Recent advances in drug targeting

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G acylasc removes only the phcnylacetyl groups. These two groups havc similar chemacal reactivity and cannot be selecnvely deprotcctcd by chemical methods. In another example, Victor Gotor (Umversldad de Ov~edo, O w e d o , Spain) reported selective protecnon o f a deoxynbonucleoside Llpase from Pseudomo,as cepacia catalysed the selective protection o f the secondary hydroxyl group, while lipase from Candida antarctica catalysed the sclecuve protecnon o f the primary hydroxyl m the same structure. Several other workers mentioned th~s new hpase from C. atttarctica (Novo N ordirsk A / S , Bagsvaerd, Denmark) as a highly sclecnve and synthetically useful enzyme. In related work, Fred van R a n t w u k (Delft Umversaty o f Technology, Delft, The Netherlands) described the selecuve acylation o f sugars for use as surfactants, while Jonathan Dordick (Umversity o f Iowa, Ames, IA, USA) reported on the selectave acylauon o f sucrose wath acrylic acid esters, followed by polymenzauon to produce h~ghly water-absorbent hydrogels.

N e w selective e n z y m e s

P,.cscarch efforts have also focuscd on Mentlfylng new enzyrne-catalysed reacnons. Several gnoups used oxymtrilases in the synthesis ofenantlomencally-pure cyanohydrins, while others used glycosidases to add a sugar group to other sugars or to orgamc fiagmcnts. Hiromlchi Ohta (Kmo University, Yokohama, Japan) used nltrilase to hydrolyse dinimles enantioselecnvely, and Dawd Crout (University o f Warwick, Warwick, UK) synthesized acyloms with pyruvate decarboxylase. More complex reactions often require whote cells as catalysts. Several posters describcd enantmselecrive hydroxylation o f hydrocarbons. Interestingly, benzoxazole protecting groups on carboxylic acids ehmmated side reactions m thesc whole-cell hydroxylations. Herbert Holland (Brock University, St Catharmes, Canada) described a pre&cnve model for hydroxTlatlon reacnons carned out by the fungus Mortierella. Many mlcroorgamsms contain an enannoselecnve cpoxide hydrolase, descnbed m several posters frorn Kurt l%ber's group (Graz University o f Tech-

nology, Graz, Austna) and a presen. tatmn by Roland Furstoss ( U R A C N R S , Marseille, France). Stefano Servi (Pohtecmco di Mllano, Mdano, Italy) dcscnbed unusual synthetic reacnons catalyscd by baker's ycast For example, the ad&tlon of benzyl mercaptan to yeast cultures captures a glucose metabolite, (s)-glycerate, isolated in >98% enantiomeric excess as the benzyl thioester. Although catalytic anubochcs promise to be a new source of blocatalysts, Andreas Pltickthun (Max Planck Insntute fiir Biochemle. Martmsned, Germany) pointed out the need to incrcase their turnover rates and stability before they can be applied to pracncal orgamc chemistry In summary, hpase and proteasecatalysed reacnons are becoming standard, predictable reagents in synthenc laboratories and industry, while researchers are wMening the potenual ofbmcatalysls by identafymg new, selective biocatalysts.

Romas J. Kazlauskas Departmellt oj ChemL~tty,3,lcCdl Umuer.~lty, 801 SherbtookeSt ~V , Alont~&I, Quebec, Camtda, H3A 2K6

Recent advances in drug targeting meetitls report

Drug targeting is a para&gm o f a concept born (early thls century) before its nine. In retrospect, it is now obvious that technology to direct drugs specifically to, or faclhtate their release m, areas o f the body m need o f pharmacological lntervcnt~on could not matenahze m the absencc of the great advances in molecular and cell biology witnessed m the past two decadcs. Even so, m spate o f the ilnpresslve progrcss made in developing potentially useful systems for drug dehvery, only a few products have been hcensed for chmcal use. The nnpctus to succeed has becn amphfied recently by the everincreasing availability of therapeuuc pepndes and proteins produced by recombinant D N A technology but, as with conventional drugs, these new cornpounds are often unablc to perfonn optnnally i, eivo without an efficlent means of-delivery. Certainly, the reasons fbr the consMerable delay in putting the target-

BTECH NOVEMBER1993 (VOL11)

mg concept into practice on a larger scale are not for the want o f systems. The armamcnt is indeed nch and includes a vast array o f antibo&es (mostly monoclonal), a number o f receptor-specific proteins, pcptldes and glycolipids, numerous natural and man-made polymers, and a collection of micropartlclcs, nanoparncles and hposomes. Such systems, incorporating drugs cither through chemical bonding or passively, can deliver thmr load to specific cells broadly on the basis o f r e c e p t o r hgand interaction, or release it opumally at predetermined rates. Unfortunately, these cosy scenarios are usually frustrated by the complex bmlogncal milicu interposed bctween the site o f application o f the targeted systern and its site o f action. For instancc, drug-loaded systems may be intercepted by the reticuloendothehal system (RES) et~ route to thmr destmanon, may exhibit poor uptake by targct cells, may be

dcstroyed in the gut after oral admlnlstranon, or may reduce unwanted nnmune rcactlons. The uncompromising nature of such facts has forced drug targeters to rethink, with the role ofthc imheu becoming a central consideration m the design o f systems. Happily, progress has already been made m solving or bypassing some of the problcms, as pamclpants of a recent N A T O Advanced Stu&cs Insntutc ~were able to appreciate A classic example o f a system that is perfect in theory but rather deficient in pracnce is the case o f m o n o clonal antibodies (mAbs). Hawng been the flagship o f expenmcnta] targeted cancer therapy for almost 20 ycars, mostly m thc fbrm of lmmunotoxms, rnAbs arc still a 'golden bullet' waiting to be fired Poor penetration into human sohd

~Thc Seventh NATO Advancedbtudleslnmtutc (ASI) 'Targeting of Drugs Advancesm System Consuucts'. directed by Grcgory Gregona&s,was held at Cape Sounlon Beach. Greece, 24June - 5July 1993

© 1993, ElsevierScience PublishersLtd (UK)

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f Or l~lrFl turnouts (e.g. less than 0.01% of the dose per gram of tissue), the paucity of turnout-specific antigens and mAb ammunogeniclty have all contributed to unremarkable results in the clinic, thus dampening initial enthusiasm 1. Although tumour penetration still remains a problem, there is now considerable opumzsm with regard to tumour-specafic determinants. Potential targets include the protean core of tumour mucins that are aberrantly or incompletely glycosylated (anucore mAbs are opcrationally tumour-specific because they do not bind to the fully glycosylated mucins on normal epithelia), antigens expressed on small-cell lung-cancer cells and oncogene products, which can serve as tumour-associated antigens following oncogene activation and ovcrexpresslon. As such mutations are found only in neoplastic tissues, overcxpresscd oncogene products are targets for antibodyguided Imaging and therapy (Agamemnon Epenetos, Royal Postgraduate Medical School, London, UK). The use ofligands that bind to ovcrcxpressed tumour surface molecules is an attracnve alternative to mAbs against such molecules (Davad FitzGerald, National Cancer Institute, Bethesda, MD, USA). For instance, transforming growth factor alpha (TGF-ot) and interleukin 6 (IL-6) have been fused with Pseudomotlas exotoxin (PE) (one of the most toxic substances known) following delenon of the binding activity of the toxin 2, and these compounds have been shown to have a curative effcct m nude-mouse models of human cancer. An immunological response to administered mAbs is hkely to neutralize their effectiveness, and the resuItmg immune complexes may lead to unwanted side-effects. Production of chimeric and 'humanized" mAbs has gone a long way towards resolving, but not completely ehmmating, the problem, but even human mAbs are known to reduce antl-idlotypic antibodies (Herman Waldmann, University of Cambridge, Cambridge, UK). A completely diff'erent and perhaps easier approach to render mAbs and other potentially therapeunc proteins tolerogemc is by coupling the highly hydrophlhc molecule monomethoxypolyethylene glycol (mPEG) to the antigen (Alec Sehon, University of Manitoba, Manitoba, Canada). Using severe combined lmmunodeficiency (SCID) rmce cngrafted with a mixture of

human T- and B- + mononuclear cells, the human antl-murme annbody response as well as the antiidIotypac antibody response to the respective murme m a b s were suppressed specifically3. Interestmgly, at is also possible to suppress ammunc responses, even to murme mabs possessing idiotypic speclficities that are distinct from those of the tolerogenie mAbs. provided that the two types ofmAbs share the same heavyand hght-cham isotypes. A group of cell-specific hgands that are likely to be nonimmunogenic are natural glycoproteins, such as deslalylated human %-glycoprotein, which can transport drugs quantitatively to hcpatlc parenchymal cells expressing the galactose receptor. Attempts to exert control over lagand-receptor interactions and the drug-loading capacity of the ligand, have given nse to neoglycoproteins (plain proteins covalcntly linked to sugars). One of the neoglycoproteins discussed (mannosylated albumin coupled with N-acetylmuramyl dipepnde) was able to eradicate Lewis lung-carcinoma metastases in mice (Michel Monsigny, Centre de BiophysIque Moleculaare, Orleans, France). To avoid the potential ammunogenicity of such proteins as a result of structural modifications, the group have designed glycosylated, presumably mcrt, polymers. One of these (lactosylated poly-Llysinc) was shown to mediate specific gene transfer into hepatoma cells 4. Polymenc carriers are thought to have advantages over proteins: they can be synthesized in large quantities in a nonimmunogenic and biodegradable form, and are cheaper. Some of these polymers have an inherent abihty to target, a good example being the polyammo aczd poly(Glu, Ala, Tyr), which is rctaincd by the lungs after intravenous injection (Ruth Duncan, Farmitalia Carlo Erba, Milan, Italy). Control over their synthesis also allows the production of a number o£ polymer versions with specific properties conducive to targeting drugs enteraUy (Jindrich Kopecek, University of Utah, Salt Lake City, UT, USA). In one such version, drug-containing (vm aromanc azobonding) N- (2-hydroxypropyl)methacrylamlde copolymers arc coupled to fucosylamine, which binds to fucose-terminating lectins in the colon. This enables the compound to be retained in the colon

long enough fbr microbial enzymes to act on the azobond and release the drug locally. Another version ISbased on pH-sensldve hydrogels that can also be degraded by microbial enzymes in the colon. By the time the hydrogels reach the colon, the degree of their swelhng has increased (because of an increased degree of ionization) to a point that allows azoreductase activity to cleave the cross links and release entrapped peptides and proteins. Polymeric systems that release drugs at a constant ratc can maintain the drug concentration within the desired therapeutic range with a single dose. However, there arc climcal situations where such an approach may not be sufficmnt [e.g the dclivcry of insulin, gastnc inhibition, ~-adrenergic blockade (through [3-blockers), birth control, etc.]. Indeed, studies on chronopharmacology indicate that the onset of certain diseases exhibits strong circadian/temporal dependency. There is, therefore, a need for system constructs that are responsive to biological rhythms. Recent work s has led to the design of the so-called open- and closed-loop systems (Joseph Kost, Ben Gurion University o f Negev, Beer-Sheva, Israel). The former are regulated externally by devices (e.g. magnetic, ultrasonic, thermal, electric, etc.) that tugger pulsatde drug delivery. (Early studies on a photoresponsive polymer-delivery system were reported by P,.on Tomcr and Alexander Florence, School of Pharmacy, University of London, London, UK). The latter, on the other hand, are controlled by feedback reformation based on enzyme substrate interactions, pH-sensmve drug solubility, competitive binding and antibody-antigen interactions. Among man-made drug-dehvery systems, hposomes (phosphohpld vesicles) have occupied a ccntral and often controversial role for over 20 years Their chief attraction is their versatlhty in physical characteristics. Liposome compositaon, bilayer fluidity, size, surface charge, mode of drug accommodation and vesicle coating wath targeting ligands, can all be tailored to produce vesicles for specific needs. These range from a wide variety of actual and potential therapeutic apphcatIons (e.g. cancer and antimicrobial therapy, cytokme-, hormone- and enzyme therapy, metal detoxlficanon, amaging, and as haemoglobin carriers) to roles m immunoregulatlon as immunological TIBTECH NOVEMBER1993 (VOL

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adjuvants aud vaccine careers 6. Most of the work with hposomes has been carried out by the intravenous route, which is the route of choice for the treatment of many important systcmlc diseases. O n injection, convennonal ]iposomes are taken up by the IKES, chiefly by the fixed macrophages of the liver and spleen These tissues are often the focus of localized disease, especially microbial, and m this respect hposomes have been a success, with the licensing of the first injectable product (AmBisome% an amphotericmB-based formulation for the treatment of disseminated fimgal disease) m several Western counmes. It has been recognized, however, that for liposomes to meet the challenges of intravenous therapy, other than that involving the IZES, their interaction with the RES must be curtailed for a period of time long enough to allow uptake at alternative sites. Tlns can n o w be achieved by combining small (~100nm diameter) vesicle size and a rigid bllayer [charactenstics shown to reduce lZES uptake dramatmally (Gregory Grcgorla&s, School of Pharmacy, University of London, London, UK)] with a 'stencally stabilized" vesicle surface by means of mPEG coating (Terry Allen, Umverslty of Alberta, Edmonton, Canada). The latter seems to improve matters further by rcndenng vesicle clearance from the circulation dose-independent (Demetrlos Papahadjopoulos, University of California, San Francisco, CA, USA). Work by these two groups, and others, indicates that the coupling oftumour-speclfiC annbodies to the stcrically stabilized vesicle surt5cc promotes (doxorublcmcontaining) vesicle locahzation in solid turnouts, with a concomitant increase in survival rates 7. An alternanvc, and perhaps preferable, type of macromolecule that could serve to increase the orculatlon time of hposomcs and proteins and also of small peptldes and conventional drugs (a role which the low molecular weight rnPEG, k n o w n to be excreted rapidly through the kidneys, is unlikely to fulfil) is naturally occurring phospholipid-containIng polyslahc a c l d ~ (PS) (Brenda McConnack, School of Pharmacy, University of London, London, UK). P, esults show that half-lifts (up to 40 hours) of PS in the circulation of m i c e injected intravenously depend on the PS used, that they can be further increased by deacylation of IBTECH NOVEMBER1993 (VOL 11)

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its phosphohpld moiety, that they decrease with shorter chain derivatives and appear to bc doseindependent~. Moreovcr, dI-ugs covalently b o u n d to PS assume the half-life of the polymer which, in theory at least, could be tailored to values that are optimal for the action of a given drug. Preliminary evidence also suggests that PS bound to a much larger moiety, namely liposomes, augments their clrculanon time, presumably through steric stabilization of the vesicle surface. The majority of research on drug targeting uses the intravenous route, which must be regarded as an invasivc mode of entry to the body. In conventional medicine this route is resorted to only when necessary, so ~t behoves scientists to continue to probe thc oral route. Controversy still reigns over the feasibility ofusmg the oral route for the delivery of peptides and macromoleculcs which arc subject to degradation in the gut and which have less than optimal properties for absorption. Bob Davis (University of Nottingham, Nottingham, UK) dIscusscd the problems and opportunme¢) of colloidal particulate systcms admitted to the Peyer's patches of the gut. Several groups, including that of Florence l°,ll have shown that the specialized M cells of the Peycr's patches are able to take up and translocate small quantities of particulate material. Davis, however, cast doubt on the veracity of these claims, believing there to be sortie artefactual influence, such as ttic mode of sectioning tissues spreading particles into the examined areas. It is wise to be critical, particularly as the oral route holds so much promise. However, much ofthc relevant work I
Drexler, looking into the future m his book E,gines o f Creation 13 rightly talks of present-day conventional drug delivery in which drugs 'dumped into the body, tumble and bump around in solunon haphazardly until they bump a target molecule, fur and stick, aft~ectingits function'. The constructs and approaches &scussed at this ASI, with their carncrs and specific hgands, go some way towards ensuring that the process is less haphazard. But we are still some way from employing Drexler's nanomachines with their powers as 'cnganes of healing' to gain access to &seased sites, recognize the appropriate target, &sassemble damaged parts, rebuild and reassemble. O u r incremental progress is, nonetheless, excmng.

References 1 Kosmas,C , Llnardou,H and Epenetos,A (1993)J Du(¢ Tagct. 1, 81 91 2 Pastan, 1, Chaudhary, V K and FitzGerald, D J 11092)Atom Rev Bwdlenl 61,331-354 3 B]toh, S, Lang, G M , Klcrck-Jaszczuk, D, Fuj1rnoto,S and Sehon, A H (1993) Hunt Anttbo@ Hybfidomas 4, 134 143 4 MMoux, P. Mendes, C, Legrand, A, B.ammnd. J. Meyer, R, Monslgny, M and Roche, A C (1993)N.cMc Aod, Re, 4, 871 878 5 Kost, J (1990) Pnl~ed and Selj-Rtiwhaed Dmf Deln,ery, CRC Press 6 Gregona&s,G fed) (ICl93)Liposo.le Ted> nolwy, 2nd edn, Vols 1-3. CP.C Press 7 Papahadjopoulos, I). Allen, T M, Gablzon, A, Mayhew, E, Matthay, K, Huang, S K, Lee, K D ,Woodle, M C andLaslc,D D (1991)Pwc NatlAcad Sol liSA 88, 11460 11464 8 Gregona&s,G, McCormick, B , Wang, Z and L1fely,11, 11093) FIqBS Lctt 315, 271-276 9 Challacombe, S J, Rahman, 1), Jef}]ey, H , Daws,S S and O'Hagan,D T 11992) hn,nmolo~y 76, 164 168 10 Jam, P u , Halbert, G W, Langndg~,j and Florence, A T (1090) J Pha*m Pham~acol 42, 821-826 11 Jam, P U, Florence,A T and McCa~tl~}, D (1992)lnt _I lqla,nateutics 84, 245 252 12 Couvreur,P and PUlSleUx,F (1993)Adv Din2 Dehv Res 10, 141 162 13 Drexler, K E (lC)92)Ellglnc~ o/ Creatlo,, Oxford UmversltyPress

Gregory Gregoriadis Alexander T. Florence Creme for Dwg Drln,ery Re.~earch, &hool of Pha.m~Ly, Unlve~stty ~¢ Londou, 29/39 Brunsu,rck Sqmlre, Lomlou, UK I/VE:IN IAX