a ttempts: a heparin/protamine-based triggered release ...€¦ · 252 y.-j.park et al. / advanced...

Advanced Drug Delivery Reviews 55 (2003) 251–265www.elsevier.com/ locate/addr

A TTEMPTS: a heparin /protamine-based triggered releasesystem for the delivery of enzyme drugs without associated

side-effects

Yoon-Jeong Park, Jun-Feng Liang, Hui Song, Yong Tao Li, Sarita Naik,*Victor C. Yang

College of Pharmacy, The University of Michigan, 428 Church Street, Ann Arbor, MI 48108-1065,USA

Received 10 April 2002; accepted 13 July 2002

Abstract

A prodrug type delivery system based on competitive ionic binding for the conversion of the prodrug to an active drug hasbeen developed for delivery of enzyme drugs without their associated toxic side-effects. This approach, termed‘‘ATTEMPTS’’ (antibody targeted, triggered, electrically modified prodrug-type strategy), would permit the administrationof an inactive drug and then subsequently triggered release of the active drug at the target site. The underlying principle wasto modify the enzyme with small cationic species so that it could bind a negatively charged heparin-linked antibody, and thelatter would block the activity of the enzyme drug until it reached the target. To provide the enzyme drug with appropriatebinding strength to heparin, a cationic poly(Arg) peptide was incorporated onto the enzyme either by the chemical7

conjugation method using a bifunctional crosslinker or by the biological conjugation method using the recombinantmethodology. Methods for drug modification, heparin-antibody conjugation, and the prodrug and triggered release features ofthe ‘‘ATTEMPTS’’ approach are described in detail in this review article. 2002 Elsevier Science B.V. All rights reserved.

Keywords: ATTEMPTS; Prodrug; Enzyme drug; Heparin; Protamine; Chemical conjugation; Biologic conjugation

Contents

1 . Introduction ............................................................................................................................................................................ 2522 . The ‘‘ATTEMPTS’’ approach .................................................................................................................................................. 253

2 .1. Description of the approach .............................................................................................................................................. 2532 .2. Components of the ‘‘ATTEMPTS’’ approach..................................................................................................................... 254

2 .2.1. Model drugs .......................................................................................................................................................... 2542 .2.1.1. Azure-A-modified trypsin .......................................................................................................................... 2542 .2.1.2. Peptide (Arg) Cys-modified t-PA................................................................................................................ 2557

2 .2.2. Antibodies ............................................................................................................................................................. 256

*Corresponding author. Tel.:1 1-734-764-4273; fax:11-734-763-9772.E-mail address: [email protected](V.C. Yang).

0169-409X/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0169-409X( 02 )00181-3

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252 Y.-J. Park et al. / Advanced Drug Delivery Reviews 55 (2003) 251–265

2 .3. Heparin- and protamine-induced prodrug and triggered release features ............................................................................... 2573 . Biologic approach to create the modified enzyme drug............................................................................................................... 258

3 .1. Hirulog-streptokinase (HSK) fusion protein ....................................................................................................................... 2583 .1.1. DNA modification.................................................................................................................................................. 2593 .1.2. Expression of HSK fusion protein ........................................................................................................................... 259

3 .2. Poly(Arg) -modified t-PA ................................................................................................................................................. 2597

4 . Optimization of the ‘‘ATTEMPTS’’: approach by computer simulation....................................................................................... 2614 .1. Identification of the incorporation sites by computer simulation........................................................................................... 2614 .2. Amino acid sequence to be incorporated by mutation.......................................................................................................... 2624 .3. Site-directed mutagenesis ................................................................................................................................................. 2624 .4. Characterization of the t-PA mutants.................................................................................................................................. 263

5 . Concluding remarks ................................................................................................................................................................ 263Acknowledgements...................................................................................................................................................................... 263References .................................................................................................................................................................................. 264

1 . Introduction difficult to achieve desirable therapeutic efficiency[17–25].

Enzymes have been widely considered as potential Providing enzyme drugs with the ability to dis-therapeutic drugs because they are potent at fairly tinguish between target and normal substrates shouldsmall doses, exceedingly specific toward their sub- be, therefore, the key to their success in clinicalstrates, relatively easy for preparation as liquid applications. Of all the approaches developed to date,dosage forms, and highly active under physiological the antibody-targeting approach appears to be mostconditions [1–5]. Despite such advantages, however, promising, because it can consistently and substan-very few enzymes have been used as therapeutic tially enhance the specificity and selectivity of thedrugs due to potential immunogenicity and their enzyme drugs [26–38]. One such method using anindiscriminate nature in reaction with both target and antibody for selective drug delivery was a two-stepnormal substrates. To alleviate the immunogenicity, approach called antibody-directed enzyme prodrugmodification of protein drugs with some functional therapy (ADEPT) [35–38]. In this approach, anmolecules has been suggested and such approaches antibody-enzyme conjugate was first administered tohave been proven effective both in vitro and in vivo deliver the enzyme selectively to the tumor deposit,[6–13]. For example, to escape immune response in followed by the administration of a prodrug that wasvivo, protein drugs have been modified with poly- specifically constructed to render it convertible, onlyethylene glycol (PEG), because PEG would repel by the action of the enzyme on the tumor target, toopsonins in the circulation and protect the adminis- the parent, active drug. The converted active drugtered protein drug from clearance in the body [6–9]. would then diffuse into the tumor, causing cellIn this regard, the more outstanding limitation of an necrosis. Despite the fact that the ADEPT approachenzyme to serve as a drug lies in the inability to offers several advantages such as high selectivity fordistinguish the target substrate from normal sub- tumor cells by the antibody, high concentration ofstrates, which subsequently leads to production of active drugs around the tumor site etc., it is notunwanted toxic effects [14–16]. While conversion of without its own limitations. The primary drawback isthe target substrate would result in therapeutic that prodrug formation and its conversion to theefficacy of an enzyme drug, conversion of the active drug are mediated by chemical conjugationnormal substrate would lead to systemic side-effects. and enzyme cleavage, respectively. Therefore, drugCurrently existing enzyme drugs including the candidates are very much limited to small moleculesthrombolytic agents (e.g. t-PA) and anticancer pro- due to the ease of controlling chemical conjugationteins (e.g. RNase, asparaginase) are all suffering and of creating a single cleavage site for the enzyme.from this drawback (i.e. indiscriminate behavior Macromolecular drugs such as proteins are, however,toward target and normal tissues) that renders it less suitable for the ADEPT system for several

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Y.-J. Park et al. / Advanced Drug Delivery Reviews 55 (2003) 251–265 253

reasons. One is that the presence of multiple func-tional groups in the protein can lead to uncontroll-able, heterogeneous chemical conjugation. Also, theconditions employed in chemical conjugation can beharsh to protein drugs, leading to significant impair-ment of their activity. In addition, protein drugs arelarge by nature and thus difficult to produce spe-cifically for the designed enzyme used in activatingthe prodrug. In this regard, macromolecular drugsincluding enzymes need a different system to achievetargeted and prodrug type delivery as seen by theADEPT approach.

A novel approach named antibody targeted trig-gered electrically modified prodrug type strategy(‘‘ATTEMPTS’’) has recently been developed in ourlaboratory for delivery of enzyme drugs. Preliminarylaboratory studies using trypsin and thrombolyticagents (e.g. streptokinase, t-PA) as the model enzymedrugs have demonstrated the feasibility and utility ofthis approach in delivering enzyme drugs [39–46].The reason for selecting trypsin as the model enzymeis because most clinically important proteases aretrypsin-like enzymes. On the other hand, the reasonfor selecting the thrombolytic agent is because theuse of t-PA or other plasminogen activators duringthrombolytic therapy is known to carry the risk ofhemorrhage as a major side-effect. This is primarilydue to the indiscriminate nature of the agent inattacking both the fibrin-bound and circulating plas-minogen [39–41]. In this regard, selection of athrombolytic agent as the model protease drug wouldhelp confirm the feasibility of utilizing the ‘‘AT-TEMPTS’’ approach in alleviating the toxic effectsassociated with the delivered enzyme drug. Herein,

Fig. 1. Schematic diagram of the proposed approach, ‘‘AT-we provide a detailed review of our recent work onTEMPTS’’ [40–46].

the development of this ‘‘ATTEMPTS’’ approach.

species. The two components are linked via a tight2 . The ‘‘ATTEMPTS’’ approach but reversible electrostatic interaction. Because the

cations used to mediate the binding of the enzyme2 .1. Description of the approach drugs are relatively small (e.g. a small positively

charged peptide), the modified enzyme in principleAs shown in Fig. 1, the ‘‘ATTEMPTS’’ delivery can retain a significant level of its catalytic activity.

system is comprised of a large complex made of two This modified enzyme, however, will be deprived ofcomponents: (i) a targeting component consisting of its catalytic activity after binding with its antibody/an antibody chemically linked with an anionic heparin counterpart, presumably due to the blockingheparin molecule; and (ii) a drug component consist- of the enzyme’s active site by these appendeding of the enzyme drug modified with cationic macromolecules. Therefore, similar to a prodrug type



approach like the ADEPT system, the enzyme drug cation to modify trypsin because it possesses awould be without its catalytic activity following the positively charged quaternary ammonium center andadministration of the large drug complex, thereby is known to bind heparin strongly. In addition, azure-alleviating its systemic side-effects. After reaching A is a blue colored dye that absorbs visible light atthe target via the attached antibody, the active, 620 nm, therefore, coupling of azure-A to trypsin canmodified enzyme drug can then be released at the be monitored visually and quantified easily using atarget site by the use of a triggering agent, spectrophotometer. To minimize both intra- (withinprotamine. Protamine is used clinically as a heparin trypsin) and inter-molecular (between two azure-A orantidote and is known to bind heparin more strongly trypsin molecules) crosslinking during the couplingthan most cationic species. The released enzyme can process, carbodiimide (EDAC) was selected as thethen be concentrated at the site of action, thereby activating agent. This was because EDAC wouldmaximizing its catalytic activity toward target sub- allow the initiation of the activation at the –COOHstrates while minimizing its toxic effects towards group of an enzyme, and therefore it could be usednormal substrates. Since the retention of the prodrug concomitantly with azure-A to activate trypsin with-status after administration and subsequently the out the risk of forming inter-molecular crosslinksconversion from prodrug to the active drug rely on between two azure-A molecules. Azure-A wasthe binding strength of the modified enzyme toward conjugated to EDAC-activated trypsin, which washeparin, the insertion of an appropriate cationic then further purified using a Sephadex G-25 column.moiety to the enzyme is critical to the success of the Results from characterization of this azure-A-modi-‘‘ATTEMPTS’’ approach. Both chemical and bio- fied trypsin are summarized in Table 1. Interestingly,logical conjugation methods can be used to insert the the azure-A-modified trypsin retained 80% of thecationic moiety in the modification of the enzyme original trypsin activity, when compared to thedrug, and a detailed discussion of such methods are control sample containing untreated trypsin that waspresented in the following sections prepared to the same protein absorbance at 280 nm.

These results suggested that incorporation of azure-A2 .2. Components of the ‘‘ ATTEMPTS’’ approach to trypsin did not seem to alter much trypsin activity.

Based on the molar extinction coefficient for azure-A2 .2.1. Model drugs and trypsin, the molar ratio of azure-A versus trypsin

in the azure-A-modified trypsin was estimated to be2 .2.1.1. Azure-A-modified trypsin |4.9:1.

Trypsin was first selected as the model enzyme to It should be pointed out that in order for theexamine the in vitro feasibility of the ‘‘AT- ‘‘ATTEMPTS’’ approach to function properly, twoTEMPTS’’ approach. This was because most clini- essential binding strengths between the modifiedcally significant proteases in the circulation are enzyme and heparin must be achieved. One is thattrypsin-like enzymes (e.g. coagulation factors). this binding strength must be stronger than heparinAzure-A dye (MW 278) was chosen as the model and antithrombin III (ATIII), so that the modified

Table 1Preparation and characterization of the azure-A-modified trypsin [40]

Properties Azure-A-modified trypsin

Recovery yield (%) 3163Molar ratio (azure-A/ trypsin) 4.9:1Retention of original trypsin activity (%) 8062

aHeparin induced inhibition toward substrate (%) 83637 21Binding constant versus heparin (3 10 M ) 0.7460.05

bTrypsin activity recovered with protamine (%) 100a Substrate: 6-phosphate dehydrogenase.b Heparin beads containing adsorbed azure-A-modified trypsin were treated with a 0.15 M protamine solution.



enzyme can remain associated with and inhibited byheparin. The other requirement is that this bindingstrength must be weaker than heparin and protamine,so that protamine can be used to trigger the releaseof the modified enzyme from heparin inhibition.According to one result of the binding strength, theprepared azure-A-modified trypsin seen in Table 1was already within these binding requirements be-

6 21tween 5.03 10 M (heparin and ATIII) and 2.538 2110 M (heparin and protamine) as all the binding

constants were accurately measured using theheparin sensor previously developed in our labora-tory [42–46].

2 .2.1.2. Peptide (Arg) Cys-modified t-PA7

Thrombolytic agents were then selected as themodel enzyme drugs to test our ‘‘ATTEMPTS’’approach simply because they are currently the mostwidely used protease drug. Instead of conjugating thecationic species individually, a polycationic peptideconsisting of seven arginine residues (poly(Arg) )7 Fig. 2. Chemical conjugation of t-PA and poly((Arg) ) using7was used to add seven positive charges to t-PA bifunctional crosslinker,N-succinimidyl 3-(2-pyridyldithio) prop-simultaneously. The use of this poly(Arg) peptide ionate (SPDP).7

offered two major advantages. One was that, accord-ing to our previous data [41–45], this cationicpeptide yielded the appropriate heparin-binding 85–90% of the original enzyme activity of native

t-PA. Furthermore, both t-PA and mtPA yielded astrength (i.e. stronger than that of ATIII but weakersimilar Michaelis-Menten kinetic profile for plas-than that of protamine) required by the ‘‘AT-minogen activation [41–46]; although theK valueTEMPTS’’ approach. The other advantage was that m

for mtPA (2.7mM) was slightly higher than that forall seven positive charges were at the same locationt-PA (1.1 mM), the K /k value for mtPA (18.2for the most effective heparin binding. A m cat

21 –1poly((Arg) )(Cys) peptide was synthesized and then mM min ) was slightly lower than that for t-PA721 –1linked to t-PA by using the heterobifunctional cross- (26.4mM min ). Aside from the above charac-

linking reagent agent N-succinimidyl 3-(2- teristics, mtPA also exhibited similar plasminogenpyridyldithio) propionate (SPDP). In this conjugation activation kinetics as that of t-PA in the presence ofreaction, the amino groups on t-PA were activated by fibrinogen, although the rate was somewhat lowerSPDP, and the SPDP-activated t-PA was then spe- than that of t-PA. It is well recognized that t-PA is acifically linked to the SH-group on the cystein thrombolytic agent superior to the others, owing toresidue of the poly((Arg) )(Cys) peptide (Fig. 2). its unique fibrin selectivity [44] and its enhanced7

Characterization of this (Arg) Cys modified t-PA, thrombolytic activity in the presence of fibrin(ogen).7

termed mtPA, clearly showed a significantly en- The fact that plasminogen activation mediated byhanced heparin-binding ability than that from native mtPA was also enhanced in the presence of fibrino-t-PA [41–46]. In addition, modification of t-PA with gen suggested that mtPA still retained a similarthe (Arg) Cys peptide did not cause significant response to the stimulation by fibrinogen as that of7

alteration in the fibrin-binding ability of the t-PA, as t-PA. It is worth noting that the strategy involved inplasminogen activation mediated by the modified the ‘‘ATTEMPTS’’ approach for enzyme deliveryt-PA (mtPA) was similarly enhanced in the presence possesses an additional advantage over the strategyof fibrinogen. Moreover, this mtPA retained up to of utilizing the antibody-enzyme conjugates. After



reaching the target site, the latter requires the dis- a site directed coupling method that would allow forsociation of the conjugate in order to exert the an end-point attachment of heparin to the F regionc

biologic functions of the conjugated enzyme or, of an antibody was designed. This method wouldotherwise, the activity of the enzyme will be ham- retain an intact heparin molecule for electrostaticpered by the steric hindrance of the attached anti- interaction with the cation-modified enzyme, as wellbody. Generally, at the target site, uptake of the as an intact F region in the antibody for targetingab

antibody-protein conjugates is via the process of function. Fig. 3 illustrates the mechanism of thisendocytosis, and dissociation of the conjugates oc- conjugation method. In general, this method wascurs primarily in the cytosol. This dissociation based on the crosslinking of the carbohydrate moietybetween the two large molecules (i.e. antibody and on the F region of the Ab and the oxidized aldehydec

protein), however, is slow and incomplete, resulting group at the terminal of the heparin molecule.in a reduced biologic activity. In contrast, drugrelease by using the ‘‘ATTEMPTS’’ approach is notbased on the chemical dissociation of the conjugatewithin the cytosol, but instead on a competitive ionicbinding between heparin and added protamine, re-sulting in complete dissociation of the enzyme drugfrom the antibody-enzyme complex. In this regard, arapid onset of biologic activity of the enzyme drugcan be achieved by using the ‘‘ATTEMPTS’’ sys-tem.

2 .2.2. AntibodiesThe antibody used in the ‘‘ATTEMPTS’’ approach

is an essential component since it ensures theselectivity and localization of the drug action. Themain requirement of the Ab-heparin conjugate usedin the ‘‘ATTEMPTS’’ system is that it must reachthe target, specifically and also with high affinity. Inother words, the antibody should have minimumbinding towards the other normal sites. Anotherrequirement in formulating the Ab-heparin conjugateis that covalent conjugation of heparin to the anti-body must not destroy the targeting ability of latter,nor should the covalent conjugation alter the bindingaffinity of heparin toward the modified enzyme drugor subsequently protamine. To examine the feasibili-ty of the ‘‘ATTEMPTS’’ system in delivering t-PA,the IgG-59D8 antifibrin antibody was selected. Thiswas because IgG-59D had been shown to be farmore specific towards fibrin than any other currentlyexisting clot-targeting antibodies. Since the targetingfunction of this antibody was well established, theonly concern of the targeting component that couldpossibly affect the overall feasibility of the ‘‘AT-TEMPTS’’ approach was whether conjugation ofheparin to this antibody would affect the designated Fig. 3. Schematic description of the synthesis of the heparin-functions of both molecules. To address this concern, antifibrin IgG conjugate [43–46].



Results obtained from ELISA assay using fibrin asthe capturing agent showed that IgG-59D8 on theheparin-Ab conjugate maintained more than 80% ofits fibrin-targeting ability. In addition, results ob-tained from the aPTT clotting assay and the anti-Xachromogenic assay indicated that heparin on theheparin-IgG-59D8 conjugate also retained over 85%of its original anticoagulant activity. Furthermore,heparin on the heparin-IgG-59D8 conjugate alsoretained its full protamine binding ability, as de-termined by using the protamine sensor and themethodology previously developed in our own lab-oratories [41–46]. In summary, conjugation ofheparin to the antibody using this newly developedmethod did not alter the designated functions of both

Fig. 4. In vitro clot lysis studies [36–39]. Wells[1–4 containmolecules required in the ‘‘ATTEMPTS’’ system. 0.025, 0.05, 0.1, and 0.2mg of t-PA, respectively; well[5

contains 0.2mg free mtPA; well[6 contains 0.2mg mtPA boundto the heparin beads; well[7 contains 0.2mg mtPA released from2 .3. Heparin- and protamine-induced prodrug andheparin beads by the addition of 50mg protamine; and well[8triggered release featurescontains buffer only.

The hypothesis of the ‘‘ATTEMPTS’’ approach isbased on the triggered release of the active enzyme Fig. 4, the heparin-bound mtPA (well[6) yieldedby competitive binding of protamine to the prodrug- significantly reduced clot lysis (, 5%) when com-behaved heparin-enzyme complex. In this regard, the paring to free mtPA (well[5). When protamine washeparin-bound t-PA should not have proteolytic added to the heparin-bound mtPA (well[7), markedactivity since heparin would block the active site of clot lysis was resumed, indicating that the heparin-t-PA by ionic interaction with the cationic peptide on induced inhibition of mtPA activity was reversed bythe t-PA surface. Results show that less than 3% of protamine. These results were consistent with thosenative t-PA were able to bind to the heparin beads obtained by using the conventional chromogenicwhereas 25% of mtPA were, apparently due to the assay [42–45]. It is, therefore, evident that heparinincorporation of the poly(Arg) peptide in the latter. induces inhibition of the catalytic activity of mtPA7

In addition, as much as 95% of the amidolytic (i.e. the pro-drug feature), and this inhibition can beactivity of the heparin-bound mtPA was inhibited by readily reversed by using the triggering agent,heparin binding, suggesting a near-complete achieve- protamine.ment of the anticipated prodrug feature. Also, as The above results obtained by using theexpected, the inhibition of mtPA by heparin was chromogenic assay and the fibrin clot lysis assaylargely reversed (|42%) by the addition of have both corroborated that heparin can induceprotamine [41–46]. blockage of mtPA activity and this blockage can be

Alleviation of the toxic effects of t-PA by the released by protamine. To further confirm theseprodrug feature of the ‘‘ATTEMPTS’’ approach was results, the heparin-Ab conjugate, instead of thefurther confirmed by the presence of virtually no heparin beads, was tested for ability in inhibiting thedepletion of the plasma levels of plasminogen,a - activity of mtPA. Results show that the heparin / IgG2

antiplasmin, fibrinogen by the mtPA-heparin com- 59D8 conjugate was also capable of blocking theplex, as opposed to significant depletion of such activity of mtPA, although its efficiency in blockingcoagulation factors by the use of free mtPA [41–46]. t-PA activity (40–60% inhibition) was somewhat

An in vitro fibrin clot lysis assay was developed to lower than that by the heparin beads (. 95%further demonstrate the usefulness of this heparin / inhibition). This lower inhibitory action by theprotamine-based t-PA delivery system. As shown in heparin / IgG conjugate may be explained by the



presence of free mtPA in the sample which couldexert plasminogen activation and, consequently, re-sult in the observed lesser inhibition of mtPA activityby the heparin / IgG conjugate. Such an assumptionwas supported by the fact that further removal ofunbound mtPA from the sample would increase thedegree of inhibition by the heparin /Ab conjugates.

3 . Biologic approach to create the modifiedenzyme drug

Biological modification by using recombinantDNA technology was employed to directly preparethe cation-modified enzyme drugs. Biological conju-gation presents several advantages over chemicalconjugation: (i) the conjugation process can be easilycontrolled to obtain a homogeneous product with ahigh yield; (ii) the recombinant technique willalleviate the risk of random and multiple incorpora-tion of the cationic peptides onto the enzyme as seenby the chemical conjugation method. In this chapter,biologic conjugation of a hirulog peptide to strepto-kinase as well as that of the poly(Arg) peptide to7

t-PA is presented.

3 .1. Hirulog-streptokinase (HSK) fusion protein

Our very first, initial attempt to create a modifiedenzyme for the ‘‘ATTEMPTS’’ system by the re-combinant method was to produce a hirulog-strepto-kinase fusion protein chimera (termed ‘HSK’) con-taining a small hirulog peptide (a potent thrombininhibitor derived from hirudin) fused to the N-ter-minus of streptokinase (a widely used clinical throm-bolytic agent). Similar to the heparin /protamine-based ‘‘ATTEMPTS’’ system, this HSK system also Fig. 5. A schematic diagram of recombinant prodrug approach

using hirulog and its triggered release by hirudin [47].utilized two components: (i) a fibrin-targeting anti-body chemically linked to an inactive thrombin and(ii) HSK which is linked via a tight and yetdissociable thrombin-hirulog interaction (Fig. 5) nase by the appended macromolecules. Since hirudin[47]. The HSK would retain its plasminogen-activat- is known to bind thrombin several orders of mag-ing activity, since the catalytic domain of streptoki- nitude more strongly than hirulog, it can be used tonase resided at the C-terminus. On the other hand, dissociate the active HSK from its counterpart. Thusthis HSK would be deprived of the proteolytic the approach would allow for the administration of aactivity after its conjugation with the Ab-thrombin fibrin-targeting but ‘inactive’ thrombolytic conjugatecounterpart, primarily due to steric hindrance pro- and, subsequently, a hirudin triggered release of thevided at the plasminogen-binding site of streptoki- ‘active’ HSK in close proximity to the fibrin deposit.



This would permit the thrombolytic drug to spe- ty of producing an active, thrombin-binding, modi-cifically attack the components of a thrombus while fied streptokinase by using the recombinant means.sparing the circulating clotting factors, thereby alle- Despite this success, the thrombin/hirudin basedviating bleeding risks. delivery system for thrombolytic agents faced a

major drawback. The problem lies primarily in the3 .1.1. DNA modification requirement of thrombin as the triggering agent. In

Amplification of the streptokinase gene (termed thrombolytic therapies where blood vessels are oc-‘sk’) was conducted by PCR using two primers cluded due to the formation of clots, the use of a(SK-A and SK-B) that flank the streptokinase coding potent coagulation factor like thrombin, even if it issequence. A SalI site was also introduced into the in an inactive form, would raise serious concerns inSK-B primer. Since hirulog was a long, synthetic clinical practice. In this regard, the use of a heparin /peptide that contained 20 amino acids with a se- protamine-based ‘‘ATTEMPTS’’ approach discussedquence of NH -FPRPGGGGNGDFEEIPEEYL, ad- previously would seem to be far more acceptable in2

dition of the coding sequence of hirulog tosk was future clinical application.conducted in two steps using these two primers [47].Results of the agarose gel electrophoresis revealed a3 .2. Poly(Arg) -modified t-PA7

1296-bp and a 1334-bp (termed ‘hsk’) fragmentfollowing the first and second step of addition, Our second attempt with a biological method tosuggesting successful incorporation of the entire create a cation-modified thrombolytic agent wascoding sequence of hirulog tosk. conducted by inserting the poly(Arg) peptide se-7

quence into the t-PA protein by using the recombi-3 .1.2. Expression of HSK fusion protein nant DNA methodology. DNA amplification, isola-

The hsk gene was transformed intoEscherichia tion, and routine enzymatic manipulations werecoli cells according to a standard procedure to carried out following standard procedures [48]. Aexpress the HSK fusion protein. The expressed schematic illustration of the procedures for construc-proteins were isolated by sodium dodecyl sulfate tion of the plasmid for expression of rmt-PA ispolyacrylamide electrophoresis (SDS–PAGE) and presented in Fig. 6. As shown, the plasmid forthen transferred to a nitrocellulose membrane for expression of t-PA,ptPA-trp12, was separately di-Western blot analysis. A protein band with a molecu- gested with restriction enzymes EcoRI1NarI,lar weight equivalent to the sum of hirulog and EcoRI1Bg/ II, or NarI1Bg/ II. The cleaved DNAstreptokinase was seen on the Western blot assay fragments were then isolated by agarose gel electro-[47], indicating successful expression of the HSK phoresis and purified using the Nucleic Acid Rapidprotein. Isolation Kit (Sigma). A 336-base-pair (bp) EcoRI-

HSK fusion protein showed that it possessed the NarI fragment for encoding prior peptides, a 1700-bpplasminogen-activating activity that could be in- NarI-Bg/ II fragment for expression of t-PA, and ahibited by the binding of added thrombin. The 4200-bp vector fragment containing trp promoter andinhibition of the plasminogen-activating activity of an encoding gene against antibiotics were collected.HSK by thrombin appeared to be dose-dependent. These three fragments and the double strandedWhen an increasing amount of thrombin (1–25 U) oligonucleotide (encoding the poly(Arg) sequence)7

was added to samples containing HSK, a gradual were linked in order using T4 ligase. After ligation,reduction in the level of plasminogen conversion was the reaction mixture was transformed into the HB101observed in these samples. When an excess amount bacterial strain by following the standard calciumof hirudin was added to the thrombin-inhibited HSK, chloride approach. The bacteria were then plated onthe plasminogen-activating activity of HSK was LB solid selective medium containing tetracycline,restored, as reflected by the significant conversion of and colonies that appeared after 12–16 h in culture atplasminogen to plasmin. 378C were collected. The corresponding plasmid for

Although it was a preliminary study, the above expression of rmt-PA was screened and identified byinvestigation nevertheless demonstrated the feasibili- digestion with restriction enzymes Bg/ II and Sa/ I.



Fig. 6. Biologic modification of t-PA with poly(Arg) using recombinant DNA technology: construction of the recombinant PtPA-(Arg)7 7

plasmid for expression of mtPA [43,44,48].

The expression of the recombinant, (Arg) -modi- that is equivalent to the molecular weight of the7

fied t-PA (termed mtPA) was confirmed by the (Arg) peptide) than that of the native t-PA. The7

presence of a protein band in the Western blot assay insertion site of poly(Arg) was specifically designed7

with a slightly higher molecular weight (| 2000 Da to locate at kringle I domain of t-PA, which is the



fibrin binding domain but is dispensable for throm- free domain by mutation would seem to be a morebolytic activity [47,48]. The mtPA, thus prepared, logical approach.yielded an almost identical initial rate in convertingplasminogen as that of t-PA, indicating that mtPA 4 .1. Identification of the incorporation sites byretained the full intrinsic catalytic activity of t-PA. computer simulationThis catalytic activity of mtPA was significantlyinhibited by the addition of heparin (| 54% of initial The selection of the incorporation site is extremelyactivity), and this inhibition was largely reversed (to important, as previous results suggest that an in-75% of the original activity of mtPA) by the addition appropriate location of peptide insertion site canof protamine. Although there still was a room for diminish heparin-induced inhibitory effect upon itsfurther improvement, these results nevertheless binding [45]. In general, the site should be spatiallyopened the possibilities of direct production of the close to the active site of t-PA such that bounddesired protein by using the recombinant DNA heparin would effectively interrupt substrate inter-method. action, and yet modification of this site should not

significantly alter any functional properties of theprotease activity in the absence of heparin. In other

4 . Optimization of the ‘‘ATTEMPTS’’: words, modification should be performed on a sur-approach by computer simulation face-exposed segment with the possibility of retain-

ing the tertiary structure of the protease. In thisIt should be pointed out that the ultimately created regard, we applied computer simulation to identify

mtPA must fulfill three essential requirements: (i) it the possible target sites that are surface-exposedmust retain the same chemical and physical prop- regions that would tolerate mutation based onerties of the original t-PA; (ii) it must possess the evolutionary and experimental evidence. The overallwell defined heparin-binding strength (i.e. greater strategy is therefore as follows: (i) identify proteinsthan that between heparin and antithrombin III but that share a high degree of sequence similarity to theweaker than that between heparin and protamine); catalytic domain of the t-PA; (ii) perform a multipleand (iii) binding of heparin must be able to block the sequence alignment of these amino acid sequences tocatalytic domain of mtPA or, in other words, to identify variable regions; and (iii) select variableinhibit the activity of mtPA. Although the previously regions that are on surface-exposed loops and also inadopted method of inserting the poly(Arg) peptide close proximity to the active sites. Using this com-7

to the kringle domain of t-PA by recombinant DNA puter simulation and multiple sequence alignment,technology showed promise, this approach neverthe- the 37- and 186-loop in t-PA were been identified asless yielded several potential pitfalls. One was that the two surface-exposed domains that should beinsertion of a peptide to t-PA could lead to structural tolerant to mutation (Fig. 7). Of the two loops, theand conformational changes of the enzyme, altering 37-loop was selected as the site for incorporation ofthe pharmacological functionality of the enzyme as the peptide because it was arginine-rich and alsowell as the pharmacokinetic profiles of t-PA. The implicated in binding with PAI-1, a circulating t-PAother concern was that the insertion site of the inhibitor. Modification of t-PA at this region couldpoly(Arg) peptide might not be surface-exposed or potentially provide an additional advantage, because7

be without any biological functions while insertion by disrupting the PAI-1 binding the circulation half-of the poly(Arg) peptide into folded areas of t-PA life of the mutated t-PA would be prolonged. The7

could render it inaccessible to heparin binding and crystal structure of t-PA revealed that the 37-loop˚subsequent activity inhibition, and conjugation of the was| 20 A from the active site catalytic triad, and

poly(Arg) peptide too close to the active site or was located at the edge of the active site cleft7

other functional sites of t-PA would impair the [49–55]. Considering that a heparin dodecasac-˚catalytic and biological functions of the protease. To charide with an average MW of| 5 kDa was 50 A in

overcome such potential problems, creation of a length [56–58], accessibility of the substrate to thepolycationic domain on a surface-exposed, function- t-PA catalytic site would likely be denied by the



combination of these two consensus heparin-bindingsequences. Another consideration was that this pep-tide sequence was relatively similar to that(KHRRSPGGR) of the 37-loop of t-PA, and shouldtherefore be readily created by minimal steps ofmutation. The third consideration was that a prolineresidue was included to retain theb-turn structure inthe modified loop, retaining this loop to be complete-ly exposed on the surface. Lastly and also mostimportantly, two cystein residues were included inthe mutation sequence to yield free –SH group, forthe purpose that in case this incorporated cationicpeptide segment did not yield the required heparin-binding strength, additional heparin-binding peptidescould be attached to these –SH groups via the SPDPchemical conjugation method.

4 .3. Site-directed mutagenesisFig. 7. Tertiary structure of human t-PA catalytic domain. Thechain length of heparin tetrasaccharide is estimated based on

Site-directed mutagenesis was employed to createliterature data for heparin dodecasaccharide [44,54].the desired cationic peptide sequence at the 37-loopof t-PA. Attempts were made to create four mutants.

binding of a heparin molecule to the mutated 37- Mutagenesis was conducted by using the mutagenicloop. oligonucleotides and the Mutagenesis Kit. Sequence

analysis of the mutated plasmids for t-PA mutants4 .2. Amino acid sequence to be incorporated by demonstrated that the 37-loop of t-PAs were com-mutation pletely changed to those as initially purposed (Fig.

8). These t-PA mutants were expressed by transfec-Further analysis of the identified incorporation site tion into the CHO cell line, and then collected and

in t-PA indicated that it contained a sequence of purified by using the previously established standardapproximately nine to ten amino acids that could be procedure. Following expression, these mutants werereplaced by mutation without disrupting the overall identified by Western blot using the hybridizationfold of this protein [50–53]. In this regard, a method with a goat anti-human t-PA antibody.nonapeptide sequence of RCRRCPRRR was selected Results showed that these mutants displayed theto be incorporated into the 37-loop by mutation. This same migration distance on the gel as that of nativeselection was based on several important considera- t-PA. In addition, the fact that these mutants re-tions. One was that this peptide contained the sponded to the anti-human t-PA antibody used in thesequence known for strong heparin binding. In cited Western blot suggested the presence of t-PA moietiespapers, Cardin and Weintraub [59,60] examined 21 in these mutants [47,48].heparin-binding proteins of peptide stretches that Purified t-PA mutants was examined by aminowere enriched in basic amino acids, and suggested acid composition analysis, and an increased argininethat the following represented two general types of content was observed in those mutants in accordance‘consensus sequences’ for heparin binding: (i) with the changed arginine residues by mutation.XBBXBX; and (ii) XBBBXXBX (whereas B is a Moreover, amino acid sequence analysis confirmedbasic amino acid such as lysine or arginine and X is that the sequence of the 37-loop in these fourany hydropathic amino acid). The proposed peptide mutants was changed from KHRRSPGER tosequence of PCRRCPRRR (BXBBXXBBB) to be KHRRCPRRR, KRRRCPRRR, KRRRRPRRR, andincorporated at the 37-loop closely mimicked a KKRRKPKKR, respectively.



heparin column clearly demonstrated the differencesin heparin-binding strength between such mutantsand the native t-PA. All these mutants were elutedfrom heparin column at a higher ionic strength thanthat of the native t-PA, indicating the presence of anenhanced heparin-binding ability for these mutants.However, there were no significant differencesamong the mutants in their binding strength towardheparin. Overall, the mutation did not seem to alterthe biologic functions of the t-PA, while offering ahigher heparin binding strength than the parent t-PA.Further in vitro and in vivo testing using thesemutants is currently under way.

5 . Concluding remarks

‘‘ATTEMPTS’’, a heparin /protamine-basedtargeting and pro-drug delivery system, was de-veloped to deliver macromolecular drugs such asenzymes without their associated toxic side-effects.By using this ‘‘ATTEMPTS’’ approach, the inactiveform of an enzyme drug can be localized specificallyat the target site via an antibody and the active formof the enzyme drug can then be released by using thetriggering agent, protamine. Both chemical andbiologic conjugation methods were applied to createthe cation-modified enzyme drug, with the posses-sion of a specific binding affinity to heparin. Neitherof these two conjugation methods altered the bio-logical function of the parent drug. Since the releaseof the active drugs was based on the ionic inter-action/competitive binding between heparin andprotamine, no dissociation of the chemical bondsFig. 8. Four mutants of t-PA having different cationic peptidewould be needed, unlike the process required in thesequences (shown in under line) incorporated into the target

37-loop of t-PA. conventional approach of utilizing an antibody-drugconjugate. In conclusion, the ‘‘ATTEMPTS’’ ap-proach could be applied to the design of other novel

4 .4. Characterization of the t-PA mutantsdelivery systems for clinically important catalyticdrugs, or even for small drugs that would possess

Characterization of these four heparin-binding t-strong toxic side-effects.

PA mutants is currently in progress in our laboratory.Preliminary studies using both the chromogenicassay and fibrin gel lysis assay showed that thebiological activities of these t-PA mutants were A cknowledgementssuccessfully retained. In addition, the activity ofthese t-PA mutants was fully inhibited by the anti- This work was partially supported by a grant fromtPA antibody. Elution of these t-PA mutants on a the National Institute of Health ([ HL55461), USA.



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