Background of the Invention Pharmaceutical conjugates and their production are described in WO 98/17319.

In particular, conjugates of albumin microparticles linked by adsorption or by means of a spacer to a RGD peptide, such as fibrinogen, are described. Their production depends on the presence of functional SH groups on albumin. It is also proposed that Factor V1II may be bound, e. g. as a second active agent, for use in treating haemophilia.

Summary of the Invention The present invention is based on the distinction between adsorption and linking for respective active agents bound to an insoluble carrier such as an albumin microcapsule, e. g. of fibrinogen and another glycoprotein such as Factor V m. Such products may have utility because one active agent is effective at the site of action and/or to have a therapeutic effect and the other acts as a targeting agent. Preferably, the targeting agent is bound and the effective agent is adsorbed.

Description of the Invention Components that can be used in the invention, and linking and adsorption procedures, are fully described and exemplified in WO-A-98/17319 and also in WO-A- 99/25383, the contents of which are incorporated herein by reference.

An illustrative glycoprotein is Factor VIII. Other suitable compounds are blood coagulation factors, proteins ofthe coagulation cascade, thrombolytic agents, antibodies or a-1 antitrypsin. Further illustrative effective agents are cytotoxics and lipid-type (steroidal) compounds. Blook-derived or recombinant products may be used.

Another illustrative active agent, especially as a targeting agent, is fibrinogen.

Other suitable compounds are RGD-containing peptides and fibrinogen y-chains. Further illustrative targeting agents are antibodies, e. g. to a bladder or other tumour.

It is a feature of the invention that, because a targeting agent can be used, products of the invention may usefully include appropriate other active agents. Such

agents will be chosen with regard to the site of action, usually a wound or other bleeding locus, and to the nature of the problem that is addressed.

The carrier that is used in the invention is preferably produced by spray-drying, under conditions that allow good control of particle size and size distribution. For example, the preferred size is up to 6 *um, e. g. 1 to 4 um, in order that the particles can pass through capillaries.

Suitable materials and procedures, and also methods for stabilising the microparticles, by heat or by chemical cross-linking, are fully described in WO-A- 92/18164, WO-A-96/15814 and WO-A-96/18388, the contents of which are incorporated herein by reference. As explained in the latter publication, the conditions that are described do not affect functional groups, such as the thiol groups in albumin, which therefore remain available for reaction with biological molecules.

The microparticles used in this invention may have the physical characteristics described in the two publications identified above, e. g. being smooth and spherical, and containing air. In order to obtain insoluble, cross-linked microcapsules, the spray-dried product may be reacted with a chemical cross-linking agent. However, heat and y- irradiation are preferred, and may also sterilise the dry powder products.

In the invention, one active agent is bound to such a carrier without a linker, by adsorption. This may be achieved by precipitation ofthe peptide or other agent on to the surface of the microspheres, e. g. by control of pH and other conditions, as will be evident to one of ordinary skill in the art. Excess/unbound material is then washed away.

As indicated above, products of the invention containing fibrinogen may act at the site of tumours. Therefore, they may be used in tumour therapy, e. g. by linking a cytotoxic agent by the particular method of this invention or by the methods described in WO-A-96/18388. Suitable cytotoxic agents include methotrexate, doxorubicin, cisplatin or 5-fluoro-2-deoxyuridine.

The targeting of drugs to tumour cells may be achieved using products of the invention as vehicles reacting directly with the cells or by participating in the aggregation and deposition of fibrin at the site of cell adhesion. The products may then be used to target the disseminated tumour cells in the circulation, by specific interactions with the cell glycoprotein receptors (seek and destroy) or by participation in the platelet

aggregation process at the site of adhesion. In both cases, the cytotoxic drug is concentrated at the site of the invading tumour cells.

Alternatively, tumour aggregation may be inhibited in the circulatory system, or even at the site of adhesion, by coating the tumour cell surface with products of the invention, and blocking the sites/mechanisms that activate platelets. This would then allow the body's natural defence mechanisms to facilitate the removal of the tumour cells.

Products containing, for example, the GPIb receptor (interacts with von Willebrands factor) or receptors for collagen or other sub-endothelial matrix components may also be delivered, to potentially block the binding sites for tumour cells by coating the sub-endothelial matrices. The product should still allow an interaction with platelets at the site of a wound, but should also restrict the invasion of vascular wall by any immobilised tumour cell.

An important advantage of the present invention is that the activity of fibrinogen (or other RGD peptide) can be substantially retained. The content of active fibrinogen can be determined by known procedures; see WO-A-98/17319.

A platelet substitute ofthe invention usually comprises at least 0.01%, preferably at least 0.015%, more preferably at least 0.02%, and most preferably at least 0.025%, active fibrinogen. The amount of fibrinogen should not be too great, in order to avoid aggregation, e. g. up to 1,1.5,2 or 2.5%. Of the fibrinogen content, it is desirable that at least 50%, preferably at least 70%, more preferably at least 90%, should be active.

This can be determined with respect to the total content of fibrinogen, which again can be measured by method such as ELISA. Total fibrinogen may also be determined by radio-labelling, e. g. with 125I, and counting, by conventional procedures.

The fibrinogen may be blood-derived, transgenic or recombinant, full-length or any active fragment thereof Fragments are disclosed, inter alia, by Coller et al, J. Clin.

Invest, 89: 546-555 (1992).

For use as a therapeutic agent, a product of the invention may be administered as is, or mixed with any suitable carrier known to those of ordinary skill in the art. The amount of the product administered will be determined largely to the severity of the wound or other condition to be treated. A typical dosage may be 1.5 x 109 microcapsules per kg body weight.

The following Examples illustrate the invention. In the Examples, the attachment of two biologically active proteins onto HSA microcapsules has been investigated.

Factor VIII and fibrinogen have been chosen for this purpose and the attachment employed is both natural adsorption and covalent crosslinking.

Two functional groups on the HSA microcapsules were targeted for covalent attachment, namely amine and free thiol moieties. Specific crosslinkers were chosen for both proteins to utilise these different groups.

The crosslinker m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) has been chosen for the purpose of covalent attachment of one active onto HSA microcapsules. The amine moieties contained within the fibrinogen or FVIII react with the NHS-ester functional group on the spacer and the derivatised protein is then reacted with HSA microcapsules allowing the maleimide group to form a stable disulphide bond with the free thiol on the HSA Cys-34 residue.

The assay used for analysis of Factor VIII loading and activity during these studies is the Quadratech Coatest VIII: C/4 Assay kit. The assay utilises the cofactor capabilities of Factor VIII.

Alphanate4}) Factor VIII was obtained from Grifols UK and used throughout.

Aliquots of 1 international unit Factor VHI/ml in distilled water were stored at-20°C until required.

The fibrinogen used in the Examples was a full-length, blood-derived, commercially available product (SNBTS) that had been doubly virally-inactived.

HSA microcapsules used in the Examples were prepared by spray-drying and were then stabilise by heating, as described in WO-A-9615814. The microcapsules were sunk with 1% Tween 80 and washed extensively with PFPW to remove Tween 80 and excipient prior to use.

In Example 1, fibrinogen is covalently bound, and Factor VIII adsorbed, to microcapsules. In Example 2, the Factor VIII is covalently bound, and fibrinogen adsorbed.

Example 1 An aliquot was removed from a 3mg/ml stock solution of MBS in DMF (81, 24gag, 72.5moles) and added to 20mM sodium phosphate pH 7.5 (1867Z1). The

solution was mixed thoroughly and fibrinogen (125p1 of 40mg/ml solution in distilled water, 14.5moles) introduced to make a total reaction volume of 2ml. Reacted for 30 minutes at room temperature with continuous stirring.

Microcapsules (100mg, 1515nmoles) were sunk in 1% Tween 80 and washed twice in distilled water, once in reaction buffer, 20mM sodium phosphate pH 7.5, prior to reaction to remove any excipients.

The fibrinogen solution was then added to the HSA microcapsules and reacted for a further 30 minutes at room temperature. After the product had been washed twice in reaction buffer to remove any excess unreacted fibrinogen, the supernatant was discarded and 1U FVIII added (1U FVIR in distilled water). Total reaction volume lml.

The FVIII was reacted for one hour before washing once in reaction buffer and reconstituting to a FVIII concentration of lU/ml.

Two controls were included in the experiment. The first contained Factor VIII and microcapsules only, the second derivatised fibrinogen and microcapsules only. These controls were used for comparison with the reaction product.

The samples were assayed using the slide test and the Quadratech Coatest VIII C: 4 assay kit.

The slide test assay records the time taken for the final product to react with thrombin to form aggregates. The final product (50go) was placed onto a microscope slide and thrombin (50z1) added. The two were stirred for 2 seconds and the time from when aggregates begin to form to when the aggregation ends is recorded.

In a repeat experiment the final product was formulated using 5 lmg/ml mannitol, 25mM sodium phosphate buffer pH 7.0. Sample counts were obtained from the Coulter Multisizer and the volume of formulating agent required to give a sample concentration of 1 SOOmillion microcapsules per ml calculated. The sample was centrifuged at 3 SOOrpm for 2 minutes and the supernatant decanted prior to resuspension in the calculated volume of formulation buffer.

Table 1 shows the slide test results, the concentration ofFactor VIII present and percentage yield obtained from the Coatest assay for the reaction sample and controls.

Table 1 Sample Unformulated Formulated Concentration Yield Slide test slide test/>Units/% /seconds/second s FVIII Control 1 FVIII + N/A N/A 0.3111 31.1 microcapsules N/A N/A 0.2189 21.9 Control 2 Fibrinogen-MBS + l/2-5 N/D N/A N/A microcapsules 1-3 4-9 N/A N/A Reaction FVIIIlFibrinogen-| N/D 0.2574 25.7 MBS/microcapsules 1-3 3-11 0.2144 21.4 The unformulated slide test activity which was similar to the control, suggests that the adsorption of Factor VIII onto HSA microcapsules does not affect the attachment of fibrinogen. On formulation, slight activity is lost in the sample containing FVIII. However, this loss is also observed with comparable products containing fibrinogen alone, on formulation.

The concentration of FVIII in uUnits obtained from the assay can be directly related to activity. Comparison of these values suggests that no activity is lost as a result of crosslinking fibrinogen to the microcapsules as a second active.

Example 2 From a stocksolutionofMBS inDMSO (5mg/ml), an aliquot (I 00gl) was added to O. 1M sodium borate buffer pH 8.0 to give a solution of 0.005mg/ml MBS concentration.

To 1U FVIII an aliquot of the dilute MBS solution was added to introduce 10 molar equivalents ofMBS to the reaction (0.549} il, 2.74ngMBS) and reactedfor4 hours at room temperature.

The derivatised FVIII was then added to formulated adsorbed HSA-fibrinogen microcapsules (AND 196) (1 OOmg) from which the formulation buffer had been removed by centrifugation. After a further reaction time of 2 hours, the sample was washed once

in distilled water and reconstituted to 1U FVIII/ml or lOOmg/ml microcapsule concentration.

A control in which derivatised Factor VIII reacted with microcapsules only was included. In a repeat experiment, the samples were formulated in Example 1.

The samples were assayed using the slide test and the Quadratech Coatest VIII C: 4 assay kit as described in Section 3.1.

Table 2 shows the results obtained from the experiments. The slide test activity of AND 196 from the vial used in the experiment was obtained as a control. The time recorded for the starting material was 1-3 seconds.

Table 2 Sample Slide test F o r m u I a t e d Concentration Yield /seconds s I i d e t e s t/IlUnits/% /seconds Control FVIII-MBS + N/A N/A 0.2648 26 microcapsules N/A N/A 0.2942 29 Reaction FVIII-MBS + 0/1-2 N/D 0.2561 26 AND196 0-1 _ 2-8 0.

The samples produced were shown to have high activity when compared to the AND 196 control. There appeared to be no loss in activity of FVIII in the presence of adsorbed fibrinogen.