WO2000029028A1 - Conjugues pharmaceutiques de glycoproteines et d'excipients insolubles - Google Patents

Conjugues pharmaceutiques de glycoproteines et d'excipients insolubles Download PDF

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Publication number
WO2000029028A1
WO2000029028A1 PCT/GB1999/003803 GB9903803W WO0029028A1 WO 2000029028 A1 WO2000029028 A1 WO 2000029028A1 GB 9903803 W GB9903803 W GB 9903803W WO 0029028 A1 WO0029028 A1 WO 0029028A1
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WO
WIPO (PCT)
Prior art keywords
fibrinogen
reaction
microcapsules
carrier
reagent
Prior art date
Application number
PCT/GB1999/003803
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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 AU10673/00A priority Critical patent/AU1067300A/en
Publication of WO2000029028A1 publication Critical patent/WO2000029028A1/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 utility of such conjugates, wherein the linking is via the carbohydrate moiety of the glycoprotein. In this way, the function of the active protein moiety may be retained after binding the glycoprotein to the carrier.
  • the illustrative glycoprotein is, for example, fibrinogen or Factor NHL
  • suitable compounds are blood coagulation factors, proteins of the coagulation cascade, thrombolytic agents, antibodies or ⁇ -1 antitrypsin.
  • another active agent may be bound, covalently or by adsorption; see WO-A-98/17319 and also WO-A-99/25283.
  • glycoproteins such as FNIQ or fibrinogen may be bound to microcapsules via a spacer. More specifically, the invention utilises the fact that a carrier such as HS A has free, SH, ⁇ H 2 or COOH groups, with which a bifunctional compound can react, the bifunctional compound having one group that may be selectively reactive with the glycoprotein to be conjugated.
  • a carrier such as HS A has free, SH, ⁇ H 2 or COOH groups, with which a bifunctional compound can react, the bifunctional compound having one group that may be selectively reactive with the glycoprotein to be conjugated.
  • controllable cross-linking can be achieved due to the specificity of one of the linking groups for the functional group available on on the carrier.
  • Controllable cross-linking is one important aspect of the present invention, since it may have a direct bearing on the activity of the attached molecule.
  • the spacer can include enzyme-cleavable peptides, acid or alkali-labile bonds and be of variable length, depending on the requirements of the application.
  • the length of the spacer may be another important aspect of this invention, as it may determine the conjugate's ability to target receptors, such as fibrinogen to GPIIb/IIIa. Suitable spacer lengths are 10 to 600 nm, e.g. 20 to 400 ran.
  • a product of the invention may have utility as a platelet substitute.
  • a product comprises an insoluble carrier, e.g. stabilised albumin, to which fibrinogen is bound, essentially without loss of the fibrinogen's activity.
  • This invention provides, inter alia, pure, robust, therapeutically-acceptable, platelet substitutes. Purity may be embodied in the absence of chemical cross-linker and/or surfactant. They are suitable for use in the treatment of thrombocytopenia. It is an additional feature of the invention that, because fibrinogen acts as a targeting agent, products of the invention may usefully have other bound 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.
  • 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.
  • the products of the invention may be useful in the treatment of haemophilia.
  • a thrombolytic drug such as urokinase
  • blood clots may be treated by the use of ultrasound.
  • the air-containing microcapsules of this invention are especially suitable.
  • a bifunctional compound (say, Y 1 - Y- Y 2 ) may be used in the invention, to link the carrier and the glycoprotein.
  • Y 1 is thiol-reactive.
  • the hydrazide method of crosslinking may be applied for the attachment of any moiety that possesses a carbohydrate functionality to HSA microcapsules. Specific examples include the ⁇ -chain of fibrinogen, GBIb and Factor Nm.
  • the carbohydrate clusters In order to attach the carbohydrate moiety to HSA microcapsules, the carbohydrate clusters should be subjected to a mild oxidation, e.g. with sodium periodate. This would cleave the cis-diol component of the sugar ring to yield reactive aldehydes.
  • a mild oxidation e.g. with sodium periodate. This would cleave the cis-diol component of the sugar ring to yield reactive aldehydes.
  • the invention preferably utilises the hydrazide concept, as part of a heterobifunctional crosslinker.
  • This linker possesses the hydrazide functionality at one end and be separated from a pyridyldithio group (S-SC 5 H 4 JSI) either by a simple carbon chain length, or a more complex aromatic structure.
  • S-SC 5 H 4 JSI pyridyldithio group
  • the hydrazide group reacts with aldehyde groups on the periodate-treated ⁇ -chain giving rise to a covalent hydrazone link.
  • the pyridyldithiol group participates in a disulfide exchange reaction with the free thiol, Cys34, on HSA microcapsules.
  • Each oligosaccharide chain contains eleven monosaccharide units in total, comprising a combination of -acetylglucose, mannose, galactose and ⁇ -acetylneuraminic acid (sialic acid) residues.
  • the carbohydrates are linked through an asparagine residue on both fibrinogen chains.
  • the carbohydrate structure of the gamma chain of human fibrinogen is shown in Figure 1.
  • a crosslinking spacer based on the hydrazide functional group has been developed for the covalent binding of fibrinogen to HSA microcapsules.
  • the fluorescent label fluorescein-5-thiosemicarbazide has been used to establish the success of the crosslinking reactions involving both fibrinogen and HSA microcapsules.
  • the carbazide moiety is similar to, and reacts in the same manner as, the hydrazide group contained within the crosslinker.
  • 3-(2-pyridyldithio)propionylhydrazide is the preferred crosslinker. It can participate in both hydrazone formation with fibrinogen and a free thiol reaction with HSA microcapsules.
  • the disulphide bond of the hydrazone-derivatised fibrinogen breaks and undergoes a free thiol exchange with the Cys-34 residue on the HSA microcapsules to form a second disulphide bond. This causes the release of pyridyl-2-dithione, the presence of which can be detected by absorbance at 343nm.
  • This crosslinking technology can be extended to include the attachment of isolated gamma chain, which had retained its glycosylation, Factor NHI and other glycoproteins.
  • the free thiol content of the microcapsules may be increased using Traut's reagent (2-iminothiolane). This reagent modifies the ⁇ -amino groups of lysine into thiol groups, resulting in an increase in the number of free thiols available to bind with the spacer and thus fibrinogen.
  • Increased loading may also be achieved by cross-linking thiol-containing amino- acids or peptides to the microcapsules prior to attaching the protein of interest (e.g. cysteine, reduced glutathione).
  • Chemical linkers such as iminothiolane can be used to introduce (as well as increase the number of) thiol groups.
  • Other proteins could be used to produce microcapsules if thiol groups were added to their surface, as an alternative to using the inherent properties of HSA (i.e. free thiol groups).
  • products of the invention containing fibrinogen may act at the site of tumours. Therefore, they may be used in tumour therapy, e.g.
  • cytotoxic agent 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.
  • Products of this invention may be loaded with cytotoxic agents or a combination of cytotoxic and targeting agents. They 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
  • active fibrinogen 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.
  • fibrinogen used in the Examples was a full-length, blood-derived, commercially available product 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.
  • PFPW pyrogen-free purified water.
  • DTNB 5,5-dithiobis(2-nitrobenzoic acid).
  • Free thiol content was measured using the Ellman assay with DTNB. This reagent participates in a thiol exchange mechanism with any free thiols present on the protein under examination, and releases (TNB) which can be measured at 412 nm using UN/NIS spectrophotometry.
  • Sodium phosphate buffer pH 7.0 was used to make the stock fibrinogen solution.
  • ImM sodium periodate at 0°C was quenched with glycerol after 30 minutes.
  • a control was set up for both reactions in which an equal volume of reaction buffer was added in place of sodium periodate.
  • the use of 0.001M sodium periodate resulted in the formation of 68.8nmoles of aldehyde (74.1% yield). Increasing this concentration to 0.002M caused other sugar residues besides sialic acid to be oxidised during the reaction as the calculated percentage +yield gave a value greater than 100%.
  • the conditions required for periodate oxidation were established as 0.001M sodium periodate at 0°C for 30 minutes followed by glycerol quenching.
  • FTSC FTSC was added to the samples in a 10 molar excess with respect to the number of moles of aldehydes per mole of fibrinogen present. The reaction was carried out in the absence of light. An aliquot was removed after 30 minutes and the remainder after 70 hours. See King et al, Biochem. (1986) 25:5774-9, and Hermanson, Bioconjugate Techniques, 1st ed., Acad. Press, USA (1996); these studies suggest that hydrazone formation can take 30 minutes or 70 hours for a 60% yield at pH 6.0, under certain conditions.
  • Excess FTSC was removed by purification of the sample through a PD-10 G25 sephadex column. After elution using 0.1M sodium phosphate, pH 7.2/0. IM sodium chloride buffer, the samples were loaded onto a reducing SDS-PAGE gel to determine whether the formation of the semicarbazone linkage had been successful.
  • Reaction conditions for semicarbazone formation comprise a 2 hour reaction time at a temperature of 37°C. Further, results show that a greater yield is obtained at pH 6.0, although the pH 5.8 values are very similar after 2 hours. Addition of salt and increase in pH may be used to eliminate buffer change during fibrinogen purification.
  • glycerol may be used to quench the oxidation reaction.
  • the number of moles of semicarbazide bound per mole of fibrinogen were calculated by firstly determining the number of moles of fibrinogen contained in the reaction sample. The number of nmoles of semicarbazide present were obtained from reading the sample absorbance against the FTSC standard curve. The control sample value was then subtracted from that of the reaction sample and divided by the number of moles of fibrinogen. The resultant value was 2.516 moles semicarbazide/ mole fibrinogen. The theoretical value for a complete reaction is 8 moles/mole.
  • the success of the free thiol reaction was determined by reacting PDPH with both HSA solution and microcapsules.
  • the absorbance at 343nm was used to detect the presence of pyridyl-2-dithione released as a side product from the reaction.
  • HSA solution (1ml at 138mg/ml, 2090nmoles) in 20 molar excess was incubated with PDPH (23.9 ⁇ g, 104 nmoles) for one hour, an aliquot was removed and the absorbance at 343nm measured. Dithiothreitol (DTT) was added to the remainder of the sample. Any remaining PDPH would react with the DTT releasing the pyridyl-2-dithione moiety, but no increase in absorbance was observed. Therefore, it was assumed that no unreacted PDPH remained and that the reaction with HSA solution was complete.
  • the crosslinker was also reacted with HSA microcapsules as above. Microcapsule slurry concentrations of both 50 and lOOmg/ml were used. The PDPH was dissolved in dichloromethane (DCM) at lOOmg/ml microcapsule concentration.
  • DCM dichloromethane
  • the conditions required for production of derivatised fibrinogen were defined as 0.001M sodium periodate in 0. IM sodium phosphate pH 5.8 for periodate oxidation and two column purification steps utilising 0. IM sodium phosphate pH 7.2/0. IM sodium chloride for elution. Addition of crosslinker in DCM at high concentration was followed by reaction at 37°C for 2 hours.
  • the absorbance readings indicate that the reaction is nearer completion after one hour when the free thiol content of the microcapsules is increased.
  • the margin of increase in absorbance once DTT is added is dramatically reduced using both 5 and 40 molar equivalents of Traut's reagent when compared to the control sample.
  • the 40 molar equivalent sample gave a higher yield of free thiol exchange, the use of 5 molar equivalents were chosen as a fewer number of washes were required to remove excess reagent. A smaller percentage of microcapsules are also lost as a result. From these experiments, an increase in the microcapsule free thiol content is preferred, for greater reaction success.
  • PDPH-derivatised fibrinogen was performed using the conditions summarised above.
  • Microcapsules (1 OOmg) were sunk in 1% Tween 80 and washed in 1 OmM sodium phosphate pH 6.0 to remove excipients. From a lOmg/ml stock solution of Traut's reagent, a volume corresponding to 5 molar equivalents was added. The total reaction volume was made to 2ml with reaction buffer, lOmM sodium phosphate pH 6.0, and incubated at room temperature for one hour. The numerous post reaction washes required, retained only 63 mg of microcapsules. The samples were then reconstituted in 2ml of buffer to give a final concentration of 31mg/ml and reacted with lmg PDPH- derivatised fibrinogen for one hour at room temperature.

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

L'invention concerne un conjugué pharmaceutique d'un excipient insoluble et d'une glycoprotéine liée à cet excipient via sa fraction hydrate de carbone.
PCT/GB1999/003803 1998-11-16 1999-11-15 Conjugues pharmaceutiques de glycoproteines et d'excipients insolubles WO2000029028A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU10673/00A AU1067300A (en) 1998-11-16 1999-11-15 Pharmaceutical conjugates of glycoproteins and insoluble carriers

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9825105.1A GB9825105D0 (en) 1998-11-16 1998-11-16 Pharamaceutical conjugates
GB9825105.1 1998-11-16

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Publication Number Publication Date
WO2000029028A1 true WO2000029028A1 (fr) 2000-05-25

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GB (1) GB9825105D0 (fr)
WO (1) WO2000029028A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2322229A3 (fr) * 2001-10-10 2012-03-07 Novo Nordisk A/S Remodelage et glycoconjugation de facteur stimulant les colonies de granulocytes (G-CSF)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5308617A (en) * 1988-08-10 1994-05-03 Halzyme Ltd. Protein heparin conjugates
WO1996018388A2 (fr) * 1994-12-16 1996-06-20 Andaris Limited Microparticules reticulees et utilisation de ces particules comme vehicules therapeutiques
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 (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5308617A (en) * 1988-08-10 1994-05-03 Halzyme Ltd. Protein heparin conjugates
WO1996018388A2 (fr) * 1994-12-16 1996-06-20 Andaris Limited Microparticules reticulees et utilisation de ces particules comme vehicules therapeutiques
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
QUASH, G.A. ET AL.: "Diagnostic and Therapeutic Procedures with Haptens and Glycoproteins (Antigens and Antibodies) Coupled Covalently by Specific Sites to Insoluble Supports", TARGETED DIAGNOSTICS AND THERAPY, vol. 2, 1989, pages 155 - 186, XP000882639 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2322229A3 (fr) * 2001-10-10 2012-03-07 Novo Nordisk A/S Remodelage et glycoconjugation de facteur stimulant les colonies de granulocytes (G-CSF)

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AU1067300A (en) 2000-06-05
GB9825105D0 (en) 1999-01-13

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