WO2000035487A1 - Conjugues pharmaceutiques comprenant deux agents actifs - Google Patents

Conjugues pharmaceutiques comprenant deux agents actifs Download PDF

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Publication number
WO2000035487A1
WO2000035487A1 PCT/GB1999/003801 GB9903801W WO0035487A1 WO 2000035487 A1 WO2000035487 A1 WO 2000035487A1 GB 9903801 W GB9903801 W GB 9903801W WO 0035487 A1 WO0035487 A1 WO 0035487A1
Authority
WO
WIPO (PCT)
Prior art keywords
agent
fibrinogen
carrier
conjugate according
microcapsules
Prior art date
Application number
PCT/GB1999/003801
Other languages
English (en)
Inventor
Roy Harris
Nicola Jane Church
Original Assignee
Quadrant Healthcare (Uk) Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Quadrant Healthcare (Uk) Limited filed Critical Quadrant Healthcare (Uk) Limited
Priority to AU10672/00A priority Critical patent/AU1067200A/en
Publication of WO2000035487A1 publication Critical patent/WO2000035487A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6925—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]

Definitions

  • This invention relates to pharmaceutical conjugates, in particular those comprising two active agents, and to their production and use.
  • 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 NHL
  • an insoluble carrier such as an albumin microcapsule, e.g. of fibrinogen and another glycoprotein such as Factor NHL
  • the targeting agent is bound and the effective agent is adsorbed.
  • An illustrative glycoprotein is Factor NHL
  • suitable compounds are blood coagulation factors, proteins of the coagulation cascade, thrombolytic agents, antibodies or ⁇ -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 ⁇ -chains.
  • Further illustrative targeting agents are antibodies, e.g. to a bladder or other tumour.
  • products of the invention may usefully include appropriate other active agents.
  • active 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.
  • the preferred size is up to 6 ⁇ m, e.g. 1 to 4 ⁇ m, 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.
  • 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.
  • the spray-dried product may be reacted with a chemical cross-linking agent.
  • heat and ⁇ - irradiation are preferred, and may also sterilise the dry powder products.
  • one active agent is bound to such a carrier without a linker, by adsorption. This may be achieved by precipitation of the 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.
  • 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.
  • 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 of the 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%.
  • 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 125 I, 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).
  • 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 10 9 microcapsules per kg body weight.
  • the following Examples illustrate the invention.
  • the attachment of two biologically active proteins onto HSA microcapsules has been investigated. Factor NHI 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 /w-maleiniidobenzoyl- ⁇ -hydroxysuccinimide ester has been chosen for the purpose of covalent attachment of one active onto HSA microcapsules.
  • the amine moieties contained within the fibrinogen or FNIII react with the HS-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 NHI loading and activity during these studies is the Quadratech Coatest NHI : C/4 Assay kit.
  • the assay utilises the cofactor capabilities of Factor VHL
  • Alphanate® Factor NHI was obtained from Grifols UK and used throughout. Aliquots of 1 international unit Factor NIH/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 (S ⁇ BTS) that had been doubly virally-inactived.
  • HSA microcapsules used in the Examples were prepared by spray-drying and were then stabilised 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.
  • Example 1 fibrinogen is covalently bound, and Factor Ni ⁇ adsorbed, to microcapsules.
  • Example 2 the Factor NHI is covalently bound, and fibrinogen adsorbed.
  • Example 1 An aliquot was removed from a 3mg/ml stock solution of MBS in DMF (8 ⁇ l,
  • 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 FVHI added (1U FNIH in distilled water). Total reaction volume 1ml.
  • the FNIH was reacted for one hour before washing once in reaction buffer and reconstituting to a FVHI concentration of lU/ml.
  • the slide test assay records the time taken for the final product to react with thrombin to form aggregates.
  • the final product 50 ⁇ l was placed onto a microscope slide and thrombin (50 ⁇ l) added. The two were stirred for 2 seconds and the time from when aggregates begin to form to when the aggregation ends is recorded.
  • 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 1500million microcapsules per ml calculated. The sample was centrifuged at 3500rpm 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 of Factor NHI present and percentage yield obtained from the Coatest assay for the reaction sample and controls.
  • the concentration of FNIII in ⁇ Units 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.
  • the derivatised FVTTI 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.
  • Example 1 A control in which derivatised Factor VIII reacted with microcapsules only was included. In a repeat experiment, the samples were formulated in Example 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.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nanotechnology (AREA)
  • Molecular Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un conjugué pharmaceutique comprenant un véhicule insoluble auquel un premier agent actif est lié par adsorption, et un deuxième agent actif lié par l'intermédiaire d'un segment de liaison à un groupe fonctionnel porté par ledit véhicule.
PCT/GB1999/003801 1998-12-17 1999-11-15 Conjugues pharmaceutiques comprenant deux agents actifs WO2000035487A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU10672/00A AU1067200A (en) 1998-12-17 1999-11-15 Pharmaceutical conjugates comprising two active agents

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9827813.8A GB9827813D0 (en) 1998-12-17 1998-12-17 Pharmaceutical conjugates
GB9827813.8 1998-12-17

Publications (1)

Publication Number Publication Date
WO2000035487A1 true WO2000035487A1 (fr) 2000-06-22

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ID=10844425

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1999/003801 WO2000035487A1 (fr) 1998-12-17 1999-11-15 Conjugues pharmaceutiques comprenant deux agents actifs

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AU (1) AU1067200A (fr)
GB (1) GB9827813D0 (fr)
WO (1) WO2000035487A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007015107A3 (fr) * 2005-08-04 2007-07-26 Haemostatix Ltd Agent therapeutique
CN108267577A (zh) * 2018-01-15 2018-07-10 广州市妇女儿童医疗中心 一种EV71病毒IgA抗体检测试纸条

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998017319A2 (fr) * 1996-10-21 1998-04-30 Quadrant Healthcare (Uk) Limited Substituts de plaquettes et procedes de conjugaison appropries pour leur preparation
WO1999025383A1 (fr) * 1997-11-14 1999-05-27 Quadrant Healthcare (Uk) Limited Conjugues comprenant deux principes actifs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998017319A2 (fr) * 1996-10-21 1998-04-30 Quadrant Healthcare (Uk) Limited Substituts de plaquettes et procedes de conjugaison appropries pour leur preparation
WO1999025383A1 (fr) * 1997-11-14 1999-05-27 Quadrant Healthcare (Uk) Limited Conjugues comprenant deux principes actifs

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007015107A3 (fr) * 2005-08-04 2007-07-26 Haemostatix Ltd Agent therapeutique
GB2429153B (en) * 2005-08-04 2010-03-10 Haemostatix Ltd Artificial platelets
CN108267577A (zh) * 2018-01-15 2018-07-10 广州市妇女儿童医疗中心 一种EV71病毒IgA抗体检测试纸条

Also Published As

Publication number Publication date
GB9827813D0 (en) 1999-02-10
AU1067200A (en) 2000-07-03

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