Pharmaceutically active conjugates having improved body tissue binding specificity

Pharmaceutically active conjugates comprising a pharmaceutically active substance for treating a disorder of the body that involves a specified body tissue conjugated directly or indirectly with at least one fragment of an adhesive glycoprotein such as fibronectin, the said glycoprotein fragment(s) having improved binding specificity compared with the parent protein for the said body tissue.

This invention relates to pharmaceutically active conjugates having 
improved binding specificity for specified body tissue. The invention 
further relates to methods of preparing these conjugates and to 
pharmaceutical preparations that contain them. In particular embodiments, 
the invention relates to pharmaceutically-active conjugates containing 
either plasminogen activators or anti-rheumatic drugs. 
It is common practice to administer large doses of various drugs to the 
body to treat conditions which may only affect a small region of the body. 
Large doses are often required in order to attain a sufficient 
concentration of the drug at the target area. However, these large and 
constantly-administered doses can produce serious side effects in patients 
being treated, which has often lead to treatment being suspended even 
though improvement in the treated condition might be taking place. This 
problem has existed in the past particularly in the administration of 
toxic anti-tumour drugs for destroying cancer cells and in the 
administration of toxic anti-rheumatic drugs, especially gold compounds. 
One further example of the administration of drugs where this problem 
exists is in the treatment of thrombotic conditions. Conventional 
treatment for certain thrombotic conditions, such as deep vein thrombosis 
or pulmonary thrombosis, involves continuous infusion of agents which 
stimulate fibrinolysis Fibrinolysis is the term given to the process of 
proteolytic degradation of a fibrin-based clot. Thrombolysis is the 
plasmin mediated proteolysis which brings about the break up of large 
vascular obstructions. It is generally accepted that the main enzyme 
responsible for fibrinolysis is plasminogen-plasmin. The zymogen, 
plasminogen, is converted by one of a range of activators to plasmin, 
which is an active protease capable of degrading a cross-linked fibrin 
clot to soluble products (FDPs). Plasmin is a serine protease, produced 
from plasminogen by limited proteolytic cleavage accompanied by a 
conformational change. 
The precise mechanism of plasminogen activation depends on the plasminogen 
activator involved. Thus, activation can also occur without proteolytic 
cleavage in the case of plasminogen activators from certain microorganisms 
which act allosterically, e.g. streptokinase. In general activation is 
more effective if it occurs on the surface of the clot, a phenomenon aided 
by the affinity that plasminogen has for fibrin. Plasminogen activators 
occur in blood, a variety of tissues and in body fluids such as urine, 
saliva and semen. As hereinbefore indicated, they are also produced by 
certain microorganisms. Small quantities of a labile plasminogen 
activator, tissue plasminogen activator (t-PA), occur in the circulation. 
Its level is raised following stimuli including exercise and venous 
occlusion. t-PA, which carries a fibrin-binding region, has been isolated 
from cadaveric plasma and from culture medium of vascular endothelial 
cells and the Bowes melanoma cell line. More recently, this plasminogen 
activator has also been prepared by recombinant DNA technology (see, for 
example, GB-A No. 2119804). 
Urokinase (UK) is a plasminogen activator present in urine. Unlike t-PA, it 
can be readily isolated in a highly purified, crystalline form. It is a 
single chain .beta.-globulin which exists in two forms of molecular 
weights 54,000 and 32,000 respectively. UK is (apparently) synthesised in 
the kidney and, unlike t-PA, it has little affinity for fibrin. UK 
activates plasminogen to produce plasmin which is subject to inhibition. 
Streptokinase (SK) is a plasminogen activator produced by .beta.-haemolytic 
streotococci and available in purified preparations. SK is a single chain 
.alpha.2-globulin having a molecular weight around 46,000. It acts as an 
activator by binding to plasminogen causing a conformational change. The 
resultant SK-plasminogen complex possesses enzymic activity, but is not 
inhibited by .alpha.2-macroglobulin. 
Both UK and SK have been used successfully as thrombolytic agents. SK is 
more commonly used since it is cheaper to produce, although UK is 
generally regarded as a better agent. SK is highly antigenic and tends to 
be effective for only one treatment, after which the number of antibodies 
raised by the body's autoimmune system render subsequent treatments much 
less effective. In addition, the titre of anti-SK antibodies varies 
between individuals depending on the previous history of Streptococcal 
infections. This makes it difficult to determine the effective safe dose. 
Antibody mediated resistance does not occur with UK. 
Although UK has no antigenicity, UK therapy suffers from the dual 
disadvantages of limited availability and high cost, consequent on the 
need to fractionate very large volumes of human urine. The huge dose of UK 
necessary to maintain a thrombolytic state is thought to be partly due to 
its poor affinity for fibrin. The result is that a complete course of 
treatment requires the fractionation of around 5000 liters of urine. In 
the case of both UK and SK therapy, there is a serious risk of 
haemorrhagic complication due to the systemic administration of large 
doses of activator. Attempts to achieve thrombolysis by local 
administration have not been encouraging. In the case of SK, it is thought 
that effective and haemorrhagic doses are almost the same, so that an 
effective dose can cause hyperplasminaemia, fibrinogen degradation, and an 
accumulation of fibrinogen degradation products which have an 
anticoagulant effect and so add to haemorrhagic complications. 
More recently, attempts have been made to overcome this problem by linking 
drugs to carriers which have a high affinity for the area of the body 
requiring treatment. These carriers target the drug on to the area 
requiring treatment and can thereby be administered in relatively low but 
still effective doses. Examples of such carriers are disclosed in 
published patent specifications EP-A No. 2-114685 and, more recently, U.S. 
Pat. No. 4,587,122, which describe the preparation and use of various 
anti-tumour, anti-bacterial and anti-inflammatory drugs covalently 
conjugated with whole fibronectin (an adhesive glycoprotein) using protein 
cross-linking agents. Although fibronectin is claimed to be an effective 
carrier which binds readily to morbid regions of the body where treatment 
by drugs might be required, fibronectin itself carries binding sites for a 
large number of body tissues and so preferential binding to a site of the 
body requiring treatment cannot be guaranteed; indeed, preferential 
binding may occur in other parts of the body in which case the amount of 
drug accumulating in the area requiring treatment may in some cases be 
negligible. Furthermore, there is some evidence to suggest that 
fibronectin normally accumulates at sites within the body by 
self-association on to initial, bound fibronectin. That is to say, 
self-association leads to non-specific amplification of fibronectin 
accumulation. Clearly, this mechanism would severely impair the 
"targetting" ability of administered fibronectin-drug conjugates since 
they could bind to any site of fibronectin accumulation within the body. 
The present invention is based on the concept of providing improved drug 
conjugates having targetting portions which have a higher relative degree 
of binding specificity for particular areas of the body where drug 
treatment is required and which have a reduced tendency to self-associate. 
The present invention thus seeks to provide a solution to the problem of 
how to fulfill this need by providing targetting portions of 
pharmaceutically-active conjugates in the form of protein fragments, e.g. 
generated by the enzymic digestion of adhesive glycoproteins, which have 
affinity for a specified body tissue involved in a bodily disorder. One 
advantage of employing these fragments is that it is possible to prepare 
adhesive glycoprotein fragments which have specificity or at least a high 
degree of selectivity for single body tissue types, whereas whole adhesive 
glycoproteins, such as intact fibronectin, usually have a broad range 
affinity for a number of body tissues. The use of protein fragments 
therefore makes it possible to prepare conjugates that will bind 
substantially only to the specified body tissue involved in the disorder 
being treated. Furthermore, it has been found using fibronectin as an 
appropriate model, that protein fragments have a reduced tendency to bind 
under physiological conditions to their parent glycoprotein, which 
enhances their specificity of binding within the whole body. 
According to a first aspect of the present invention, therefore, there is 
provided a pharmaceutically active conjugate comprising a pharmaceutically 
active substance for treating a disorder of the body that involves a 
specified body tissue characterised in that said pharmaceutically active 
substance is conjugated directly or indirectly with at least one fragment 
of an adhesive glycoprotein having improved binding specificity compared 
with the parent protein for the said body tissue. 
The glycoprotein will normally possess binding sites specific to at least 
two different tissues. 
Protein fragments suitable for a conjugate according to the present 
invention may be derived for example by protease digestion of a 
naturally-occurring adhesive glycoprotein or portion thereof, in 
glycosylated or non-glycosylated form, or from a genetically engineered 
equivalent thereof having the same amino acid sequence or the same amino 
acid sequence apart from one or more changes which do not affect binding 
specificity. Moreover, it will be appreciated that suitable protein 
fragments for a conjugate of the present invention may alternatively be 
prepared directly by recombinant DNA technology or chemical synthesis. The 
pharmaceutically active substance will preferably be covalently conjugated 
to the chosen protein fragment or fragments directly or indirectly through 
a cross-linking reagent or via a carrier molecule which may carry a number 
of molecules of an active substance as well as one or more fragments of an 
adhesive protein. 
A pharmaceutically active conjugate of this type can be administered by 
normal means (usually orally or intraveneously), and yet is more readily 
and efficiently targetted on to particular sites of the body which are 
involved in a specified bodily disorder. Such a conjugate will tend to 
bind with a high degree of selectivity to a site of interest readily and 
rapidly as it is carried past the site within the blood stream or another 
body fluid and so the tendency for competing side reactions to occur 
between the active substance in the conjugate and other body tissues will 
be reduced. This has the dual advantage of decreasing the dosage 
requirements for treating a specified disorder whilst at the same time 
reducing the extent of undesirable side effects that are frequently 
associated with the administration of large quantities of 
inefficiently-utilised pharmaceutically active substances to the body. 
Adhesive glycoproteins and certain digestion fragments thereof are known to 
have a strong affinity for certain proteins produced by the body. 
Preferred conjugates of the present invention are therefore of a type that 
are effective for treating disorders of the body involving an accumulation 
of tissue containing or consisting of protein matter. In this case, the 
pharmaceutically active substance may preferably comprise an agent for 
bringing about the proteolytic degradation of the accumulated protein. 
Where the accumulated protein consists of a fibrin-based clot, a suitable 
conjugate of the present invention will comprise a plasminogen activator, 
for example t-PA, streptokinase (SK) or urokinase (UK), conjugated to a 
protein fragment with a high degree of selectivity for binding to fibrin, 
preferably a fibronectin fragment with binding affinity for fibrin, but 
which unlike whole fibronectin substantially lacks gelatin or collagen 
binding affinity. Indeed, conjugates of the present invention have been 
found to be particularly effective when the pharmaceutically active 
substance is in the form of a protein or enzyme because these can be 
readily conjugated through covalent bonding to glycoprotein fragments. 
Further examples of preferred conjugates according to the present invention 
are anti-rheumatic drug-protein conjugates suitable for selectively 
targetting the chosen drug to articular tissue in joints affected by 
rheumatoid arthritis. This condition is characterised by chronic synovial 
inflammation leading to release and activation of collagenase with the 
result that collagen in the connective tissue of affected joints is broken 
down and denatured to form gelatin. Thus, a conjugate of the present 
invention for treatment of rheumatoid arthritis will comprise an 
anti-rheumatic drug such as gold, a gold compound or penicillamine, bound 
directly or indirectly to one or more protein fragments with a high degree 
of selectivity for gelatin-binding in the body. As hereinbefore indicated, 
fibronectin has a gelatin-binding domain and moreover has a higher 
affinity for gelatin (denatured collagen) than native collagen. 
Consequently, it is most preferred to employ as the protein fragment 
component of an anti-rheumatic drug conjugate of the present invention one 
or more fibronectin fragments comprising a gelatin-binding domain, but 
having substantially no fibrin-binding affinity. 
Other pharmaceutically active substances which may be conjugated with 
protein fragments to form a conjugate of the present invention are 
anti-tumour agents, anti-inflammatory agents and anti-bacterial agents 
which are in the main antibiotics. Examples of substances which can be 
conjugated are given in EP-A No. 2-0114685 and U.S. Pat. No. 4,587,122 and 
include daunomycin, mitomycin, cephalothin, penicillin G and secretin. Yet 
further examples of pharmaceutically active substances which may be 
conjugated with protein fragments to form conjugates according to the 
present invention are protein growth factors to promote, for example, 
localised wound repair and antibodies (e.g. of the IgM or IgG class). 
The one essential characteristic which is possessed by pharmaceutically 
active substances suitable for incorporation within conjugates of the 
present invention is their ability to combine with proteins, especially 
adhesive glycoprotein fragments, or non-toxic carrier molecules such as 
dextran either directly or via a linking group. They may, for example, 
contain a functional group such as an amino, carboxy or hydroxyl group. 
One particular active substance of interest for incorporation in 
conjugates of the present invention is gold, which can be conjugated 
directly with sulphydryl groups present in proteins. 
In order to increase the efficiency of delivery of a pharmaceutically 
active substance to a specified body tissue, a conjugate of the present 
invention may be prepared in which the active substance is loaded on to a 
non-toxic carrier molecule such as a dextran, preferably a protein such as 
fibronectin or albumin or a portion thereof, and conjugated directly or 
indirectly with one or more targetting protein fragments. The carrier will 
generally be covalently conjugated. Clearly, many adhesive protein 
fragments, for example from 1 to 20, especially from 1 to 5, may be 
conjugated to a single carrier molecule. 
Thus, it will be appreciated that where the chosen carrier is fibronectin 
or a high molecular weight portion thereof having a range of different 
binding specificities, these will be largely negated by the attachment of 
the active substance and protein fragments and will thus be prevented from 
substantially reducing the selectivity of the conjugate. Use of such a 
carrier is especially preferred for the preparation of anti-rheumatic 
conjugates of the present invention wherein gold or a gold compound is 
employed together with one or more gelatin-binding fibronectin fragments. 
Conventional administration of a toxic gold compound for treatment of 
rheumatoid arthritis has the disadvantage that undesirable side effects 
are liable to occur, particularly as a result of accumulation of gold in 
the liver and kidneys. However, by employing an anti-rheumatic conjugate 
of the present invention wherein gold or a gold compound is bound to both 
a carrier molecule and at least one targetting fibronectin fragment having 
a high degree of selectivity for gelatin binding in the body, a high 
concentration of gold can be achieved at arthritic joints with a 
substantially reduced risk of liver or kidney damage. Particularly 
preferred are conjugates of this type wherein gold per se, derived for 
example from aurothiomalic acid or a salt thereof, is directly conjugated 
to sulphydryl groups of a carrier protein. In such a conjugate, the 
targetting protein fragment(s), preferably one or more fibronectin 
fragments having gelatin-binding affinity, but substantially lacking 
fibrin-binding affinity, will be conjugated to the carrier by means of a 
cross-linking agent, e.g. cyanamide. Because of its high capacity for 
binding gold via sulphydryl groups, fibronectin or a portion thereof is 
especially preferred as the carrier molecule for a conjugate of this type. 
During preparation of such an anti-rheumatic conjugate employing whole 
fibronectin or a portion thereof having a gelatin binding domain, 
precautionary measures may be taken to protect the gelatin-binding 
affinity of the carrier protein of the final conjugate Thus, direct 
covalent conjugation of the carrier protein with gold may be carried out 
in the presence of gelatin, e.g. soluble gelatin or gelatin bound to an 
agarose-based support such as Sepharose*. Moreover, binding of the 
targetting protein fragment(s) may be carried out under mild conditions 
which do not destroy the gelatin-binding ability of the carrier protein. 
By taking such protective measures to ensure retention of a gelatin 
binding site in the carrier protein, the selectivity of the final 
conjugate will not be reduced and indeed it may be enhanced. .noteq.* 
trade mark 
The preparation of fragments of an adhesive glycoprotein having enhanced 
specificity compared with the parent protein for a single tissue type can 
be achieved by adopting the following procedural steps: 
(a) Select an adhesive glycoprotein having the desired tissue binding 
specificity; 
(b) Fragment the selected protein, preferably by enzymic digestion with a 
protease, e.g. trypsin, thrombin or cathepsin D; and 
(c) Select those fragments which have specific affinity or at least a high 
degree of selectivity compared with the parent protein for the body tissue 
involved in the disorder to be treated. 
In the case of preparation of a conjugate of the present invention wherein 
the pharmaceutically active substance is non-proteinaceous, e.g. gold or a 
gold compound, it will be understood that the active substance may be 
conjugated directly or via a cross-linking reagent to the adhesive protein 
selected in step (a) prior to proteolysis, the conjugation conditions 
being chosen so that the adhesive protein retains binding affinity for the 
tissue of interest. 
Step (c) is preferably conducted by affinity chromatography. Thus, the 
protein fragments resulting from proteolysis in step (b) may be subjected 
to affinity chromatography on an affinity support having specific binding 
affinity for the tissue binding site of interest or a closely associated 
non-tissue binding site. The bound fragments may then be eluted and, if 
necessary, one or more further affinity chromatography steps subsequently 
carried out to remove fragments which carry in addition to the tissue 
binding site of interest one or more additional tissue binding sites. The 
initial affinity chromatography step and any subsequent affinity 
chromatography steps may, for example, be carried out on an affinity 
support having immobilized thereon appropriate body tissue or simulated 
body tissue. For such an affinity support, the tissue or simulated tissue 
may be conveniently immobilized on an agarose-based support, e.g. 
Sepharose. 
Where the chosen protein for proteolysis has more than one tissue binding 
specificity, alternatively in order to obtain fragments having the desired 
enhanced specificity for a particular tissue type, step (c) may begin with 
subjecting the protein fragments from step (b) to one or more separation 
steps in which protein fragments having affinity for body tissues other 
than the tissue of interest are selectively removed. Each such separation 
step may be conveniently performed, for example, by affinity 
chromatography on immobilised body tissue (or simulated body tissue) for 
which binding affinity is not required in the fragments of interest. 
Finally, the desired fragments may be isolated by affinity chromatography 
employing a further affinity support with immobilised body tissue or 
simulated body tissue having affinity for the tissue binding site of 
interest or by employing an affinity support with specific binding 
affinity for a non-tissue binding site closely associated with the 
required tissue binding site. 
The size of the selected fragments will depend upon the size of the protein 
from which the fragments are generated, the method of protein 
fragmentation (which is preferably by proteolytic enzyme digestion), and 
on the extent and severity of the fragmentation method employed. 
One protein from which the fragments can be derived is the 
naturally-occurring adhesive glycoprotein fibronectin (m.w. 440,000), 
which is known to possess a wide range of binding sites, including binding 
sites for gelatin and fibrin, and can be readily digested into fragments 
by proteolytic enzymes. It is found in plasma and other body fluids and is 
associated with connective tissues, cell surfaces and basement membranes. 
Its wide variety of biological functions is attributed to a series of 
specific binding sites which bind it not only to gelatin and fibrin, but 
also to cell surfaces, glycosaminoglycans and other macromoleculaes. 
Plasma fibronectin is composed of two very similar, but non-identical 
polypeptide chains which are connected by a disulphide bond at the 
COOH-terminus and is more susceptible to proteolysis than other basement 
membrane and plasma proteins. Serine proteases cleave intact fibronectin 
initially at two preferential sites, releasing a short COOH-terminal 
fragment containing the interchain disulphides and an NH.sub.2 -terminal 
fragment, mw 27,000-30,000. This carries binding sites for fibrin, actin, 
S. aureus and heparin and a cross-linking site for plasma transglutaminase 
(factor XIIIa). The digestion proceeds yielding the binding domains shown 
below: 
______________________________________ 
Protein(1)(2)(3)(4)(5)(6)(7) 
Fragment 
##STR1## 
______________________________________ 
Fragment 
1 Binds to fibrin, heparin, actin and S. 
2 Binds to gelatin, collagens and fibronectin 
4 Binds to cells 
5 Binds to heparin 
6 Bind to fibrin 
Fibronectin fragments having binding affinity for fibrin in the absence of 
gelatin-binding affinity or gelatin-binding affinity in the absence of 
fibrin-binding affinity may be conveniently isolated from a mixture of 
fibronectin fragments, e.g. a protease digestion mixture of whole 
fibronectin, by an appropriate two stage affinity chromatography procedure 
employing a fibrin monomer-affinity support, e.g. fibrin 
monomer-Sepharose, and a gelatin-affinity support e.g. gelatin-Sepharose. 
Since the fibrin-binding sites of fibronectin are closely associated with 
heparin-binding sites, the fibrin monomer-affinity support in the 
above-indicated fragment purification procedures may be advantageously 
substituted by a heparin-affinity support, e.g. heparin-Sepharose, which 
has a higher capacity than fibrin monomer-sepharose and can be more 
readily prepared. Indeed, heparin-Sepharose may be obtained from 
commercial sources, e.g. Pharmacia A.B. 
The preferred molecular weight range of fibronectin fragments having fibrin 
binding specificity is from 25 to 400 kDa as measured by HPLC (high 
performance liquid chromatography) or from 25 to 200 kDa as measured by 
SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis), 
whereas the molecular weight range of fibronectin fragments which have 
gelatin binding specificity is preferably in the range 40 to 500 kDa as 
measured by HPLC or 40 to 200 kDa as measured by SDS-PAGE, the smaller 
fragments in these ranges having increased specificity. The higher 
molecular weights recorded when using HPLC are probably due to association 
of fragments before or during measurement which give rise to an increase 
in apparent molecular weights. 
Thus, according to a further aspect of the present invention, we provide a 
method of preparing a pharmaceutically active conjugate of the present 
invention which comprises conjugating a pharmaceutically active substance 
directly or indirectly to at least one fragment of an adhesive 
glycoprotein having improved binding specificity for a body tissue 
involved in the disorder to be treated compared with the whole adhesive 
protein or, where the pharmaceutically active substance is not a protein, 
conjugating the pharmaceutically active substance to the whole adhesive 
glycoprotein or a portion thereof, optionally with protection of binding 
sites specific to the said body tissue, followed by proteolysis to produce 
protein fragments carrying conjugated pharmaceutically active substance 
and selection of at least one of said fragments having improved binding 
specificity compared with the said protein or portion thereof. 
Thus, for example, an anti-rheumatic conjugate according to the present 
invention may be prepared by directly conjugating gold to sulphydryl 
groups of an adhesive protein possessing gelatin-binding affinity, e.g. 
whole fibronectin, followed by protease digestion and selection of a 
conjugate with improved selectivity over the parent conjugate for 
gelatin-binding in the body. Where fibronectin or a gelatin-binding 
portion thereof is chosen as the starting adhesive protein, gold 
conjugation will be carried out in the presence of gelatin, e.g. soluble 
gelatin or gelatin-bound to an agarose-based support, so as to protect the 
gelatin-binding site of the adhesive protein. 
In the case of a process according to the present invention where one or 
more fragments of an adhesive protein having improved binding specificity 
compared with the parent protein for the tissue of interest are employed 
for conjugation to a pharmaceutically active substance, the 
pharmaceutically active substance may be bound, preferably covalently, to 
a carrier prior to conjugation directly or indirectly with the targetting 
protein fragment(s) or such carrier binding may be carried out subsequent 
to protein fragment binding, in which case conditions will be chosen so 
that the required tissue binding capability of the protein fragment(s) is 
retained. Where a conjugate of the present invention comprising as the 
pharmaceutically active substance a non-proteinaceous species is generated 
by protease digestion of a larger adhesive protein-containing conjugate, 
it may also be feasible to subsequently bind the selected conjugate to a 
carrier without substantially reducing the desired tissue binding 
specificity. 
The activity of conjugates of the present invention increases with 
increasing loading of active substance bound to the protein fragment(s) 
and so the active substance is preferably present in molar excess within 
the conjugate. However, too high a loading of active substance will tend 
to mask the required tissue binding site on the tissue-binding substrate 
and may give rise to poor conjugation efficiency. For this reason, a 
conjugate of the present invention preferably contains from 1 to 100, more 
preferably from 1 to 50, moles of active substance (usually a single 
compound or element) per mole of protein fragment. When the active 
substance consists of a protein or enzyme or other high molecular weight 
substance, the molar ratio of protein fragment to active substance in the 
conjugate is preferably from 1:1 to 1:10. On the other hand, active 
substances of low molecular weight, for example gold atoms, are preferably 
present at higher loading ratios, for example 1:10 to 1:50. 
In the preparation of a conjugate according to the present invention, other 
than a gold-protein fragment anti-rheumatic conjugate as hereinbefore 
described, the protein fragment or fragments are most preferably bound 
with the pharmaceutically active substance using a known protein 
cross-linking agent such as a carbodiimide, a dialdehydo derivative of a 
dicarboxyliic acid, a diisocyanate, or an oxidised dextran having an 
aldohexopyranose ring-cleaved structure. 
Suitable carbodiimides include any of those disclosed in U.S. Pat. No 
4,046,871 and may be selected from any of the following: 
1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride, 
1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene-sulphonate, 
1-cyclohexyl-3-(4-diethylaminocyclohexyl) carbodiimide metho-p 
toluene-sulphonate, 
1-cyclohexyl-3-(.beta.-diethylamino-ethyl)-carbodiimide, 
1-ethyl-3-(2-morpholino-ethyl)-carbodiimide hydrochloride, 
1-ethyl-3-(2-morpholino-ethyl)-carbodiimide sulfate and cyanamide. 
Where the cross-linking agent employed consists of a dialdehydo derivative 
of a dicarboxylic acid, the acid is preferably a carboxy-terminated 
C.sub.2 -C.sub.5 straight chain alkane and is most preferably glutaric 
acid, the dialdehydo derivative of which is glutaraldehyde. A suitable 
method of conjugation employing glutaraldehyde is given by J. W. Payne in 
Biochem J (1973) 135, 867-873. 
An example of a suitable diisocyanate is hexamethylene diisocyanate. 
Suitable oxidised dextrans and methods of conjugation employing the same 
are disclosed in U.S. Pat. No. 4,587,122. 
Covalent coupling of a pharmaceutically active substance with one or more 
protein fragments for preparation of a conjugate of the present invention 
may, for example, be carried out by (a) mixing and reacting simultaneously 
the protein fragment(s), cross-linking agent and pharmaceutically active 
substance, (b) reacting the cross-linking agent with the 
pharmaceutically-active substance, and then reacting the product with the 
protein fragment(s), or (c) reacting the cross-linking agent with the 
protein fragment(s), and then reacting the product with the 
pharmaceutically active substance. 
Covalent coupling of a pharmaceutically active substance with one or more 
protein fragments to form a conjugate of the present invention will 
generally be conducted in an aqueous solution of pH 5 to 9, preferably 6 
to 8, and preferably in a buffer solution. Preferred reaction temperatures 
are 10.degree. C. to 30.degree. C., particularly room temperature. The 
reaction time is preferably from 0.5 to 15 hours, more preferably from 0.5 
to 5 hours. The preferred molar ratio of pharmaceutically active substance 
to protein fragment in the reaction mixture is preferably from 1:1 to 
100:1, with, if appropriate, sufficient or excess cross-linking agent 
being present to effect conjugation of the two. 
Active substance-protein fragment conjugates produced by the methods 
described above are preferably separated from the reaction mixture in 
which they are produced by affinity chromatography on immobilised body 
tissue or simulated body tissue for which the protein fragments have 
affinity. 
Subsequent covalent coupling of an active substance protein fragment 
conjugate to a high molecular weight carrier, e.g. fibronectin, albumin or 
a high molecular weight portion thereof, may also, for example, be carried 
out by employing a cross-linking reagent under mild conditions as 
specified above. Preferably, however, when it is desired to prepare a 
conjugate according to the present invention with a carrier, the carrier 
will be covalently coupled to the pharmaceutically active substance, 
either directly or via a coupling reagent, prior to protein fragment 
binding. Thus, for preparation of a preferred anti-rheumatic conjugate of 
the present invention wherein gold is conjugated to a carrier protein, an 
appropriate gold compound, e.g. aurothiomalic acid or a salt thereof such 
as sodium aurothiomalate, will preferably initially be reacted with the 
sulphydryl groups of the carrier so as to directly couple gold via these 
groups. As hereinbefore indicated, where the protein chosen for this step 
is fibronectin or a gelatin binding portion thereof, the carrier 
conjugation may be carried out in the presence of gelatin, e.g. soluble 
gelatin or gelatin bound to an agarose based support, e.g. Sepharose, in 
which case the carrier protein will retain a gelatin-binding site. 
Alternatively, fibronectin may, for example, conveniently be directly 
coupled with gold when conjugated itself to an agarose-based support such 
as Sepharose or when adsorbed on to fibronectin thus bound. For the 
preparation of a carrier containing, anti-rheumatic conjugate of the 
present invention, gold may also conveniently be directly conjugated to 
albumin via sulphydryl groups of the protein by, for example, reacting an 
appropriate gold compound such as sodium aurothiomalate with albumin bound 
to an agarose-based support, e.g. an albumin-Sepharose column. If in the 
preparation of an anti-rheumatic conjugate of the present invention gold 
is initially conjugated to a protein which is itself covalently conjugated 
on a support, e.g. Sepharose, it will be understood that the required 
gold-carrier protein conjugate may be released by appropriate protease 
digestion, i.e. the carrier protein of the final conjugate will be derived 
from a larger pre-carrier protein. 
According to a third aspect of the present invention, there is provided a 
pharmaceutical composition which comprises a pharmaceutically active 
conjugate according to the first aspect dissolved or dispersed in a 
pharmaceutically acceptable diluent or carrier, for example saline 
solution. Administration of the composition may be by intravenous 
injection or by oral ingestion of the composition in the form of a tablet 
or ingestible .liquid. A typical dose of aqueous composition may contain 
10-300 mg of conjugate in 0.05-10 ml of composition. 
According to a further aspect of the present invention, we provide a 
conjugate according to the present invention for use in therapeutic 
treatment of a human or non-human animal, e.g. conjugates of the present 
invention wherein a plasminogen activator is bound to a fibronectin 
fragment having predominantly fibrin targetting capability for use in 
treatment of a thrombotic condition or use of a conjugate according to the 
present invention wherein an anti-rheumatic substance, e.g. gold, is 
conjugated to a fibronectin fragment having predominantly gelatin 
targetting capability for use in the treatment of rheumatoid arthritis. 
We also provide as a still further aspect of the present invention, use of 
a conjugate according to the present invention for the preparation of a 
composition for use in the treatment of a disorder involving a specified 
body tissue for which said conjugate has predominant targetting 
capability. 
As yet another aspect of the present invention, we additionally provide a 
method of delivery of a pharmaceutically active substance to a tissue 
involved in a disorder of the body wherein said pharmaceutically active 
substance is administered in the form of a conjugate according to the 
present invention having predominant targetting capability for said 
tissue. 
The following non-limiting examples are intended to illustrate the present 
invention.

EXAMPLE 1 
PREATION OF ANTI-THROMBOTIC CONJUGATES 
A. Materials 
Al. Preparation of Physiological Fibrin Monomer (PFM) 
The PFM used in the following examples consisted of fibrinogen which was 
purified by precipitation from blood plasma cryoprecipitate supplied by 
the Plasma Fractionation Laboratory, Churchill Hospital, Oxford, England 
(GB). This PFM contained 68 wt.% fibrinogen, 10% fibronectin (a natural 
contaminant) with traces of factor VIII:RAg and factor XIIIa. Since normal 
fibrin blood clots invariably incorporate fibronectin, this PFM was 
subsequently used as a simulated fibrin blood clot. 
A2. Preparation of Fibronectin-Free Fibrin Monomer (FFFM) 
FFFM was prepared by removing fibronectin from the fibrinogen within PFM by 
subjecting the fibrinogen to gelatin-sepharose affinity chromatography. 
The preparation of columns used in this chromatographic procedure is 
described below. 
B. General Procedures 
B1. Preparation and Use of Gelatin-Sepharose Affinity Chromatography 
Columns 
Gelatin-Sepharose columns were prepared following the coupling procedure 
outlined in the manufacturer's (Pharmacia, Uppsala, Sweden) recommended 
procedure for adsorbing materials on to CNBr-activated Sepharose 4B. A 
coupling ratio of 15 mg gelatin per g moist weight Sepharose gel was used 
to prepare the columns. 
The running conditions for the prepared columns were adapted from Vuento 
and Vaheri, J. Biochem (1979) 183, p.331 with the following changes:-the 
running buffer consisted of a 10 mM phosphate, 10 mM citrate, 150 mM NaCl 
solution pH 7.5 and elution was achieved with 1 M arginine in phosphate 
buffered saline. The columns were used at 4.degree. C. 
B2. Preparation and Use of Fibrin Monomer-Sepharose Affinity Chromatography 
Columns 
Fibrin monomer columns were prepared following the method of Heene and 
Matthias, Throm. Res. (1973) 2, p.137. Two types of column were prepared, 
in which the monomer used was either Physiological Fibrin Monomer (PFM) or 
Fibronectin Free Fibrin Monomer (FFFM). PFM--Sepharose and FFFM--Sepharose 
columns were prepared by coupling either PFM or FFFM to CNBr-activated 
Sepharose 4B by the manufacturer's (Pharmacia) recommended procedure. The 
running conditions for both types of column were the same as those used in 
General Procedure B1 described above. 
B3. Preparation and use of Heparin-Sepharose Affinity Chromatography 
Columns 
Heparin-Sepharose columns were prepared following the procedure recommended 
by the manufacturer for coupling materials on to CNBr-activated Sepharose 
4B. Such columns are also available `ready-coupled` from the same 
manufacturer (Pharmacia, Uppsala, Sweden). Running conditions were as 
described above for gelatin-Sepharose, except that the running buffer 
consisted of a 10 mM phosphate, 20 mM NaCl, 0.5 mM EDTA solution, pH 7.5 
and elution was achieved using a 10 mM phosphate, 0.5 M NaCl, 0.5 mM EDTA 
solution, pH 7.5. Columns were used at room temperature. 
B4. Separation of Protein Fragments on Sephacryl*S200 
Further resolution of protein fragments selected by affinity chromatography 
was carried out by gel permeation chromatography using Sephacryl S200. The 
running buffer was 10 mM phosphate, 150 mM NaCl, pH 7.5. Approximately 10 
mg were applied to a 320 ml bed volume column (2.2.times.84 cm) at a flow 
rate of 12 ml/hr. .noteq.* trade mark 
B5. Assay for Plasminogen Activators 
The concentrations of plasminogen activator in solutions were determined 
using the Kabi Diagnostica "Initial Rate of Reaction" method for the 
Determination of Plasminogen in Plasma, using S-2251 chromogenic substrate 
(Kabi Vitrium, Sweden). The protocol for this method is summarised below: 
(1) Dilute human blood plasma with assay buffer solution consisting of 50 
mM Tris-HCl, pH 7.4 containing 12 mM NaCl in the volumetric ratio of 
plasma to buffer solution of 1:20 and add 200 microliters of the diluted 
plasma to a reaction tube. 
(2) Incubate the tube at 37.degree. C. for 2 to 6 minutes. 
(3) Add either a known number of units (e.g. 10 or 25 units) of plasminogen 
activator or a test conjugate containing plasminogen activator, made up to 
100 microliters with assay buffer solution, to the tube. 
(4) Incubate the tube at 37.degree. C. for 10 minutes. 
(5) Add 700 microliters of substrate solution consisting of S-2251 diluted 
to 0.86 mM working solution with assay buffer. 
(6) Mix, transfer the contents of the tube to a micro-cuvette and measure 
the change with time of the absorbance (A) of the mixture at 37.degree. C. 
to 405 nm wavelength light (.DELTA.A.sub.405). 
(7) Calculate .DELTA.A.sub.405 per minute and plot this result against the 
units of plasminogen activator per ml present in the buffer solution. 
An alternative plasminogen activator assay was used to test for such 
activity on PFM - Sepharose. An end-point assay had to be used because 
Sepharose could not be tested by the "initial rate of reaction" assay 
described above. The assay protocol is summarised below: 
(1) Partly dry the test PFM - Sepharose by suction filtration on a sinter 
funnel. 
(2) Weigh 100mg of test PFM - Sepharose (moist weight) into a reaction 
tube. 
(3) Add 200 microliters of diluted human blood plasma. 
(4) Mix 
(5) Incubate the tube for 10 minutes at 37.degree. C. 
(6) Add 700 microliters of S-2251 chromogenic substrate (Kabi) solution at 
37.degree. C., mix and incubate for exactly 180 seconds. 
(7) Add 100 microliters of 50% acetic acid and mix immediately to stop the 
reaction. 
(8) Measure the absorbance of the resulting solution to 405 nm within 4 
hours and relate the result to a standard curve for known concentrations 
of plasminogen activator against absorbance. 
C. Generation Of Protein Fragments By Digestion Of An Adhesive Protein With 
Proteolytic Enzymes 
Fibronectin was chosen as the starting adhesive protein. Prior to 
digestion, the fibronectin was checked for purity by size exclusion High 
Performance Liquid Chromatography (HPLC) using an Ultropac TSK G 4000 SW 
column, fractionation range from 1000kDa down to 5kDa. This was necessary 
in order to standardise the fibronectin fragments generated by proteolytic 
enzyme digestion, since the presence of fibronectin aggregates or 
fragments in the fibronectin prior to digestion could have affected the 
subsequent digestion process. 
C1. Digestion of fibronectin with trypsin 
10 mg of fibronectin in 10 ml of PBS (PBS =phosphate buffered saline pH 7.5 
containing 10 mM sodium phosphate and 0.15 M NaCl) was digested with 0.05 
mg of trypsin for 2 hours at 37.degree. C. Trypsin is an endopeptidase 
capable of specifically cleaving peptide bonds adjacent to an arginine or 
lysine residue. Samples of the reaction mixture were removed at the times 
stated in Table 1 below. The reaction was stopped after 2 hrs by the 
addition of 0.1 mg of soya bean inhibitor. 
The molecular weights of the fibronectin fragments produced by digestion 
were determined by gel permeation high performance liquid chromatography 
(HPLC) analysis using a TSK G 3000 SW column. The running buffer was a 0.1 
M phosphate solution pH 6.8 containing 0.05% sodium azide, flow rate 0.4 
ml/min, with a 70 minute elution time. The absorbance of the eluate was 
monitored at 280 nm for the presence of fibronectin fragments. 
TABLE 1 
______________________________________ 
Digestion of Fibronectin with Trypsin 
Incubation time 
(minutes) Protein Fragment Molecular Weight (kDa) 
______________________________________ 
0 439 -- -- -- -- -- 
10 -- 358 -- 140 56 42 
30 -- 358 181 140 56 42 
60 -- 358 181 140 56 42 
120 -- -- 181 140 56 42 
______________________________________ 
Extensive digestion of fibronectin by trypsin (that is, for more than 10 
minutes) dramatically reduced its gelatin binding activity, as measured by 
a competitive, enzyme-linked assay (Doran et al, Vox Sang (1983) 45, 
243-251). Consequently, the principal of using the minimum effective 
digestion time of 10 minutes was adopted. 
C2. Digestion of fibronectin with thrombin 
The procedure of C1 was repeated, except that the fibronectin was digested 
with 250 international units (iu) of thrombin instead of trypsin. Thrombin 
is a serine protease responsible for the final conversion of fibrinogen to 
fibrin during blood clot formation. The digestion was stopped after 120 
minutes by the addition of benzamidine to a final concentration of 8mM in 
the reaction mixture. Samples of the reaction mixture were removed at the 
times stated in Table 2 below and were analysed using the same procedure 
as outlined in C1. 
TABLE 2 
______________________________________ 
Digestion of Fibronectin with Thrombin 
Incubation time 
(minutes) Protein Fragment Molecular Weight (kDa) 
______________________________________ 
0 439 -- -- -- -- -- 
10 468 218 83 -- -- -- 
30 462 218 83 -- -- -- 
60 468 -- 83 50 27 23 
120 470 218 110 45 28 -- 
______________________________________ 
D. Selection and Purification of Protein Fragments 
Products of fibronectin digestion with trypsin or thrombin comprising a 
mixture of fragments with either or both gelatin-binding affinity and 
fibrin-binding affinity or neither of these two binding specificities were 
used as the starting material to select out fibronectin fragments having 
fibrin-binding affinity in the absence of gelatin-binding affinity or vice 
versa using a dual column affinity chromatography procedure. 
D1. Use of thrombin generated fibronectin fragments 
The method of C2 was repeated on a larger scale with the quantities of 
reagents and solution volume increased proportionally. The product, 
consisting of a solution of fibronectin fragments obtained by fibronectin 
digestion for 120 minutes at 37.degree. C. with thrombin, was divided into 
several aliquots containing known amounts of fibronectin fragments. These 
aliquots were applied directly to a 15 ml column of gelatin-Sepharose (see 
the general procedure B1 described above) in order to remove those protein 
fragments with an affinity for gelatin possessing both gelatin and 
fibrin-binding sites or a gelatin binding site in the absence of a 
fibrin-binding site. The unbound fractions of these aliquots, which 
contained protein fragments with an affinity for fibrin in the absence of 
gelatin-binding affinity and protein fragments having neither fibrin nor 
gelatin-binding affinity, were then applied to a 15 ml column of 
FFFM-Sepharose (see the general procedure B2 described above). The eluates 
from the FFFM-Sepharose column contained purified protein fragments with 
binding affinity for fibrin but no binding affinity for gelatin. The 
results of this two-stage affinity-chromatography procedure are given in 
Table 3 below in terms of product yields. 
TABLE 3 
__________________________________________________________________________ 
2-stage affinity chromatography of fibrin-binding protein fragments 
Sample 
Gelatin-Sepharose FFFM-Sepharose 
Applied 
Affinity Chromatography 
% Protein 
Affinity Chromatography 
% Protein 
(mg) mg bound 
mg unbound 
Recovery 
mg bound 
mg unbound 
Recovery 
__________________________________________________________________________ 
33.0 2.84 22.94 78 1.19 3.79 11.5 
15.0 6.4 6.5 86 1.72 2.44 68.4 
7.2 2.64 2.56 72 0.74 1.40 81.0 
__________________________________________________________________________ 
The recoveries of protein fragments at each stage given in Table 3 above 
were based on the absorbance of fractions at 280 nm using the extinction 
coefficient for fibronectin 
##EQU1## 
The eluates from the FFFM-Sepharose and gelatin-sepharose columns were 
analysed by HPLC to determine the molecular weights of both gelatin 
binding and fibrin binding fragments. The sizes of fragments eluted from 
the gelatin column were found to be 435 and 296 kDa. The sizes of the 
fragments eluted from the FFFM column were found to be 382, 110 and 45 
kDa. 
It will be understood that the above 2-stage affinity chromatography 
procedure can be reversed (FFFM-adsorption followed by gelatin adsorption) 
to afford eluates from the second adsorption stage that contain purified 
protein fragments with binding affinity for gelatin in the absence of 
fibrin binding affinity. 
D2. Use of Trypsin generated fibronectin fragments 
(a) The procedure of D1 was repeated using fibronectin digested for 10 
minutes with trypsin in a larger scale version of the procedure described 
in C1 above. The eluates from the affinity columns were analysed by HPLC. 
This revealed that protein fragments were eluted from the gelatin column 
of sizes 358, 140, 119 and 69 kDa and protein fragments of 56 kDa were 
eluted from the FFFM column. 
(b) The close association between the fibrin binding sites and heparin 
binding sites in fibronectin was exploited by using heparin-Sepharose 
instead of FFFM-Sepharose in an alternative dual column affinity 
chromatography procedure to isolate fibronectin fragments having 
fibrin-binding affinity in the absence of gelatin-binding affinity. 
A solution of tryptic fragments of fibronectin was prepared as in D2(a) 
above and the solution divided into several aliquots containing known 
amounts of fibronectin fragments. Aliquots were applied to a 15 ml column 
of heparin-Sepharose by the general procedure previously described in B3 
above. Bound fractions which contained protein fragments with an affinity 
for heparin and fibrin were then applied to a 30 ml column of 
gelatin-Sepharose by the procedure described in B1 Unbound fractions 
contained purified protein fragments with an affinity for heparin/fibrin. 
Those binding to gelatin-Sepharose, which were those with both gelatin and 
heparin/fibrin binding activity, were discarded. Results of this two stage 
affinity chromatography procedure are given in Table 4 below. 
The eluates from the adsorbent columns were analysed by HPLC. This revealed 
that the sizes of the protein fragments from the two-stage purification 
procedure were 360, 181, 140 (minor), 56 and 42 kDa. 
While all of these fragments retained heparin/fibrin binding activity and 
did not bind to gelatin, the different fragment sizes generated by this 
method could be further resolved by separation on Sephacryl S200 as 
described in B4 above. The proportions of the fragments in the heparin 
binding, non-gelatin binding fraction are given in Table 5. 
The proportion of protein fragments at each stage which were fibrin-binding 
was determined by applying aliquots to a PFM-Sepharose column by the 
procedure of B2 above. 
TABLE 4 
__________________________________________________________________________ 
Sample 
Heparin-Sepharose Gelatin-Sepharose 
Applied 
affinity chromatography 
% Protein 
Affinity Chromatography 
% Protein 
(mg) mg bound 
mg unbound 
Recovery 
mg bound 
mg unbound 
Recovery 
__________________________________________________________________________ 
23.7 11.2 13.7 105.0 1.8 9.1 97.3 
6.0 3.22 2.72 99.0 0.32 2.85 98.4 
__________________________________________________________________________ 
TABLE 5 
______________________________________ 
Recovered % Fibrin- 
Protein % Starting 
binding 
Purification Stage 
(mg) material activity 
______________________________________ 
Fibronectin trypsin digest 
23.7 100 23 
Heparin-Sepharose 
11.12 45.6 
(bound fractions) 
Gelatin-Sepharose 
9.10 37.3 63 
(unbound fraction) 
Sephacryl S200 
Fractions: 
360K 2.61 11.0 23 
181K 1.49 6.3 39 
(140K - minor peak) 
N.D. N.D. 
56K 1.75 7.4 
68 
42K 1.19 5.0 
______________________________________ 
N.D. -- Not determined 
It will be appreciated that if the two types of affinity chromatography 
column are used in opposite order, i.e. the bound fraction of a 
gelatin-Sepharose column is applied to a heparin-Sepharose column, this 
will result in isolation of fibronectin fragments having gelatin-binding 
affinity in the absence of fibrin and heparin binding affinity. 
E. Conjugation of selected fibronectin fragments having fibrin-binding 
affinity to streptokinase 
An aqueous solution of a commercial streptokinase preparation (trade name 
"Kabikinase" supplied by Kabi Vitrum AB Haematology, Stockholm, Sweden) 
containing by weight 4% streptokinase, 50% albumin, 44% sodium salts and 
2% H.sub.2 O was passed through a column of Cibacron Blue-Sepharose CL6B 
adsorption resin at ambient temperatures in order to adsorb the albumin, 
thereby separating it from the streptokinase. The unbound fractions were 
assayed for plasminogen activator activity. 
2 mls of an aqueous solution of the unbound fractions, containing about 600 
units of albumin-depleted streptokinase per ml, were prepared by the 
procedure described above. An equal volume of an aqueous solution of 
fibrin-binding fibronectin fragments prepared in accordance with the 
method of D1 (0.12 mg per ml) was added to the streptokinase solution. The 
pH of the mixture was adjusted to 7.0 by the addition of a small amount of 
0.1 N hydrochloric acid. To the resulting solution was added, with 
stirring and at ambient temperature, a 0.588 M 
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride solution 
until the content of the carbodiimide hydrochloride in the resulting 
solution reached 0.5% (w/v). Stirring was continued for 2 hours to produce 
a conjugated streptokinase-protein fragment product in the reaction 
mixture. The desired product was recovered from the mixture by adsorption 
on to FFFM-Sepharose followed by elution with 0.2M NaCl solution. The 
concentration of the conjugate in the resulting eluate was, where 
necessary, increased by pressure ultra-filtration. 
The activity of the streptokinase within the conjugate at various conjugate 
concentrations in solution was measured by the procedure described in B5 
above against equivalent concentrations in solution of unconjugated 
streptokinase. The results of these measurements are given in Table 6 
below. 
TABLE 6 
______________________________________ 
SK Activity (.DELTA. A.sub.405 min.sup.-1) 
concentration SK-protein 
(units/ml) SK alone fragment conjugate 
______________________________________ 
10 0.019 0.010 
20 0.036 0.014 
30 0.053 0.021 
______________________________________ 
These results show that streptokinase activity in the conjugate varied from 
38%-52% of that present in its unconjugated form. 
F. Conjugation of selected fibronectin fragments having fibrin-binding 
affinity to urokinase 
2240 units of urokinase (UK) were dissolved in 2 ml of pH 7.3 buffer 
solution (20mM Tris HCl, 0.15 M NaCl) containing 0.12 mg/ml of 
fibrin-binding fibronectin fragments prepared in accordance with the 
method of D1. The total content of protein fragments within the solution 
was equivalent to 3.27.times.10.sup.-6 M. To the resulting solution was 
added 80 microliters of 5% (w/v) glutaraldehyde solution in water (final 
concentration 0.2%). The resulting solution was mixed on a vortex mixer 
while the glutaraldehyde was added. Mixing was continued for 1 minute and 
the solution was then incubated at room temperature (20.degree. C.) for 24 
hours. The desired product was recovered using FFFM-Sepharose as described 
in procedure E above. The concentration of the conjugate in solution was 
adjusted as necessary either by pressure ultrafiltration concentration or 
by dilution with buffer solution. 
The activity of the urokinase within the conjugate at various conjugate 
concentrations in solution was measured by the procedure described in B5 
above against equivalent concentrations in solution of unconjugated 
urokinase. The results of these measurements are given in Table 7. 
TABLE 7 
______________________________________ 
UK Activity (.DELTA. A.sub.405 min.sup.-1) 
concentration UK-protein 
(units/ml) UK alone fragment conjugate 
______________________________________ 
10 0.020 0.006 
20 0.0475 0.010 
30 0.0575 0.013 
______________________________________ 
The results given in Table 7 above show that the plasminogen activity of 
the urokinase within the conjugate is 21%-30% of its activity when in its 
original, unconjugated form. 
G. Alternative method for conjugation of selected fibronectin fragments 
having fibrin-binding affinity to urokinase 
2240 units of UK were dissolved in 2 ml of pH 7.3 buffer solution (20mM 
Tris-HCl, 0.15M NaCl) containing 0.12 mg/ml (3.27.times.10.sup.-6 M) of 
fibrin-binding fibronectin fragments prepared as in D1 above. The pH of 
the solution was adjusted to 5 by the addition of small amounts of 0.1 N 
hydrochloric acid. To the resulting solution was added, with stirring and 
at ambient temperature, a 0.588 M 
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride solution 
until the content of the carbodiimide hydrochloride in the resulting 
solution reached 1.0% (w/v). Stirring was continued for 1 hour to produce 
a conjugated urokinase-fibronectin fragment product in the reaction 
mixture. A mild form of carbodiimide cross-linking was also suitable using 
0.5% (w/v) carbodiimide for 2 hours at pH 7.0 (i.e. no HCl added). The 
desired conjugated product was recovered from the mixture by adsorption on 
to FFFM-Sepharose followed by elution with 0.2 M NaCl solution. The 
concentration of the conjugate in the resulting eluate was, where 
necessary, increased by pressure ultrafiltration. 
The activity of urokinase within the conjugate at various conjugate 
concentrations in solution was measured as in the procedure F above. The 
results of these measurements are given in Table 8 below. 
TABLE 8 
______________________________________ 
UK Activity (.DELTA. A.sub.405 min.sup.-1) 
concentration UK-protein 
(units/ml) UK alone fragment conjugate 
______________________________________ 
10 0.0225 0.015 
20 0.050 0.033 
30 0.060 0.042 
______________________________________ 
It may be seen from Table 8 above that the plasminogen activator activity 
of the UK within the conjugate varies from 64%-67% of its activity when in 
its original, unconjugated form. 
H. Conjugation of selected fibronectin fragments having fibrin-binding 
affinity to urokinase 
The method of G above was repeated except that the concentration of 
fibrin-binding fibronectin fragments in the Tris-HCl-saline solution was 
reduced to 0.06 mg/ml. 
I. Conjugation of selected fibronectin fragments having fibrin-binding 
affinity to urokinase 
The method of G above was repeated except that the concentration of 
fibrin-binding fibronectin fragments in the Tris-HCl-saline solution was 
reduced to 0.03 mg/ml. 
J. Binding plasminogen activator-protein fragment conjugates to simulated 
blood clot tissue (fibrin Monomer) 
In order to test the efficiency of the conjugates prepared by the 
procedures described in E and G-I above, solutions of each conjugate were 
contacted with immobilised simulated blood clot tissue and the amount of 
plasminogen activator adsorbed by the tissue was measured. It is known 
that there is a direct correlation within the body between the uptake of 
plasminogen activator by fibrin clots and their rate of subsequent 
fibrinolysis and so this test provides a useful indication of the 
pharmacological value of these conjugates. 
The simulated tissue selected was physiological fibrin monomer (PFM) 
preparation which is known to include many of the components of a normal 
blood clot. Crude fibrin monomer (Kabi Diagnostica), herein referred to as 
PFM, was immobilised on to Sepharose 4B beads (Pharmacia, Sweden) by the 
procedure described in B2 above. Plasminogen activator-fibronectin 
fragment conjugates were made up into separate solutions of known 
concentration containing from 16 to 160 units/ml of plasminogen activator 
activity. These solutions were then roller mixed for 2 hours at 4.degree. 
C. with 100 mg (moist weight) quantities of the immobilised PFM described 
above. The immobilised PFM was then washed thoroughly with 50 mM TrisHCl 
solution, pH 7.6 to remove any unbound activity. The quantity of bound 
conjugate was then determined by direct assay of the PFM-Sepharose using 
the end point assay described in B5 above and aliquots of the affinity 
adsorbent in suspension. The activity of the plasminogen activator in both 
the starting material (plasminogen activator-fibronectin fragment 
conjugate) and in the washed PFM-Sepharose after binding was determined 
(in terms of units of plasminogen activator (PA) per ml in solution) as a 
measure of fibrin-binding fibrinolytic agent in the test conjugates. The 
results of these determinations are given in Table 9 below. 
TABLE 9 
__________________________________________________________________________ 
conjugate 
Units/ml of PA 
preparation 
within conjugate added 
Units/ml of PA 
% of PA activity 
Units of PA reacted per 
procedure 
to immobilised PFM 
bound to Sepharose 
bound to Sepharose 
mg of protein fragment 
__________________________________________________________________________ 
E 16 0.42 2.6 133 
32 3.2 10.0 267 
80 23.0 28.0 667 
160 41.6 26.0 1333 
G 25 6.0 23.6 208 
50 10.4 20.7 417 
100 15.3 15.3 833 
150 17.8 11.9 1250 
H 50 18.13 36.3 833 
100 32.0 32.0 1667 
150 31.6 21.1 2500 
I 50 25.6 51.2 1667 
100 34.2 34.2 3111 
150 42.9 28.6 5000 
__________________________________________________________________________ 
The results of Table 9 are illustrated diagrammatically in FIGS. 1 and 2. 
FIG. 1 is a graph showing the correlation between the amount of conjugate 
added to the immobilised PFM and the amount of PA subsequently bound to 
it. FIG. 2 illustrates the relationship for conjugates prepared by the 
procedures of E and G-I above between the amount of plasminogen activator 
(in units) per mg fibronectin fragment used in the cross-linking reaction 
mixture that produced the conjugates and the amount of fibrin-binding 
plasminogen activator which was produced in the conjugates. Using 
urokinase there is a two-phase slope to the increase in conjugate PA 
content as more plasminogen activator was employed within the 
cross-linking reaction mixture. The slope with streptokinase is much 
steeper indicating more effective binding. 
K. Binding of Plasminogen activator-fibronectin fragment conjugates to 
Sepharose 4B 
In order to establish that the plasminogen activator-fibronectin fragment 
conjugates were binding selectively to the fibrin monomer rather than to 
its support matrix, the product of method G above was applied to 
unsubstituted Sepharose 4B gel using the binding procedure outlined in J. 
The resultant fractions were assayed for plasminogen activator activity. 
The only activity to be found was in the unbound fractions. Recovery was 
100%. Since the conjugate of method G had negligible affinity for 
unsubstituted Sepharose, it was concluded that a fibrin-substituted 
Sepharose behaved as a simulated fibrin clot and the conjugate bound 
entirely to the protein portion of the simulated clot. 
L. Binding of unconjugated plasminogen activator to simulated blood clot 
tissue 
2016 units of urokinase were made up into 2 ml of pH 7.3 buffer solution 
(20 mM Tris HCl containing 0.15 M NaCl) and applied to PFM-Sepharose 4B. 
The mixture was held at 4.degree. C. for 2 hours. It was found that only 
5% of the original UK activity present in solution became bound to the 
simulated clot, indicating that basal binding of unmodified urokinase to 
the simulated blood clot tissue was minimal. 
M. Dissolution of fibrin-based clot tissue in response to plasminogen 
activators and plasminogen activator-fibronectin fragment conjugates 
The pharmacological efficacy of the plasminogen activator-fibronectin 
fragment conjugate product of method G was further tested by examining the 
lysis of fibrin-based clots in response to the selective binding of the 
conjugate. The influence of targetted plasminogen activator conjugates was 
assessed by measurement of .sup.125 I-FDPs released from .sup.125 
I-labelled clots into the clot bathing medium. 
Fibrinogen was iodinated by the chloramine T method (Green et al, Biochem. 
J., (1963), 89, p.114) and activated using 100 U thrombin per gram 
fibrinogen. PBS - washed clot tissue was blotted into the form of a 
protein sheet from which small discs (10-20 mg) could be readily excised. 
.sup.125 I-Fibrin clot discs (20 mg) were incubated for 2hrs at room 
temperature in 0.25 ml, Tris buffer solution, pH 7.4 (50 mM Tris, 110 mM 
NaCl) containing either 800 U/ml urokinase or 800 U/ml 
urokinase-fibronectin fragment conjugate. 
The clot discs were washed twice with 2 ml of Tris buffer solution, pH 7.4 
to remove non-specifically bound urokinase. Control clot discs were 
incubated in Tris buffer solution, pH 7.4, without urokinase or conjugate 
and washed in parallel with test samples. 
Clot discs were transferred to a chamber (10 ml vol.) through which the 
clot bathing medium (25 ml) was circulated from a reservoir at a flow rate 
of 2 ml/min. The bathing medium consisted of Tris buffer solution, pH 7.4, 
containing 0.03mg/ml plasminogen and 1 mg/ml human albumin. 
Samples (0.5 ml) of the bathing medium were taken at intervals over a 24 hr 
period and solubilised radioactivity determined. The results of these 
measurements are represented diagrammatically in FIG. 3, which is a graph 
showing the time course of .sup.125 I radioactivity released from fibrin 
clot 
of discs in response to urokinase activity. Dissolution of the clots 
incubated with conjugate was up to 3.3 times faster (for example at 18 
hrs: control =nil activator-stimulated lysis) than that with the 
equivalent activity of plasminogen activator alone. This indicated that 
specific and preferential binding of conjugate can enhance the activation 
of plasminogen in the vicinity of a clot and hence fibrinolytic breakdown 
of the clot. 
EXAMPLE 2 
PREATION OF ANTI-RHEUMATIC CONJUGATES 
A. Direct Covalent Coupling of Gold to Fibronectin or Albumin 
A1. Direct covalent coupling of gold to fibronectin fibronectin-Sepharose 
or albumin-Sepharose 
1 ml of fibronectin-Sepharose slurry [1-2 parts coupling buffer (50 mM 
Tris-HCl, pH 7.5, 1 M urea) plus 1 part wet settled volume of gel 
consisting of 0.7 gm fibronectin/gm of CNBr-activated Sepharose 4B] plus 1 
ml of coupling buffer containing 50 .mu.M of sodium aurothiomalate were 
incubated with roller mixing for 4 hours at 37.degree. C. The Sepharose 
was then washed with a suitable solution, for example 50 mM Tris-HCl, pH 
7.5, to remove unreacted gold compound and protein. A moist pellet of gel 
was recovered either by centrifugation or by vacuum filtration through a 
glass sinter and incubated with trypsin at an enzyme to protein substrate 
ratio of approx 1:200 in 50 mM Tris-HCl, pH 7.5 for 15-120 mins at 
37.degree. C. Released gold-fibronectin fragment conjugate was separated 
from the Sepharose by centrifugation. 
Albumin-Sepharose was substituted for fibronectin-Sepharose in the above 
procedure to obtain gold-albumin fragment conjugates. 
Gold conjugates obtainable by the above procedures are suitable as carrier 
protein-gold conjugates for preparation of anti-rheumatic conjugates by 
cross-linking to gelatin-targetting adhesive protein fragments. 
Conjugation of gold was also tested over a range of conditions 
(temperature, incubation time, ionic strength). The gold component of 
sodium aurothiomalate was followed specifically by atomic absorption 
spectroscopy and protein was measured by the Folin-Lowry assay. Table 10 
below shows how the amount of bound gold varied. Binding (in .mu. moles of 
gold per unit volume of fibronectin-Sepharose) was significant but low 
(6-8 .mu.M) in low ionic strength Tris-HCl. Addition of 1 M urea increased 
binding by between 50%-150% with the optimum at 4 hrs and 37.degree. C. 
Table 11 below shows the relative effect of increasing levels of urea on 
binding gold to fibronectin-Sepharose. Increasing urea concentration 
between 0.1 and 1.0 M increased the gold incorporation by almost four fold 
(in this case gold binding was measured relative to the protein content of 
the conjugate). In fact, these are very high levels of binding of the 
order of 1.2.times.10.sup.4 moles gold/mol fibronectin. When 
albumin-Sepharose was substituted for fibronectin-Sepharose, gold binding 
was approximately 35%, on a protein weight basis, of the minimum level for 
fibronectin-Sepharose. Although coupling was at low ionic strength, the 
final conjugate was largely stable to physiological NaCl levels. Loss of 
gold from the immobilised complex was constant at 21% to 23% between 0.1 M 
and 0.75 M NaCl. 
TABLE 10 
______________________________________ 
Effect of reaction conditions on gold coupling 
to solid phase fibronectin 
0.15 mM NaCl 
50 mM Tris 1M Urea 
50 mM Tris % of % of 
.mu.M Au.sup.(1) 
.mu.M Au 
Basal.sup.(2) 
.mu.M Au 
Basal 
______________________________________ 
2 hr reaction 
+4.degree. C. 
6.00 4.65 77.5% 9.15 152.5% 
+37.degree. C. 
7.20 3.60 50% 12.75 177% 
4 hr reaction 
+4.degree. C. 
6.75 6.30 93% 12.00 178% 
+37.degree. C. 
7.80 6.30 81% 19.50 250% 
______________________________________ 
(1) Figures refer to total Sepharose-bound gold (by atomic absorption) 
recovered from 2 ml of gel. 
(2) Basal binding is taken as value in Tris buffer only, using 
corresponding conditions and taken as 100% 
TABLE 11 
______________________________________ 
Influence of urea concentration on conjugation of gold 
to fibronectin-Sepharose 
Urea Mean Gold incorporation 
% increase over 
Concentration 
(.mu.M/.mu.g protein)* 
control level 
______________________________________ 
0 0.05 -- 
0.1M 0.098 96% 
0.5M 0.133 165% 
1M 0.20 390% 
2M 0.193 287% 
______________________________________ 
*Mean of 2 experiments; incubation of 4 hrs at 37 C. 
A2. Direct covalent coupling of gold to fibronectin or fibronectin 
fragments immobilised on fibronectin-Sepharose 
Fibronectin-Sepharose binds large quantities of soluble fibronectin at low 
ionic strength which can be recovered by raising the buffer NaCl 
concentration. 
1 ml of fibronectin-Sepharose slurry [1-2 parts buffer (50 mM Tris-HCl, 
pH7.5) plus 1 part wet settled volume of gel consisting of 0.7mg 
fibronectin/gm of CNBr-activated Sepharose 4B] was mixed with 2 ml of 
purified plasma fibronectin (approx. 1 mg/ml) in the same buffer and mixed 
for 15-30 mins. The gel was washed (same buffer) to remove unbound 
fibronectin and incubated with roller mixing for 4 hrs at 37.degree. C. in 
50 mM Tris-HCl pH7.5 containing 1 M urea and 25 u mole of gold as sodium 
aurothiomalate per mg of fibronectin loaded. The gel was then again washed 
and resuspended in high ionic strength buffer (50 mM Tris-HCl pH7.5: 0.5 M 
NaCl) to dissociate the adsorbed fibronectin-gold conjugate. Mixing was 
carried out at room temperature for 15-60 mins. The gold-fibronectin 
conjugate in solution was de-salted by dialysis, ultrafiltration or gel 
filtration or a combination of such techniques and stored frozen or dry. 
Using this technique for gold conjugation, 37% of the gold applied was 
conjugated to give a specific binding value of 9.8.times.10.sup.3 moles 
gold/mole of fibronectin. After cross-linking to gelatin-targetting 
thrombin fragments of fibronectin (see section C), the specific gold 
binding was reduced to 1.1.times.10.sup.3 mole gold/mole of fibronectin. 
In the above procedure, soluble fibronectin may be replaced by fibronectin 
fragments to also provide carrier protein-gold conjugates suitable for 
preparation of anti-rheumatic conjugates by cross-linking to gelatin 
targetting adhesive protein fragments. 
A3. Direct covalent coupling of gold to fibronectin in the presence of 
gelatin 
Using the gold conjugation methods described in A1 and A2 above, the 
resulting gold-fibronectin or gold-fibronectin fragment conjugates do not 
retain gelatin-binding activity. In this third method used for direct 
coupling of gold to fibronectin or fibronectin fragments, the 
gelatin-binding sites of the protein employed in the coupling reaction 
were protected by binding to gelatin either in the form of soluble gelatin 
or gelatin-Sepharose. 
2 mls of a 1 to 5 mg/ml solution of gelatin (prepared by denaturation of 
purified skin collagen) in 50 mM Tris-HCl pH7.5 was mixed with 2 mg of 
intact fibronectin or gelatin-binding fibronectin fragments in the same 
buffer. The mixture was rolled for 1 hour at room temperature. 
Alternatively, the fibronectin or gelatin-binding fibronectin fragments 
were adsorbed on to gelatin-Sepharose in the same buffer. 50 .mu.moles of 
sodium aurothiomalate were added and the solution or 
gelatin-Sepharose-fibronectin slurry made up to 1 M with urea and mixed at 
room temperature for 4 hours. 
Where soluble gelatin was used, the gelatin-fibronectin binding was 
disrupted by adding 4M urea, dialysing into 20 mM sodium phosphate 
containing 4 M urea at pH 5.2 and mixing with 1 gm of pre-swollen 
carboxymethyl cellulose ion-exchanger (Whatman CM52*). Soluble gelatin was 
bound to the ion-exchanger and removed by centrifugation leaving the 
soluble conjugate. .noteq.* trade mark 
Where gelatin-Sepharose was used, the fibronectin-gold conjugate was made 
on the gel and unreacted drug or protein fragment removed by washing with 
the same buffer free of gold compound. The final fibronectin-gold 
conjugate was recovered by washing the gelatin-Sepharose adsorbent either 
with 4 M urea or 1 M arginine in 50 mM Tris-HCl, pH 7.5. 
Using a "gelatin binding protection method" as above with whole 
fibronectin, it was found possible to conjugate about 50% of the gold to 
give fibronectin-gold conjugate with 2.3.times.10.sup.3 M gold/M 
fibronectin. 
From gelatin-binding gold-fibronectin fragment conjugates thus isolated, 
conjugates having an fibronectin fragment with gelatin-binding affinity, 
but lacking fibrin-binding affinity may be selected by FFFM-Sepharose or 
heparin-Sepharose affinity chromatography suitable for direct use as 
antirheumatic conjugates. It will be appreciated that such anti-rheumatic 
conjugates may also be obtained by subjecting gelatin-binding gold-whole 
fibronectin conjugates isolated by a procedure as above to protease 
digestion, with subsequent selection of gold-fibronectin fragment 
conjugates lacking fibrin-binding affinity. Alternatively gold-fibronectin 
conjugates, as well as gold-fibronectin fragment conjugates prepared by a 
"gelatin-binding protection method" as above, may be employed as carrier 
protein-gold conjugates for preparation of anti-rheumatic conjugates by 
cross-linking to gelatin-targetting adhesive protein fragments, e.g. 
fibronectin fragments having gelatin-binding affinity, but substantially 
lacking fibrin-binding affinity. 
B. Preparation of Gelatin-Binding Fragments of Fibronectin Lacking 
Fibrin-Binding Affinity 
Fibronectin was digested with trypsin or thrombin as described in Sections 
C1 and C2 of Example 1 or with cathepsin D in conventional manner to yield 
a mixture of fragments with either or both gelatin-binding affinity and 
fibrin-binding affinity or neither of these two binding specificites. 10 
ml of a 1 mg/ml solution of protease digested fibronectin in 50 mM 
Tris-HCl, pH 7.5 was applied to a heparin-Sepharose column. Unbound 
material (free of fragments with a heparin or fibrin binding site) was 
applied to a gelatin-Sepharose column at 1 mg/ml in 50 mM Tris-HCl, pH7.5 
containing 0.5 M NaCl. After washing off unbound protein, gelatin-binding 
fragments were eluted with either 4 M urea or 1 M arginine in running 
buffer and desalted by dialysis. 
C. Cross-linking of Carrier Protein-Gold Conjugates to Gelatin Targetting 
Fibronectin Fragments 
5 ml of fibronectin carrier-gold conjugate (prepared by a procedure as 
described in A1, A2 or A3 above) in 10 mM sodium phosphate buffer pH 5.2 
was made up to a final concentration of 1% (0.24 M) cyanamide and 
incubated at 22.degree. C. for 1 to 4 hours with 5 ml of gelatin-binding 
fibronectin fragments prepared as in B above, at the same protein 
concentration as the fibronectin-gold conjugate. Excess reactants were 
removed by dialysis, ultra-filtration or gel filtration. 
D. Testing for Gelatin Binding Activity of The Final Conjugate 
The final conjugate was assayed for gelatin binding activity by applying it 
to a gelatin-Sepharose column as in B above. Bound material was eluted and 
assayed for total protein and for gold content. This gave a measure of 
gelatin-binding as .mu.M of gold/.mu.g gelatin-binding protein.