Advanced Drug Delivery Reviews, 13 (1994) 311 323 311

ADR 00155

'Carbohydrate handles' as natural resources in drug delivery

E d u a r d o P a l o m i n o Walker Cancer Research Institute, Detroit, MI, USA

(Received April 11, 1993 (Accepted April 20, 1993)

Key words: Carbohydrates; Delivery; Lectins; Polyvalency; Glycosides; Receptors; Conjugates; Neoglycoproteins

Contents

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

II. The stereochemical complexity of carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

III. Lectins: the carbohydrate receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

IV. The concept of delivery by polyvalency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316

V. Delivery through simple carbohydrate handles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317

VI. Delivery through neoglycoprotein conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

VII. Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322

Summary

Carbohydrates, perhaps the most ubiquitous chemicals in living systems, have been traditionally associated with energy production and as building blocks. Increasingly, however, the role of carbohydrates is seen as more pervasive and sophisticated. Their involvement in and with lectins, the high affinity carbohydrate- binding non-immune proteins or glycoproteins, makes them attractive as 'handles' in drug delivery. As such, carbohydrates could partake as (1) organ or site delivery. by making use of their particular physicochemical properties, and (2) specific cell

Correspondence: Dr. Eduardo Palomino, Walker Cancer Research Institute, 110 E. Warren, Detroit, MI, USA.

312 E. PALOMINO

delivery, using the idea of carbohydrate receptors to route useful drugs to cell targets. The latter concept has been re-ascribed to the selectivity found in carbohydrate conjugates of ellipticine, 8-hydroxyquinoline, 6-mercaptopurine, etc. Recently, polymeric carbohydrates have been used in the development of polyvalent drugs, those that bind to a target site through multiple interactions, and in the delivery of xenobiotics through neoglycoprotein conjugates.

I. Introduction

Despite advancements in carbohydrate research and the fact that carbohydrates are now recognized as important players in numerous cell-cell interactions such as cell recognition, cell migration, and immune defense, the use of carbohydrates as direct drug delivery systems remains virtually unexplored.

Carbohydrates are ubiquitous entities in living systems present in most cell surfaces as free polysaccharides, or as conjugates with proteins or lipids. Outside the cell in mammals, simple carbohydrates, especially glucose, circulate freely providing, after their absorption, energy and building materials for metabolic operations. This process of absorption is cell-selective and sometimes limited to a few saccharides. For example, of the dozens of simple carbohydrates theoretically possible, glucose remains the staple food or 'clean fuel' for most cells; in fact, other carbohydrates are routinely transformed to glucose prior to their storage as glycogen in the liver. This specificity is used to advantage for cellular binding by some invasive bacteria such as Shigella flexneri and Escherichia coli. In fact, the colonic epithelial cells of guinea pigs were found to have an intestinal lectin active in binding and specific for glucose and fucose [1].

The regulation of glucose transport in humans is complex and involves two classes of membrane proteins [2]: (1) Na+-dependent glucose transporters which are involved in the active transport of glucose from the lumen of the small intestine and from the proximal tubule of the kidney in the process of reabsorption; and (2) the facilitative glucose transporters, formed by not fully characterized proteins, appear to be present on all cells. These transporters possess distinct physiological and biochemical properties which endow them with specific functions in the tissues in which they are expressed. These glucose-selective proteins and other proteins which exhibit selectivity for a variety of simple carbohydrates are the preferred choice for an emerging new form of cell-specific drug delivery system.

II. The stereochemical complexity of carbohydrates

Since Fischer's investigations on sugars' geometric isomerism, the stereochemical complexities of carbohydrates have long been recognized. As an academic exercise, the positional and spatial configurations of sugars have been assessed and their numbers compared to other key biological components such as amino acids and nucleosides [1]. In its simplest form, that is using geometric isomers, the dimeric conjugation of similar amino acids or nucleosides generates one peptide or one nucleotide, respectively. Such conjugations are shown schematically in Fig. 1 (a,b). A similar approach using a pyranose as the base monomer can give rise to 11 distinct disaccharides through diverse ether linkages (Fig. lc).

CARBOHYDRATE HANDLES 313

In addition to the easily accountable isomeric combinations, a number of conformers are possible (Fig. ld), stemming from the flexibility of the pyranose ring of which the boat and chair conformations can be construed as limiting extremes. These conformational changes may influence such physical properties as solubility and polarity of the carbohydrate or carbohydrate conjugate. For example, the 3- and 6-glucuronide derivatives of morphine have been found to possess great lipophilicity. In fact, it has been postulated that much of the analgesic effect of morphine is due to the morphine 6-glucuronide (1) rather than to the parent drug [3].

The increased lipophilicity of 1 (Fig. 2) is explained through a combination of conformational changes in the sugar triggered by hydrogen bonding between the carboxylic group and the 6-OH alcohol. The net effect is a masking of the hydrophilic hydroxyl groups with concomitant decrease of water solubility. This

a.

Nucleoside Dinucleotide

b. ) Dipeptide

I Aminoacid 6

Pyranose Disaccharide Disaccharide

Fig. I. Schematic representation of geometric isomers of (a) nucleosides, (b) aminoacids, (c,d) sugars, including conformers.

314 E. PALOMINO

H ,\

HO 0 HO

( ~ F T "N~-CH3 OH \o/N

1

Fig. 2. Structure of morphine 6-glucuronide showing the intramolecular hydrogen bondings.

behavior, however, is pH-dependent and extended water-soluble conformations can be achieved at pH below 5.

The impressive number of geometric and stereochemical configurations that a pyranose ring can generate indicates the potential of such structures to encode biological information [4]. However, despite efforts to correlate the fact that, in some cases, carbohydrates modify the activities of their attached proteins, no clear evidence has emerged to confirm the hypothesis that the specificity of natural polymers depends more on their carbohydrate residues than on their sequence of amino acids or nucleotides [1].

BnO--~ / O

OBn~ OMe

OH

q ~ O M c

o,y.. s

Fig. 3. Schematic representation of the chemical conversion of D-xylose to 2-deoxy-xylose in 8 steps with loss of anomeric specificity.

C A R B O H Y D R A T E HANDLES 315

The extensive diversity of carbohydrate structures based on a single molecule might very well indicate that for each structure there exists a specific receptor. This theoretical specificity, based on carbohydrate stereochemical diversity, fits well with the classical concept of lock-and-key complementarity. F rom a synthetic point of view, however, this represents a t remendous challenge for its application in a model of cell-specific drug delivery system. Extensive protection-deprotection steps are thus required to preserve the stereochemical integrity of the sugar residue. For example, the modification of the simple sugar D-xylose (2) to 2-deoxy-xylose (3) (Fig. 3) requires a total of 8 steps, has an overall yield of 30% and renders a product that has lost its anomeric specificity [5].

It is based on the ever-increasing knowledge of the physiological behavior of carbohydrates that limited but significant progress is being made in the use of specific carbohydrates as cell adhesion inhibitors in a variety of cell-pathogen interactions [6], and, more significantly, at developing carbohydrate-based cell- specific drug delivery systems.

It is becoming more evident that the absorption of simple carbohydrates as well as the at tachment of carbohydrate-containing molecules to animal and human cells is mediated by carbohydrate-specific receptors, or lectins, present in the cell membrane.

III. Lectins: the carbohydrate receptors

The first mammal cell surface binding protein, or lectin, with high affinity for D- galactose and N-acetyl-D-galactosamine but no other carbohydrate was reported in hepatocytes by Ashwell and Morell in 1974 [7]. However, under the name 'phytohaemagglutinins ' plant lectins have been known for over 100 years and are still used extensively as biochemical tools in cell biology [8,9]. At the present stage of knowledge, isolated lectins are defined as proteins or glycoproteins of non- immune origin with high specific carbohydrate-binding affinity that have the ability to agglutinate cells and precipitate complex carbohydrates. Since the agglutination activity is based on specific carbohydrate recognition, it is usually inhibited by simple monosaccharides, but for some lectins, di-, tri-, or even higher carbohydrates are required.

Lectins are isolated from a number of sources in plants (seeds, bark, root, etc), fungi, bacteria, molluscs, body fluids of invertebrates, and from mammal ian cell

TABLE I

SOME C O M M O N L Y USED PLANT LECTINS

Lectin Source Affinity

Concanavalin A Black bean Glycine max Soybean Triticum vulgaris Wheat germ Tetragonolobus purpurea Lotus seed Viscum album Mistletoe Vigna radiata Mung bean Lens culinaris Lentil Lathyrus odoratus Sweet pea

~-D-Glucose, c~-D-mannose ~-Galactose N-Acetylglucosamine Fucose fl-D-Galactose C~-D-Galactose C~-D-Mannose ~-D-Mannose

316 E. PALOMINO

TABLE II

SOME ANIMAL LECTINS

Lectin source Sugar affinity Function

Human macrophages Peritoneal lymphocytes Chicken thymus Mouse peritoneal macrophages Rat cerebellum B 16 melanoma

Mannose Mannose-6-phosphate fl-Galactosides Galactose Mannose fl-Galactosides

Lectinophagocytosis Homing of lymphocytes Thymocyte maturation Killing of tumor cells Myelin compaction Metastasis

membranes. Their uses include a variety of in vitro applications such as blood grouping, mitogenic stimulation, fractionation of cells, and studies of normal versus pathological conditions in human tissues. Table I lists a few of the most commonly used plant lectins with their carbohydrate affinities.

In addition to the lectins present in hepatocytes, a number of lectins involved in cell recognition have been identified in viruses, protozoa, bacteria and animals [10]. A partial list of important animal lectins is presented in Table II with their best- characterized function.

Although the precise role of most lectins in nature is still unknown, in pathogenic organisms they serve as carbohydrate receptors for binding to the surface of host cells [ 11 ].

In most eukaryotic cells, lectins are present as glycoproteins and proteoglycans embedded in the glycocalyx, the peripheral carbohydrate-rich zone on the outside surface of the membrane. The complexity of some of these carbohydrate conjugates seems to be one of the forces behind such important processes as cell-cell and cell- matrix recognition [9].

The high concentration of cell-surface carbohydrates, the complexity and uniqueness of some of these oligosaccharide conjugates, and the fact that strong indirect evidence exists to link them as specific saccharide receptors warrant their acceptance as carbohydrate recognition elements.

IV. Tile concept of delivery by polyvalency

It has been suggested that in cell-cell interaction, lectins may not be the only receptors responsible for cell migration, immune defense, and many pathogen-host cell interactions that result in infection. The concept of polyvalency, or the binding to a target site through multiple interactions [12], has been proposed as a way to partially explain the enhanced affinity of some glycoprotein ligands. The idea is based on the potent anti-adhesive properties of a few natural polyvalent compounds such as equine ~2-macroglobulin, orsomucoid, fetuin (the glycoprotein of fetal serum), and mucin, the mucoprotein of ovine submaxillary glands [13]. As a result, the so-called polyvalent drugs are being developed, especially in the area of pathogen-cell adhesion, to arrest the infectivity of viral infections [14,15].

Most polyvalent drugs tested thus far are based on specific saccharide residues attached to polymeric materials, and some positive in vitro results have been

CARBOHYDRA T E HANDLES 317

obtained. For example, using everted sacs of guinea pig small intestine and colon it was demonstrated that the bioadhesion of methacrylamide co-polymers containing side-chains of fucosylamine was selective to the colonic tissue [16].

However, the additional non-specific forces such as hydrogen bonding and dipole-dipole interactions that are generated after the initial contact between the carbohydrate and its corresponding lectin - forces that may be responsible for the low dissociation rate of the complex and the success of the in vitro experiment - may be a hindrance in a multicellular in vivo experiment and could result in poor activity or undesirable effects. Regardless of their still-unknown practicality as drugs, multivalent ligands are proving to be extremely valuable as research tools [13].

It has been suggested that the lack of tight binding of the monosaccharides to the receptors is the reason behind the failure of simple carbohydrates to significantly inhibit cell-pathogen adhesion [13]. In order to decrease the dissociation constant of this interaction, chemical modifications of the simple carbohydrates can be implemented, eliminating the risk of diminishing specificity through multivariated binding. This approach has been utilized with some success in a number of drug delivery systems.

V. Delivery through simple carbohydrate handles

A number of pharmacologically important natural products contain carbohy- drate moieties attached as glycosides. Examples include anticancer agents such as the anthracycline antibiotics, bleomycins, and epipodophyllotoxin [17], cardioactive glycosides, antihemorraghic bioflavonoids, etc. The properties which these carbohydrates confer on the active species have been regarded as purely physical, and as such carbohydrate appendages have been used to improve water solubility and absorption. However, it is known that sugar moieties strongly influence drug bioavailability, permeability and selective toxicity toward tumor cells.

As delivery systems, carbohydrates could be involved in two general modes of action: (1) organ or site delivery, and (2) specific cell delivery. The former makes use of such biological parameters as the blood-brain barrier, for delivery of anti-AIDS agents through a non-glycosidic redox system [18], or the intestinal microflora for a glycosidic colon-specific drug delivery system using the high concentration of glycosidases produced by the symbiotic host [19]. The latter mode uses the idea of carbohydrate receptors to route useful drugs to cell targets. Obviously, this method is more far-reaching and has the potential of becoming the choice for reducing side effects as well as increasing drug effectiveness. Although a relatively new concept, this method may have been used inadvertently by researchers trying to take advantage of the hydrophilic properties of saccharides. In this light, the results of previous work, not conjectured originally to be the effect of selective cell uptake, will be analyzed.

An interesting study has been performed on ellipticine (4), and 9-methoxyellipti- cine (5), natural alkaloids with antitumor properties (Fig. 4) [20]. Although not targeted to elucidating carbohydrate receptors as much as improving bioavailability, the reported activity for 49 glycosides derivatives can be used to interpret the degree of binding to sugar receptors and the selectivity acquired by the new conjugates. Using two leukemia cell lines (L1210 and P388), a melanoma (B16), and a colon

318 E. PALOMINO

I

H

HO.....~. ~ ~

I

Rl

11o ]---" • tI0 OII O11 Oil

4- R=H I I O ~ O HO--'I .O. [

5- R--OCH 3 9 -R I= ~ 10-R l= W ()11

Fig. 4. Glycosides of ellipticine displaying the geometric variations of the sugars attached.

carcinoma (colon 38), it was found that pentoxides and deoxyhexosides with three hydroxy groups displayed more in vivo anti-L1210 activity than compounds with four hydroxy groups. This was more important than the configuration of the alcohol groups around the ring (compare glycoside configurations in 6 to 10). Among active species, the ~-u-arabinopyranoside (6) was slightly more active than its ~-L-lyxo counterpart (7), and the latter was more active than the t-rhamnopyranoside (8). Similarly, the D-lyxofuranoside (9) was more active than the fl-D-xyloconjugate (10). The presence of amido or acetate protecting groups rendered the molecules much less active, a fact that indicates the importance of free hydroxy groups for binding as well as for increasing the hydrophilicity of the active compound.

In comparative assays, the 5 selected ellipticine glycosides displayed more selectivity towards P388 than L1210, remarkably high inhibition values against colon 38 but poor affinity for melanoma B16. Given the fact that the only variation was the modification of the sugar, the selectivity and toxicity of ellipticine derivatives could be found in their affinities for cell surface lectins on the cells tested.

Similar results were partially disclosed for the variation of the in vitro activity of 8-hydroquinoline glucoside against two cancerous (L1210 and AML) and one normal cell line (CFU). Selectively higher activity was seen in L1210 cells than in CFU. However, 8-hydroxyquinoline alone was more toxic to AML than to L1210 [211.

Selectivity was also observed with the glucuronide of 6-mercaptopurine (11) (Fig. 5) which displayed no activity against a normal lung fibroblast but was an inhibitor of L1210 under the same in vitro conditions. Construed as the result of increased/% glucuronidase in the medium, the effect can be interpreted as the result of selective binding, given the fact that/%glucuronidase is not ubiquitous in all cancerous cells and is less prevalent in vitro than in vivo [22].

In vivo selectivity, shown by decreased toxicity to bone marrow cells, was obtained when mitomycin C was elaborated into the glucopyranosyl carbothio-

CARBOHYDRATE HANDLES 319

HO ~ 0 N ~

H H N ~ N

OH 11

O

; . r-o NH2 II II ,, oMo

J~ / ~ H r--OH

,5- R = OH 13 - R = NHAc R

Mc

~ ~ 14 - X = O

o 1 5 - X = S

- o

M e O ~ O M e

OMe

Fig. 5. Structures of 6-mercaptopurine glucuronide (il), mitomycin thioderivatives (12,13), and etoposides (14,15).

amides 12 and 13. Here, the introduction of an acetamido substituent in the sugar handle increased antitumor activity; in fact, 13 was 3 times as active as 12 against leukemia P388 [23]. This observation can be interpreted as the result of a better fit of the sugar to the cell receptors.

Slight changes in the sugar composition can produce substantial alterations in activity. Etoposide (14), for instance, when modified into the enzymatic resistant thioglycoside (15) becomes more active in two leukemia models (P388 and L1210) without changing its activity profile against a solid tumor (B16) [24,25]. This discrimination cannot be ascribed to variations in lipophilicity alone but also to a combination of increased resistance to hydrolysis with alterations in the geometrical disposition of the sugar moiety about the aglycone that changes the binding profile with respect to the receptor.

Carbohydrate appendages can be linked to the active molecules through C-C bonds (C-glycosides), C -N bonds (N-glycosides), C-S bonds (thioglycosides), or C- O bonds (O-glycosides). The most abundant natural glycosides are of the C-O type for which there are specific hydrolytic glycosidases. Modifying the type of linkage of a particular glycoside can improve the effectiveness of the drug by reducing its hydrolytic breakdown, and, in some cases, the inherent increase in hydrophobicity enhances binding to the lectin receptor.

Using the above-mentioned concept, a series of C-c~-mannopyranosyl derivatives (16-19) with affinity for the lectin receptor of Escherichia coli were synthesized and

320 E. P A L O M I N O

HO

HO

16 - R = CH 3

17 - R = COOH

18 - R = NH3 +

19- R = CH2OH

HO HO

o

H N ~ N H

20 21

Fig. 6. Several C-c~-mannopyranosyl derivatives.

their inhibition of pathogenic adhesion to yeast cells measured in an in vitro assay (Fig. 6) [26].

Compared to methyl-~-D-mannopyranoside, a known inhibitor, compound 16 was one order of magnitude more active. The inhibitory activity of the other derivatives decreased in the order 19 > 18 > 17, a result that is inversely related to the polarity of the molecules. The delicate stereochemical requirements of the carbohydrate receptor, on the other hand, were suggested by the lack of activity of 20, the/~-isomer of compound 16.

The selective activity found in 16, assigned by the authors as the side-chain hydrophobic effect, encouraged the design of 21, a biotin-linked derivative of mannose, to be used as a secondary receptor for avidin and streptavidin. The bifunctional derivative 21 showed the same activity as 16. However, the conjugate formed between 21 and avidin displayed the highest inhibitory activity at a concentrat ion three orders of magnitude lower than that of methyl-a-D- mannopyranoside.

VI. Delivery through neoglycoprotein conjugates

Conjugates of bovine serum albumin (BSA) and carbohydrates have attracted some attention as drug delivery systems. Based on the principle of carbohydrate recognition, delivery of xenobiotics is believed to proceed by cell endocytosis. This system has been used especially in host macrophages and liver cells.

In one methodology, BSA was joined through a thioether linkage to galactose, glucose, and mannose, and their uptake and disposition in liver cells was measured in an in vivo assay [27]. Selectivity was observed between parenchymal cells and galactose and glucose residues, whereas non-parenchymal cells recognized mannose. This method was extended to the production of glycosylated superoxide dismutase and dextran derivatives.

Another BSA-carbohydrate methodology makes use of the specific mannose

C A R B O H Y D R A T E HANDLES 321

selectivity of macrophages [28]. The conjugate formed from BSA, mannose and methotrexate (MTX) was tested in vitro on Leishmania-infected macrophages obtaining a 100-fold increase in inhibition compared to MTX alone. Similar results were obtained in vivo when the treatments were given after infection. However, the effectiveness of the therapy decreased when treatment was administered 15 days after infection.

A new method for delivery of antisense oligonucleotides takes advantage of the internalizing abilities of the BSA mannose conjugate in macrophages [29]. A 20-fold increase in oligonucleotide uptake was observed using labeled conjugates when compared to the oligonucleotide alone. However, no data on the activity of the actual antisense material was provided for this system which relies on an undisclosed mechanism for release of the oligonucleotide after internalization.

VII. Perspective

Despite efforts from different laboratories, there is not yet an established method of cell-specific drug delivery. Some success has been achieved in creating systems for organ-specific drug delivery ii.e. brain, colon), but these are based on the general physicochemical properties of the prodrugs involved, such as solubility, differential rates of degradation, etc., and not on the inherent characteristics of the target cells.

The use of monoclonal antibody conjugates, a promising method of drug delivery, relies on a strong knowledge of cell biology and presents obvious theoretical limitations to the type of materials that can be coupled to the delivery model. The potential of carbohydrates as cell-specific drug delivery systems, on the other hand, is unlimited since it combines cell recognition patterns with wide chemical applicability.

Carbohydrate-bound xenobiotics can be viewed as prodrugs when bound through a fissionable C-O bond. However, contrary to the concept of prodrugs, it has been shown that many of these O-glycosides possess selective activity. The drugs can be linked to the sugar through C N, C-C, or C S bonds, making the modified compound more resistant to enzymes and changes in pH without affecting the main activity of the underlying drug.

Two main obstacles to this method hinder its development: (1) limited knowledge about carbohydrate cell receptors, and (2) complex and limited synthetic methodologies [21]. The first limitation can be partially addressed by measuring the uptake of carbohydrate-bound xenobiotics in different types of isolated cells.

The second limitation represents a major hindrance to the development of the system. Unlike protein or oligonucleotide syntheses, there is not an automated or simplified method for synthesizing glycosides. Thus, each modified drug must be specifically designed and its synthesis executed in a stereochemically selective fashion. This limitation has made the use of semisynthetic bio-organic methods more attractive and valuable to the point that even slight modifications to a relatively known technique have merited patent rights [30].

In spite of the limitations, newly emerging synthetic methodologies coupled with developments in cell biology surely warrant a promising future for carbohydrates as cell-specific drug delivery systems.

322 E. PALOMINO

References

1 Sharon, N. and Lis, H. (1989) Lectins as cell recognition molecules. Science 246, 227 246. 2 Burant, C.F., Sivitz, W.I., Fukumoto, H., Kayano, T., Nagamatsu, S., Seino, S., Pessin, J.E. and Bell,

G.I. (1991) Mammalian glucose transporters: Structure and molecular recognition. Recent Progr. Hormone Res. 47, 349 388.

3 Carrupt, P.A., Testa, B., Bechalany, A., E1Tayar, N., Desca, P. and Perrisoud, D. (1991) Morphine 6- glucuronide and morphine 3-glucuronide as molecular chameleons with unexpected lipophilicity. J. Med. Chem. 34, 1272 1275.

4 Sharon, N. (1980) Carbohydrates. Sci. Am. 243 (Nov.), 90-116. 5 Dyatkina, N.B. and Azhayev, A.V. (1984) Aminonucleosides and their derivatives. XII. A new

synthesis of 1-O-methyl-3-azido-2,3-dideoxy-D-ribofuranose. Synthesis 961 963. 6 Rao, B.N.N., Moreland, M. and Brandley, B.K. (1991) The potential of carbohydrates as cell

adhesion inhibitors. Med. Chem. Res. 1-8. 7 Neufeld, E.F. and Ashwell, G. (1980) Carbohydrate recognition systems for receptor mediated

pinocytosis. In: W.J. Lemarz (Ed.), The Biochemistry of Glycoproteins and Proteoglycans, Plenum Press, New York, pp. 241 261.

8 Kennedy, J.F. and White, C.A. (1983) Bioactive Carbohydrates, Wiley & Sons, New York, pp. 209- 227.

9 Alberts, B., Bray, D., Lewis, J., Raft, M., Roberts, K. and Watson, J.D. (1989) Molecular Biology of the Cell. Garland Publ., New York.

10 Shen, T.Y. (1987) Saccharide determinants in selective drug delivery. Ann. NY Acad. Sci. 272-280. 11 Sharon, N. and Lis, H. (1989) Lectins. Chapman & Hall, London. 12 Matrosovich, M.N. (1989) Towards the development of antimicrobial drugs acting by inhibition of

pathogen attachment to host cells: a need for polyvalency. FEBS Lett. 252, 1-4. 13 Pritchett, T.J. and Paulson, J.C. (1989) Basis for the potent inhibition of influenza virus infection by

the equine and guinea pig ~2-macroglobuline. J. Biol. Chem. 264, 9850-9858. 14 Sparks, M.A., Williams, K.W. and Whitesides, G.M. (1993) Neuraminidase-resistant hemagglutina-

tion inhibitors: Acrylamide copolymers containing a C-glucoside of N-acetylneuraminic acid. J. Med. Chem. 36, 778 783.

15 Nagy, J.O., Wang, P., Gilbert, J.H., Schaefer, M.E., Hill, T.G., Callstrom, M.R. and Bednarski, M.D. (1992) Carbohydrate materials bearing neuraminidase-resistant C-glycosides of sialic acid strongly inhibit the in vitro infectivity of influenza virus. J. Med. Chem. 35, 45014502.

16 Rathi, R.C., Kopeckova, P., Rihova, B. and Kopecek, J. (1991) N-(2-Hydroxypropyl) methacryl- amide copolymers containing pendant saccharide moieties: Synthesis and bioadhesive properties. J. Polym. Sci. A 29, 1895 1902.

17 Cassady, J.M. and Duros, J.D. (Ed.) (1980) Anticancer Agents Based on Natural Product Models. Academic Press, New York.

18 Palomino, E., Kessel, D. and Horwitz, J.P. (1992) A dihydropyridine carrier system for delivery of 2',3'-dideoxycytidine (DDC) to the brain. Nucl. Nucl. 11, 1639-1649.

19 Friend, D.R. and Chang, G.W. (1984) A colon-specific drug-delivery system based on drug glycosides and the glycosidases of colonic bacteria. J. Med. Chem 27, 261 266.

20 Honda, T., Kato, M., lnone, M., Shimamoto, T., Shima, K., Nakanishi, T., Yoshida, T. and Nogushi, T. (1988) Synthesis and antitumor activity of quaternary ellipticine glycosides, a series of novel and highly active antitumor agents. J. Med. Chem. 31, 1295 1305.

21 Palomino, E., Walker, E.H. and Blumenthal, S.L. (1991) Carbohydrate prodrugs: potential use for in situ activation and drug delivery. Drugs Future 16, 1029 1037.

22 Parker, A. and Fedor, L. (1982) Ammonium 7H-purin-6-yl-l-thio-/~-D-glucopyranosiduronate, a latent, selective anticancer agent. J. Med. Chem. 25, 1505-1507.

23 Talebian, A., Green, D., Hammer, C.F., McPherson, E. and Schein, P.S. (1990) New sugar mitomycin C analogues: preparation, routine P388 antitumor activity, and leukopenia induction. J. Pharm. Sci. 79, 1105 1108.

24 Showalter, H.D.H., Winters, R.T., Sercel, A.D. and Michel, A. (1991) Facile synthesis of thioglucose analogs of the anticancer agent etoposide. Tetrahedron Lett. 32, 2849 2852.

25 Rose, W.C., Basler, G.A., Mamber, S.W., Saulnier, M., Casazza, A.M. and Carter, S.K. (1988) Antitumor activity profile of a novel water-soluble etoposide analog (BMY-40481). Proc. Am. Assoc.

CARBOHYDRATE HANDLES 323

Cancer Res. Ann. Meet. 29, 353. 26 Bertozzi, C. and Bednarski, M. (1992) C-Glycosyl compounds bind to receptors on the surface of

Escherichia coli and can target proteins to the-organism. Carb. Res. 223, 243 253. 27 Ohtsubo, Y., Ohno, J., Nishikawa, M., Fujita, T., Hashida, M. and Sesaki, H. (1991) Hepatic

targeting of proteins utilizing carbohydrate recognition mechanism. Drug Delivery Syst. 6, 13-17. 28 Chakraborty, P., Basu, N., Seth, R. and Das, P.K. (1991) Macrophage directed delivery of

antileishmanial drug through a specific receptor system. Indian J. Chem. 30B, 127-132. 29 Bonfils, E., Depierreux, C., Midoux, P., Thuong, N.T., Monsigny, M. and Roche, A.C. (1992) Drug

targeting: synthesis and endocytosis of oligonucleotide neoglycoprotein conjugates. Nucl. Acid Res. 20, 4621 4629.

30 Borman, S. (1993) Patent grants broad protection to enzymatic carbohydrate synthesis. C&E News, March 29, 24 27.