615th MEETING, BELFAST
39 1
Table 3. Half-life of elimination
Compound
Dose (mg/kg)
Rat
Dog
ICI 125,436
1CI 116,258
1
0. I
50
26
57
26
0-methyltransferase, confirming that the metabolic
deactivation pattern was identical to that observed for
the natural catecholamines.
The stability of ICI 1 16,258 was examined in considerable detail, both in aqueous or buffered aqueous media
and in organic solvents. The main degradative route in
aqueous formulations concerned ester hydrolysis, which
was minimal at increasing in both the acid and the alkaline
direction. Satisfactory shelf-lives, with less than 10%
decomposition in 2 years at 23”C, were easily achieved by
such aqueous formulations. In organic solvents an oxidative, probably peroxide-mediated, decomposition
pathway was also encountered. Satisfactory storage
characteristics were observed in the untreated alcohols
ethanol, isopropanol and t-butanol; however, in order to
realize satisfactory stability in benzyl alcohol and carbitol
it was necessary to free these solvents from peroxide
contaminants. Thus, the prodrug showed more than
adequate stability profiles in marked contrast to those
observed for the parent drug.
The penetration of H3-labelled ICI 125,436 through
stratum corneum in vitro was determined directly using a
skin cell (Scheuplein & Ross, 1974). The rate of penetration was formulation dependent with a basal rate of
0.68 ng/cm2 per h from a methanol deposited film being
observed. The penetration was also characterized by a
lag phase of up to 109 h. Percutaneous absorption rates
for ICI 116,258 increased by up to 20-fold were observed
in the presence of the penetration enhancer I-dodecylhexahydro-2H-azepin-2-one (azone) at 5% concentration.
In a comparative experiment between ICI 116,258 and
[‘4C]fluocinolone acetonide, (‘Synalar’) ICI 1 16,258
showed a superior percutaneous absorption rate which
was 3.5 times that of the steriod.
As all the biological data so far reported have been
accumulated in rodents, it was of considerable interest
to investigate the anti-inflammatory properties of the
p-stimulants in species, whose skin was more akin to
human skin; to this end the domestic pig was selected
(Bilski & Thomson, 1984). Five female domestic pigs
were used for each experiment; six pairs of sites were
identified on either sides of the backbone of each pig. The
drug or control vehicle was applied in a properly randomized fashion to the test sites; 24 h later, increasing
doses ( l % , 5% and 10% in acetone) of oxazolone were
applied to both the standard and experimental sites. This
was immediately followed by reapplication of the formulation or vehicles to their respective sites. Thirty hours
later, the lesions were assessed for erythema, area and
transepidermal water loss.
Using the Wilcoxon Rank Test for statistical analysis of
the results, the various treatment regimes were compared. Thus, on the severe and very severe lesions treatment with ‘Synalar’ or with a 1% formulation of ICI
1 16,258 containing 3.5% of azone were indistinguishable.
The cardiovascular safety of the /?-stimulant drugs was
also investigated in pigs, a topical dose of the drug in an
appropriate occluded formulation was administered, and
any cardiovascular response monitored by remote
telemetry. ICI 116,258, when applied topically to porcine
skin under conditions promoting maximal penetration of
the drug, failed to elicit an increase in heart rate.
Bilski, A. J. & Thomson, D. S. (1984) Br. J. Dermatol. 111 (Suppl. 27),
143- 144.
Bourne, H. R., Lichtenstein, L. M., Melmon, K . L., Henney, C. S.,
Weinstein, Y. & Shearer, G. M. (1974) Science 184, 19-28
Das, N. S., Chowdary, T. N., Sobhanadri, C. & Rao, K. V. (1978) Br.
J . Dermatol. 99, 197-200
Djurup, R. (1981) Allergy 36, 289-307
Evans, D. P., Hassack, M. & Thomson, D. S. (1971) Br. J. Pharmacol.
43, 403408
Glenn, E. M., Bowman, B. J. & Rohloff, N. H. (1978) Agents Actions
8, 497-503
Green, A. P., Mangan, F. R., Thomson, M. J., Randall, K. E. &
Boyle, E. A. (1984) J. Pharm. Pharmacol. 36, 314-317
Harkonen, M., Hopsu-Havu, V. K. & Raij, K. (1974) Acta. Derm.
Venereol. 54, 13-18
Kaplan, R. J., Daman, L., Shereff, R., Rosenberg, E. W. & Robinson,
H. (1976) Arch. Dermatol. 112, 88C881
Kaplan, R. J., Daman, L., Rosenberg, E. W. & Feigenbaum, S.
(1978) Arch. Dermafol. 114, 6 M 2
Keahey, T. M. &Greaves, M. W. (1980) Arch. Dermafol. 116,174-177
Lowe, N. J., Stoughton, R. B., McCullough, J. L. & Weinstein, G. D.
(1981) Arch. Dermatol. 117, 394-398
Scheuplein, R. J. & Ross, L. W. (1974) J. Invest. Dermatol. 62,
353-360
Sniegoski, P. J. (1976) J. Org. Chem. 41, 2058-2061
Stawiski, M. A,, Powell, J. A,, Lang, P., Shork, A,, Duell, E. A. &
Voorhees, J. J. (1975) J. Invest. Dermatol. 64, 124-127
Voorhees, J. J., Kelsey, W., Stawiski, M., Smith, E., Duell, E. A.,
Haddox, M. & Goldberg, N. (1973) in The Role of Cyclic Nucleotides
in Carcinogenesis (Schultz, J. & Gratzner, H. K., eds.), pp. 325-373,
Academic Press, New York
Yoshikawa, K., Adachi, K., Halprin, K. M. & Levine, V. (1975) Br. J.
Dermatol. 93. 253-258
Synthetic polymers as carriers for chemotherapeutic agents
JOHN B. LLOYD,* RUTH DUNCAN*
and J. KOPECEK?
*Biochemistry Research Laboratory, University of
Keele, Keele, Stagordshire ST5 5BG, U.K., and
?Institute of Macromolecular Chemistry,
Czechoslovak Academy of Sciences, 16206 Prague,
Czechoslovakia
Ten years have passed since the publication of Ringsdorf s
seminal article on pharmacologically active polymers. In
that paper (Ringsdorf, 1975) he forsaw in outline most of
the developments that have since been made in this now
burgeoning area, recently the subject of a major symposium (Tirrell et al., 1985). The entrapment of drugs
Vol. 14
within insoluble bioerodible matrices has led to elegant
approaches to controlled and sustained drug release
(Heller, 1985; Sanders, 1985), and some macromolecules
such as the polyanions appear to have biological activity
in their own right (Regelson, 1979; Ottenbrite & Kaplan,
1985). Neither of these developments, however, has a
legitimate place in a colloquium on prodrugs. In contrast, Ringsdorf s concept of the soluble drug-polymer
conjugate, equipped with a homing device to seek out its
target cells and deliver its charge of drug there, is a
sophisticated subset of the prodrug.
Macromolecules introduced into the body are handled
very differently from small molecules. Unlike the latter
they cannot in general pass across cell membranes;
392
nor, if their molecular size is above the so-called renal
threshold, are they lost from the bloodstream into the
glomerular filtrate. Their movement into cells or from
one body compartment to another, although severely
limited by these considerations, is achieved however by
means of vesicular transport. This ubiquitous
phenomenon begins with the formation of small vesicles
by the invagination of a cell’s plasma membrane. A
stream of these vesicles, each containing the fluid bathing
the cell, together with any contained solutes, is constantly
entering most cell types, a process known as pinocytosis
or endocytosis. The vesicles have two principal destinations. In some cases they traverse the cell, fuse with the
plasma membrane on the opposite side, and thus disgorge
their contents into the ambient fluid there. This translocation of vesicles and their contained fluid and solutes
is known as transcytosis, and is of particular importance
in the capillary endothelium. It explains how soluble
macromolecules can find their way out of the circulation
and into the extracellular space of many organs and
tissues. In most cells, however, the vesicles forming at the
plasma membrane fuse with the lysosomes, where their
contents are subject to attack by the lysosomal acid hydrolases. Physiological macromolecules such as proteins,
polysaccharides and polynucleotides are digested in the
lysosome to their monomer units, which like many small
molecules are able to pass across its membrane into the
general cytoplasm. Non-digestible macromolecules remain
within the lysosome, usually for the lifetime of the cell.
Ringsdorf (1975) recognized the possibility of harnessing these concepts of cell biology in the cause of
targeted drug delivery. By attaching potent drugs to an
inert macromolecule by a ‘spacer’ cleavable only in the
lysosome, one could restrict their uptake to ‘those cells
which display high endocytic activity’. Recent work
on the substrate-specificity of pinocytosis has further
increased the potential of this approach. The incorporation of certain chemical features into a macromolecule
can enormously enhance its rate of uptake by cells, by
causing it to adhere to the plasma membrane being
internalized (Lloyd & Williams, 1984; Lloyd et al.,
1984~).Of still greater importance is the recognition that
this adsorptive uptake of macromolecules is in some
cases cell-type-specific. The clearest example known is
the specific uptake of asialoglycoproteins by mammalian
hepatocytes. Other cell-types in the body appear not to
possess the galactose-recognizing receptor in high density. It is obvious how discoveries like this could become
the basis for effective ‘homing devices’ for drug-macromolecule conjugates.
In recent years we have developed a targetable drugconjugate based on the soluble polymer polyhydroxypropylmethacrylamide. This molecule can be synthesized
with some of the hydroxypropyl groups replaced with
drug and some with targeting moieties. The parent
homopolymer has little affinity for cell membranes, and
so is not captured by adsorptive pinocytosis unless
substituted with appropriate groups. Hydrophobic sidechains lead to non-specific uptake by cells, whereas
substitution with a galactos(amin)e-terminating sidechain leads to specific uptake into the lysosomes of the
parenchymal cells of the liver. Drug analogues and, more
recently, anti-cancer drugs have been attached to the
polymer through oligopeptide ‘spacers’. Spacer design
aims to achieve stability in plasma but drug release on
exposure to the lysosomal peptidases. Satisfactory
spacers, mostly tri- and tetra-peptides, have been devised,
with the precise composition varying with the nature of
the drug bound. The conjugates have rather low immunogenicity. This work has been reviewed fully recently
(Duncan et al., 1984; Lloyd et al., 1984a,b; KopeEek
BIOCHEMICAL SOCIETY TRANSACTIONS
et al., 1985), and so is not described here in more detail.
Work in progress aims to achieve selective toxicity
against tumour cells in vitro and in vivo, using antibodies
or antibody fragments as the targeting moiety.
Although Ringsdorf (1975) developed his concept of
the drug-macromolecule conjugate in the context of synthetic polymers, it is no less applicable using natural
macromolecules. Schneider et al. (1984) have conjugated
daunorubicin to human serum albumin by a tetrapeptide
spacer and achieved targeting to both hepatocytes and
some hepatomas, by attaching galactose residues (in the
form of lactose) to the protein. Human serum albumin
to which an anti-cancer agent and a tumour-directed
monoclonal antibody are attached is discussed in another
contribution to this Colloquium (Embleton, 1986). Synthetic and natural macromolecules each have their advantages and disadvantages, their advocates and detractors.
Synthetic polymers can be tailor-made to pre-determined
chemical specifications and are easier to prepare in
quantity. In general the chemistry of drug and targeting
moiety attachment is simpler with synthetic macromolecules, and they are also more robust and probably less
immunogenic than natural macromolecules such as polypeptides. On the other hand, they are less biodegradable,
although we have described how limited biodegradability
may be conferred on polyhydroxypropylmethacrylamide
copolymers (KopeEek et al., 1985).
There has been considerable progress in realizing the
concept of the targeted macromolecular drug-conjugate,
but its clinical utility is not yet assured. The design of
suitable drug-macromolecule linkages has been achieved,
and problems of biotolerance do not appear serious. The
feasibility of targeting remains the major uncertainty.
Cell-specific determinants for pinocytosis are not yet
known for most cell-types in the body, and even the
specificity of so-called tumour-specific antigens is far
from complete. Nevertheless, since most drugs in use
today are totally untargeted, even partial succcess would
be a considerable advance, particularly in cancer chemotherapy where side-effects are notoriously severe.
We thank the Cancer Research Campaign for their support of our
work, and the Royal Society and the British Council for several travel
grants.
Duncan, R., KopeEek, J. & Lloyd, J. B. (1984) Biochem. Soc. Trans.
12, 913-915
Embleton, M. J. (1986) Biochem. Soc. Trans. 14, 393-395
Heller, J. (1985) Ann. N . Y . Acad. Sci. 446,51-66
KopeEek, J., Rejmanova, P., Duncan, R. & Lloyd, J. B. (1985) Ann.
N . Y. Acad. Sci. 446,93-103
Lloyd, J. B. & Williams, K. E. (1984) Biochem. Soc. Trans. 12,
527-528
Lloyd, J. B., Duncan, R. & KopeEek, J. ( 1 9 8 4 ~ )Pure Appl. Chem. 10,
1301-1 304
Lloyd, J. B., Duncan, R., KopeEek, J. & Rejmanova, P. (19846) in
Receptor-Mediated Targeting of Drugs (Gregoriadis, G . , Poste, G.,
Senior, J. & Trouet, A., eds.), pp. 417425, Plenum Press, New
York, London
Lloyd, J. B., Pratten, M. K., Duncan, K., Kooistra, T. & Cartlidge,
S. A. (1984~)Biochem. Soc. Trans. 12, 977-978
Ottenbrite, R. M. & Kaplan, H. M. (1985) Ann. N . Y . Acad. Sci. 446,
16C168
Regelson, W. (1979) J . Polymer Sci.Polymer Symp. 66, 483-538
Ringsdorf, H. (1975) J . Polymer. Sci.Polymer Symp. 51, 135-153
Sanders, H. J. (1985) Chem. Eng. News (1 April), 3 W 8
Schneider, Y.-J., Abarca, J., Aboud-Pirak, E., Baurain, R., Ceulemans,
F., Deprez-de Campaneere, D., Lesur, B., Masquelier, M., OtteSlachmuylder, C., Rolin-van Swieten, D . & Trouet, A. (1984) in
Receptor-Mediated Targeting of Drugs (Gregordiadis, G . , Poste, G.,
Senior, J . & Trouet, A,, eds.), pp. 1-25, Plenum Press, New York,
London
Tirrell, D. A,, Donaruma, L. G. & Turek, A . B. (eds.) (1985) Macromolecules as Drugs and as Carriers for Biologically Active Materials.
Ann. N . Y . Acad. Sci.vol. 446
1986