymer(car-modi?ed peptides acting as reversible inhibitors, bomer),still displays its inhibitory activity[25].such as phosphoramidon,bestatin,puromycin and Because of the immobilisation to an unabsorbable amastatin,are also presented in Fig.3.

drug–carrier matrix,systemic toxic side effects

should be excluded,but this has to be veri?ed by 5.4.Polypeptide protease inhibitors

detailed toxicological studies.

Furthermore,dipeptides and tripeptides display a Due to their comparably high molecular mass, weak and unspeci?c inhibitory activity towards some polypeptide protease inhibitors can be more easily exopeptidases[27].By analogy with amino acids,kept concentrated on delivery systems based on a their inhibitory activity can be improved by chemical drug–carrier matrix.The advantage of the slow modi?cations.For example,phosphinic acid dipep-release of an inhibitor from the delivery system was tide analogues are also‘transition-state’inhibitors already demonstrated by Kimura et al.[47].In their with a strong inhibitory activity towards amino-study they generated a mucoadhesive delivery sys-peptidases.They have already been used to stabilise tem exhibiting a release rate of the protease inhibitor nasally administered leucine enkephalin.Notably,the aprotinin of only approximately10%per hour,which phosphinate inhibitor VI(Fig.3)has10-and100-was additionally almost synchronous with the release times greater inhibition than bestatin and puromycin,rate of a polypeptide drug.In vivo studies with this respectively[68].delivery system showed an improved bioavailability

inhibitory agent

¨

10A.Bernkop-Schnurch/Journal of Controlled Release52(1998)1–16

Fig.3.Molecular structure of modi?ed peptide protease inhibitors.

inhibitory agent

¨

A.Bernkop-Schnurch/Journal of Controlled Release52(1998)1–1611 of the drug[47].For this reason,and due to their low inhibition of the endoproteases trypsin,chymotrypsin toxicity as well as strong inhibitory activity,poly-and elastase by complexing agents does not seem to

21

peptide protease inhibitors have so far been used to be feasible.In contrast,many Zn-dependent exo-the highest extent as auxiliary agents to overcome proteases such as carboxypeptidase A,B and amino-the enzymatic barrier of perorally administered thera-peptidase N,can very effectively be inhibited by peutic peptides and plexing agents[11,17,90].As the dissociation The basic trypsin inhibitor aprotinin(5bovine constant of many chelators,such as EDTA and pancreatic trypsin inhibitor;58amino acids)was one DTPA,is much higher towards zinc than towards of the?rst enzyme inhibitors used as an auxiliary calcium,a disturbance of inhibition by calcium ions agent for peroral(poly)peptide administration.Ap-should be negligible.However,as the inhibition rotinin is a bovine pancreatic kallikrein inhibitor with mainly depends on the concentration of the complex-a molecular mass of6.5kDa.In several reports it ing agent,its extensive dilution in the intestine has to was shown that the co-administration of aprotinin be avoided in order to achieve a suf?cient protective leads to an increased bioavailability of peptide and effect.Representatives of this class of inhibitory protein drugs,due to its inhibitory effect towards agents are EDTA,EGTA,1,10-phenanthroline and trypsin as well as chymotrypsin[7,32,47].hydroxychinoline[11,19,91,92].Due to the com-The two main families of soybean trypsin inhibitor plexation of divalent cations,they display additional are the Bowman–Birk inhibitor(71amino acids)and penetration-enhancing properties[93].

the Kunitz trypsin inhibitor(soybean trypsin inhib-

itor;184amino acids).They have a molecular mass

of8and21kDa,respectively,and are known to7.Mucoadhesive polymers exhibiting enzyme inhibit human trypsin as well as chymotrypsin.inhibitory activity

Moreover,these inhibitors display also an inhibitory

activity towards elastase,whereas carboxypeptidase In contrast to complexing agents and inhibitors of A and B cannot be inhibited[33,40].low molecular mass,unintended dilution effects in Further inhibitors of this class are chicken egg the intestine followed by the already mentioned white trypsin inhibitor(5chicken ovomucoid;186undesired side effects can be excluded by the use of amino acids)[33],chicken ovoinhibitor(449amino mucoadhesive polymers exhibiting enzyme inhibiting acids)[50]and human pancreatic trypsin inhibitor properties per se.Poly(acrylate)derivatives,such as (56amino acids),which have already been produced poly(acrylic acid)and polycarbophil,can affect the in Escherichia coli[51].activity of various luminal proteases.It is suggested

that the inhibitory effect of these polymers is based

21

on the complexation of divalent cations such as Ca

21

plexing agents and Zn[56].Detailed analysis of the inhibitory

effect towards luminally secreted and brush border Complexing agents display an inhibitory activity,membrane-bound proteases has,so far,been done by as they are capable of depriving divalent cations,Lueßen et al.[59].They revealed an inhibitory effect which are co-factors for many proteases,of their of poly(acrylate)derivatives towards the proteases enzyme structure.A complexation of these divalent trypsin,chymotrypsin,carboxypeptidase A and B. cations,as listed in Tables3and4,however,does Whether the protective effect of these polymers is not always lead to an inhibition of the corresponding suf?cient to prevent luminal enzymatic degradation enzyme.For instance,a concentration of7.5%(w/v)of polymer-embedded peptide and protein drugs,will of the complexing agent EDTA is not suf?cient to mainly depend on the dosage form used.However,it inhibit trypsin activity in vitro[59].Moreover,as the is very likely that simple formulations with poly-concentration of calcium ions in gastric and intestinal(acrylate)derivatives will not be suitable to exert a ?uids was determined to be0.4–0.7mM[89],it is suf?cient protective effect[94].Nevertheless,these likely that calcium ions might additionally disturb polymers are of high practical relevance,as they are this type of inhibition in vivo.Hence,a successful generally regarded as safe,exhibiting additional

inhibitory agent

¨

12A.Bernkop-Schnurch/Journal of Controlled Release52(1998)1–16

advantages for the peroral administration of peptide Recently our research group demonstrated that and protein drugs.Due to their mucoadhesive[95,96]mucoadhesive polymers exhibiting strong complex-as well as penetration-enhancing properties[97,98],ing properties are also capable of inhibiting brush they provide a prolonged residence time of the border membrane-bound enzymes,even when they dosage form in the intestine and enhanced drug are separated from the polymer by a mucus layer absorption.Moreover,besides inhibitory and pene-[25].This‘far distance inhibitory effect’[99],with-tration-enhancing properties,mucoadhesive polymers out any direct contact through the mucus layer, are also able to reduce the enzymatic barrier by seems to be a very promising alternative to the sticking to the mucus layer at the site of drug enzyme inhibitors of low molecular mass,thus far absorption,thereby decreasing the distance between used for the inhibition of brush border membrane-the released therapeutic(poly)peptide from the dos-bound proteases.

age form and the absorptive tissue which leads to a Additionally,the inhibitory activity of mucoadhe-reduced drug metabolism by luminally secreted sive polymers can even be extensively improved by proteases.the covalent attachment of enzyme inhibitors.As Attempts to improve the inhibitory effect of shown in several studies,the mucoadhesive prop-anionogenic mucoadhesive polymers led to the de-erties of polymers are not markedly in?uenced by velopment of chitosan–EDTA conjugates.Due to the such slight chemical modi?cations.The immobilisa-high binding capacity,as well as binding af?nity,tion on unabsorbable matrix systems,which provide towards multivalent cations,these polymers display a an intimate and prolonged contact to the absorbing strong inhibitory effect towards the enzymatic activi-membrane in the intestine,might offer several ad-ty of zinc-dependent proteases such as aminopeptid-vantages for the co-administration of enzyme in-ase N and carboxypeptidase A.However,in contrast hibitors.The reduced distance should minimize a to poly(acrylate)derivatives,no inhibitory effect presystemic metabolism of the(poly)peptide drug on 21

towards Ca-dependent proteases could be observed the route between delivery system and absorbing by chitosan–EDTA conjugates,although they dis-membrane(I).Covalently bound inhibitors remain

21

play a higher binding af?nity towards Ca ions than concentrated on the drug delivery system,which carbomer and polycarbophil[61].As these polymer should guarantee the exclusion of undesired dilution conjugates additionally offer excellent mucoadhesive effects of inhibitors,consequently eliminating an properties and can be used as carrier matrix for unintended disturbance of digestion of nutritive controlled drug release systems,they seem to be proteins(II)as well as systemic toxic side effects promising alternatives to well-established auxiliary(III).Moreover,it is likely that the amount of co-agents for overcoming the enzymatic barrier.administered inhibitor can be reduced due to the

Table5

Mucoadhesive polymer–inhibitor conjugates

Polymer–inhibitor conjugate Inhibitory activity towards Carboxymethylcellulose–pepstatin[20]Pepsin

Poly(acrylic acid)–Bowman–Birk inhibitor[94]Chymotrypsin

Poly(acrylic acid)–chymostatin[100]Chymotrypsin

Poly(acrylic acid)–elastatinal[101]Elastase

Carboxymethylcellulose–elastatinal[101]Elastase

Polycarbophil–elastatinal[101]Elastase

Chitosan–antipain[21]Trypsin

Poly(acrylic acid)–bacitracin[25]Aminopeptidase N

Chitosan–EDTA[17,61]Aminopeptidase N,carboxypeptidase A Chitosan–EDTA–antipain,–Trypsin,chymotrypsin,elastase, chymostatin,and elastatinal[102]carboxypeptidase A,carboxypeptidase B,

aminopeptidase N

inhibitory agent

¨

A.Bernkop-Schnurch/Journal of Controlled Release52(1998)1–1613

¨

[4]A.Bernkop-Schnurch,Strategien zur peroralen Applikation exclusion of dilution effects(IV).The use of such

von Peptid-und proteinwirkstoffen,Sci.Pharm.65(1997) modi?ed polymers allows not only the inhibition of

61–82.

luminally secreted proteases,but also of brush border[5]S.Fujii,T.Yokoyama,K.Ikegaya, F.Sato,N.Yokoo,

membrane-bound enzymes,as recently shown by our Promoting effect of the new chymotrypsin inhibitor FK-448

research group[17].Currently established

on the intestinal absorption of insulin in rats and dogs,J.

Pharm.Pharmacol.37(1985)545–549. mucoadhesive polymer-inhibitor conjugates are listed

[6]ngguth,H.P.Merkle,G.L.Amidon,Oral absorption of in Table5.

peptides:the effect of absorption site and enzyme inhibition

on the systemic availability of metkephamid,Pharm.Res.11

(1994)528–535.

[7]A.Yamamoto,T.Taniguchi,K.Rikyuu,T.Tsuji,T.Fujita,

8.Conclusions

M.Murakami,S.Muranishi,Effects of various protease

inhibitors on the intestinal absorption and degradation of Besides various other strategies,the co-administra-

insulin in rats,Pharm.Res.11(1994)1496–1500.

[8]I.Morishita,M.Morishita,K.Takayama,Y.Machida,T. tion of inhibitory agents seems to be a promising

Nagai,Hypoglycemic effect of novel oral microspheres of alternative to overcome the enzymatic barrier to

insulin with protease inhibitor in normal and diabetic rats, perorally administered peptide and protein drugs.So Int.J.Pharm.78(1992)9–16.

far,an enhanced bioavailability of these therapeutic[9]J.F.Woodley,Enzymatic barriers for GI peptide and protein

agents after oral dosing with enzyme inhibitors could

delivery,Crit.Rev.Ther.Drug11(1994)61–95.

[10]J.F.Woodley,Peptidase activity in the G.I.tract:Distribution be demonstrated by various in vivo studies,and there

between luminal contents and mucosal tissue,Proc.Int. is no doubt that the ef?cacy of delivery systems

Symp.Control.Release Bioact.Mater.18(1991)337–338. containing inhibitors can be widely improved.A[11]K.Ikesue,P.Kopeckova,J.Kopecek,Degradation of pro-

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edge of the potential and possible risks of these

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[16]Wissenschaftliche Tabellen Geigy,Ciba-Geigy AG,Basel, tact to the mucosa,and the immobilisation of in-

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[17]A.Bernkop-Schnurch,Ch.Paikl,C.Valenta,Novel bioadhe-

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from degradation by aminopeptidase N,Pharm.Res.14

(1997)917–922.

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