MXPA99003677A - Platelet substitutes and conjugation methods suitable for their preparation - Google Patents
Platelet substitutes and conjugation methods suitable for their preparationInfo
- Publication number
- MXPA99003677A MXPA99003677A MXPA/A/1999/003677A MX9903677A MXPA99003677A MX PA99003677 A MXPA99003677 A MX PA99003677A MX 9903677 A MX9903677 A MX 9903677A MX PA99003677 A MXPA99003677 A MX PA99003677A
- Authority
- MX
- Mexico
- Prior art keywords
- further characterized
- fibrinogen
- albumin
- microcapsules
- separator
- Prior art date
Links
- 210000001772 Blood Platelets Anatomy 0.000 title claims abstract description 14
- 230000021615 conjugation Effects 0.000 title abstract description 5
- 238000002360 preparation method Methods 0.000 title description 7
- 108010049003 Fibrinogen Proteins 0.000 claims abstract description 84
- 229940012952 Fibrinogen Drugs 0.000 claims abstract description 84
- 102000008946 Fibrinogen Human genes 0.000 claims abstract description 84
- 229940019698 Fibrinogen containing hemostatics Drugs 0.000 claims abstract description 84
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 32
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 32
- 239000000969 carrier Substances 0.000 claims abstract description 5
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 28
- 108091006822 Human Serum Albumin Proteins 0.000 claims description 28
- 239000003981 vehicle Substances 0.000 claims description 14
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 13
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- 101710027066 ALB Proteins 0.000 claims description 11
- 229940050528 albumin Drugs 0.000 claims description 11
- 239000000126 substance Substances 0.000 claims description 8
- 230000001588 bifunctional Effects 0.000 claims description 7
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- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- JDNTWHVOXJZDSN-UHFFFAOYSA-N Iodoacetic acid Chemical class OC(=O)CI JDNTWHVOXJZDSN-UHFFFAOYSA-N 0.000 claims description 5
- AOJJSUZBOXZQNB-TZSSRYMLSA-N ADRIAMYCIN Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 claims description 4
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- 239000004094 surface-active agent Substances 0.000 claims description 3
- ODKNJVUHOIMIIZ-RRKCRQDMSA-N Floxuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ODKNJVUHOIMIIZ-RRKCRQDMSA-N 0.000 claims description 2
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 claims description 2
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- LXZZYRPGZAFOLE-UHFFFAOYSA-L transplatin Chemical compound [H][N]([H])([H])[Pt](Cl)(Cl)[N]([H])([H])[H] LXZZYRPGZAFOLE-UHFFFAOYSA-L 0.000 claims description 2
- 230000035848 Platelet Aggregation Activity Effects 0.000 claims 1
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- 125000000896 monocarboxylic acid group Chemical group 0.000 claims 1
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- FBPFZTCFMRRESA-KAZBKCHUSA-N D-Mannitol Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KAZBKCHUSA-N 0.000 description 3
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Abstract
Platelet substitutes, comprising fibrinogen, or analogous products useful in therapy, comprise an insoluble carrier to which is bound an essentially non-degraded active protein including the sequence RGD. Such conjugates can be made by a conjugation process comprising 0.01 to 2.5%by weight active fibrinogen, and no more than 50%inactive fibrinogen.
Description
SUBSTITUTES OF PLATELETS AND METHODS OF CONJUGATION ADEQUATE FOR PREPARATION
FIELD OF THE INVENTION This invention relates to platelet substitutes, ie, compositions comprising fibrinogen, and also to conjugation methods that can be used, inter alia, to bind fibrinogen to a particulate carrier.
BACKGROUND OF THE INVENTION
The covalently linked conjugates, comprising an active drug and a vehicle, are useful as a means for depositing a drug, for example, to a specific site of action. Albumin has been proposed as a vehicle for this purpose. The microparticles of albumin, its production and use as a vehicle, are described in WO-A-9618388. The covalent binding of large peptides and proteins to microcapsules of human serum albumin (HSA) can cause numerous problems. There may not be enough binding sites for entanglement due to poor contact between the protein and the surface of the microcapsule. Similarly, it is difficult to control intramolecular entanglement, rather than intermolecular entanglement, when short or null interlayers are used, such as glycoaldehyde or EDC, respectively. This can lead to low microcapsule loading and protein invactivation. Agam and Livne, in a series of articles, Blood 55: 186-191 (1983)
Thromb. Haemostasis 51: 145-9 (1984) and 59: 504-6 (1988) and Eur. J. Clin. Invest. (1991), showed that fibrinogen coated on fixed platelets increased platelet aggregation, and that fibrinogen-coated erythrocytes reduced bleeding times in thrombocytopenic rats. The fixation involved the use of formaldehyde. Coller et al., J. Clin. Invest ,. 89: 546-555 (1992), describe "thromboerythrocytes", a semi-artificial autologous alternative to platelet transfusions. To avoid limitations and drawbacks by the use of fresh platelets, erythrocytes were harvested to peptides containing the RGD cell recognition sequence, using a bifunctional interleaver. In a summary presented at the fifteenth International Society Congress on thrombosis and hemostasis, in Jerusalem, Israel, from June 11 to 16, 1995, Yen et al. Reported the hemostatic potential of "thromboespheres", ie, microspheres of HSA. interlaced, with an average diameter of 1.1-1.3 μm, with human fibrinogen covalently bound to their surfaces. In a thrombocytopenic rabbit model, the bleeding times of the ear were reduced. HSA microspheres prepared by the process described in US-A-5069936, ie, a solution / desolvation process using glutaraldehyde as an entanglement agent, ethanol to cause precipitation, and a surfactant to modify the surface were reported. of the interlaced protein molecules. These steps do not provide control of the size of the microspheres, they can cause the bound protein to be degraded, and they are inconvenient for the manufacture of platelet substitutes on a large scale. US-A-5069936 describes the covalent attachment of several biological molecules, but not fibrinogen. A polyaldehyde is proposed as a covalent binding agent. Examples 12 and 14 use glutaraldehyde to bind antibody and enzyme (alkaline phosphatase), respectively. WO-A-9639128 (published December 12, 1996) also describes "thrombospheres". Again, no specific preparation is given. Fibrinogen is an adhesive glycoprotein that contains the sequence RGD (S). This molecule and other glycoproteins (including fibronectin and collagen, among others) can mediate the adhesion of tumor cells to subendothelial layers. These glycoproteins interact with the integrins present in the tumor cells, for example, the fibronectin receptor, and the GPIIb / llla receptor on platelets; see Dardik et al., Int. J. Cancer 70: 201 -7 (1997).
One of the main problems in the surgical treatment of cancers is the increased risk of tumor cells being released into the circulatory system. This is a reason for increased morbidity in patients with prostate cancer, after surgery. It would be convenient to remove the circulating metastatic tumor cells, or inhibit their deposition on vascular surfaces.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with one aspect of the present invention, a novel method for joining peptides (by which any peptide, polypeptide, protein or conjugate thereof) such as fibrinogen to microcapsules is understood, allows a separator (eg, fatty acid) or small peptide) is inserted between the protein and the microcapsule. More specifically, the invention utilizes the fact that a vehicle such as HSA has free thiol groups, with which a bifunctional compound can react, the bifunctional compound having a group selectively reactive with the active component (drug) to be conjugated. By virtue of the invention, a controllable entanglement can be achieved due to the specific character of one of the linking groups for the free thiol group available in vehicles such as HSA. Controllable entanglement is an important aspect of the present invention, since it can have a direct relationship on the activity of the bound molecule.
The separator may include digestible peptides with enzyme. labile bonds to acid or alkali, and be of variable length, depending on the requirements of the application. The length of the separator may be another important aspect of this invention, since it can determine the ability of the conjugate to send receptors, such as fibrinogen, to GPIIb / Illa. In accordance with a second aspect of the invention, for example, by the use of the novel method, a novel pharmaceutically acceptable product has utility as a platelet substitute. Said product comprises an insoluble carrier, for example, stabilized albumin, to which the fibrinogen binds, essentially without loss of fibrinogen activity. The linkage can be non-chemical, for example, by adsorption or chemistry, for example, using a linker of at least 10 nm in length. This invention provides, in the first instance, pure, robust and therapeutically acceptable platelet substitutes. The purity can be described in the absence of chemical interlayer and / or surfactant. They are suitable for use in the treatment of thrombocytopenia. It is a further feature of the invention that, because fibrinogen acts as an identification agent, the products of the invention can usefully have other active binding agents. Said agents will be selected with respect to the site of action, usually a wound or other bleeding site, and the nature of the problem to be treated.
DETAILED DESCRIPTION OF THE INVENTION
The vehicle 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 μm, for example, from 1 to 4 μm, so that the particles can pass through capillaries. Suitable materials and methods, and also methods for stabilizing the microparticles, by heat or by chemical entanglement, are described in detail in WO-A-9218164, WO-A-9615814 and WO-A-9618388, the contents of which are incorporated in the present as a reference. As explained in the last publication, the conditions 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, for example, being smooth and spherical, and containing air. In order to obtain insoluble insoluble microcapsules, the spray-dried product can be reacted with a chemical interlacing agent. However, heat and gamma irradiation are preferred, and dry powder products can also be sterilized.
In one embodiment of the invention, fibrinogen (or other RGD peptide) can be attached to said carrier without a linker, for example, by adsorption. This can be achieved by precipitating the peptide on the surface of the microspheres, for example, by controlling the pH and other conditions, as will be apparent to those skilled in the art. The excess / unbound fibrinogen is then removed with water. In another embodiment, the fibrinogen is bound using a conventional bifunctional reagent such as a polyaldehyde. Glycolaldheido is preferred.
Another example of a spacer is sulfosuccinimidyl 4- (iodoacetyl) aminobenzoate (which is soluble in water). Its length is approximately 1 nm. In the conjugation method that was defined above as the first aspect of the invention, the bound peptide preferably comprises at least fibrinogen. Other examples are factor VIII, factor IX, or other blood factors, proteins of the coagulation cascade, thrombolytic agents, antibodies and antitrypsin a-1. By providing a combination of, say, fibrinogen and factor VIII, the products of the invention may be useful in the treatment of hemophilia. In addition, or as an alternative to the use of a thrombolytic drug such as urokinase, blood clots can be treated by the use of ultrasound. For this purpose, the mciroccapsules of this invention containing air are especially suitable.
The bifunctional compound (ie, Y1-Y-Y2) which is used in the invention can be generated by reaction of simpler Y1-Y3-Y4 and? 5-Y6-Y2 compounds, wherein Y1 is the specific reactive group of thiol, Y4 and Y5 react together, so that Y3 and Y6 together are the separator Y, and Y2 is the group reactive to the drug. Thus, for example, Y1 is group i reactive to thiol, and / or Y4 is COOH, as in ICH2COOH. More specifically, the iodoacetic acid is activated by the addition of EDC (N-hydroxysuccinimide may be included to facilitate the EDC-catalyzed amidation reaction, resulting in the formation of an active succinimidium ester). The activated species is then incubated with a peptide or molecule having amino (Y5) and carboxyl (Y2) free end groups. A suitable peptide comprises, say, from 3 to 6 amino acids, such as Gly or Ala. The carboxyl group of this intermediate conjugate is then activated with EDC. The activated separator is incubated with the protein and a peptide bond is formed with the side chains of the amino acid lysine of the protein. Only one end of the separator can be bound to the protein and, therefore, entanglement can not occur on this occasion. Most plasma proteins do not contain free thiol groups, with HSA being an exception and, therefore, intramolecular entanglement of the protein is avoided. By way of example, the separator has an iodoacetyl N-terminus functionality which will react selectively with the free thiol located in the Cys-34 residue of the HSA microcapsules. The spacer may also have a free carboxylic acid which can be activated, for example, using 1-ethyl-3,3-dimethylamino propylcarbodiimine (EDC), and attached to the amine groups in a peptide. The activation of the separator with EDC can be carried out at pH 6 in pH buffer of 0.05 M 4-morpholinoethane sulfonic acid (pH regulator MES). To prevent any fibrinogen adsorption from occurring, once the fibrinogen has been reacted with the activated N-iodoacetyl peptide, for example, Gly-Leu-Phe, the pH regulator is changed to sodium borate pH regulator at 0.1 M at pH 8. Adsorption of fibrinogen does not occur at this higher pH. Any binding of fibrinogen to the HCA microcapsules will occur solely as a result of the free thiol of the HCA microcapsules that react with the carbon atom adjacent to the iodine molecule at the N-terminus of the separator. The optimum pH for the thiol-iodine interaction is 8. To increase the binding of fibrinogen (by way of example) by means of the separator on the microcapsules, the free thiol content of the microcapsules can be increased using Traut reagent (2-iminothiolane). This reagent modifies the e-amino groups of lysine in thiol groups, resulting in an increase in the number of available free thiols to bind with the separator and thus with fibrinogen. An increased charge can also be achieved by linking amino acids or peptides containing thiol to the microcapsules before binding to the protein of interest (eg, cysteine, reduced glutathione). Chemical linkers such as minothiolane can be used to introduce (as well as to increase the number of) thiol groups. Other proteins can be used to produce microcapsules if, thiol groups were added to their surface, as an alternative to using the inherent properties of HSA (ie, free thiol groups). The protein plus the separator are then incubated with HCA microcapsules containing free thiol groups. The preparation of said microcapsules by spray drying, without loss of functional groups, is described in WO-A-9615184 and WO-A-9618388. The molar ratio of separator: protein must be high enough to allow enough groups to be bound to the protein for interlacing with microcapsules, but low enough not to deactivate the protein. This specific procedure generates the intermediate conjugate ICH2CO (A) nCOOH, where (A) n represents n residues of the same amino acids or different amino acids. The choice of n length controls of the separator, for example, 10 to 600 nm (1 to 60 Angstroms), depending on the application required, is often at least 50 nm. The steps of the process described above can be carried out in solvent systems based mainly on aqueous solutions. The reaction by-products can be easily removed.
The entanglement technology should allow the controllable binding of peptides and proteins to the microcapsules, better retention of the activity of the protein, and the ability to modify the separator in terms of length and cutting capacity. As indicated above, the products of the invention containing fibrinogen can act at the site of the tumors. Therefore, they can be used in tumor therapy, for example, by linking a cytotoxic agent by the particular method of this invention, or by the methods described in WO-A-9618388. Suitable cytotoxic agents include methotrexate, dixorubicin, cisplatin or 5-fluoro-2'-deoxyuridine. Delivery of drugs to tumor cells can be achieved using products of the invention as vehicles that directly react with the cells, or participating in the aggregation and deposition of fibrin at the cell adhesion site. The products of this invention can be loaded with cytotoxic agents or a combination of cytotoxic and indicator agents. They can then be used to identify tumor cells disseminated in the circulation, by specific interactions with the cell's glycoprotein receptors (by search and destruction), or by participating in the process of platelet aggregation at the adhesion site. In both cases, the cytotoxic drug is concentrated at the site of the invading tumor cells.
Alternatively, tumor aggregation can be inhibited in the circulatory system, or even at the site of adhesion, coating the surface of the tumor cell with products of the invention, and blocking the sites / mechanisms that activate the platelets. This would then allow the body's natural defense mechanisms to facilitate the removal of tumor cells. Products containing, for example, the GPIb receptor (which interacts with von Willebrands factor) or collagen receptors or other components of the subendothelial matrix can also be released to potentially block the tumor cell binding sites by coating the matrices. subendothelial The product must still allow an interaction with the platelets in the site of a wound, but it must also restrict the invasion of the vascular wall by any immobilized tumor 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 ELISA test for fibrinopeptide A (FPA). In a test for FPA, incubation of a constant amount of fibrinopeptide A antibody in excess with the sample (or standard), leads to the formation of an antigen / antibody complex. The concentration of the residual excess antibody is inversely proportional to the amount of FPA in the sample (or standard).
To determine the antibody concentration, aliquots of the incubation mixture are transferred, for subsequent incubation, into reaction vessels coated with excess FPA. The antigen-antibody complexes obtained attached to the wall form sandwich complexes with anti-IgG antibodies labeled with peroxidase. The amount of these complexes provides a direct measurement of the concentration of FPA in the sample. The obtained sandwich complexes are determined by enzymatic reaction of peroxidase with H2? 2 / orthophenylenediamine (chromogen) and spectrophotometric measurement subsequent to 492 nm. Due to the inverse relationship between the antigen concentration and the bound enzyme activity, the measured absorbances decrease as the concentration of FPA in the sample increases. The results are evaluated by constructing a reference curve with standards of known concentrations. 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%, of active fibrinogen. The amount of fibrinogen should not be too large, to avoid aggregation, for example, of 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%, be active. This can be determined with respect to the total fibrinogen content, which can again be measured by methods such as the ELISA test. The total fibrinogen content can also be determined by radiolabeling, for example, with I 125, and counting, by conventional procedures. The fibrinogen can be derived from blood, transgenic or recombinant, of full length, or any active fragment thereof. The fragments are described, inter alia, by Coller et al., Cited above. For use as a therapeutic agent, a product of the invention can be administered as is, or can be mixed with, any suitable vehicle known to those skilled in the art. The amount of the product administered will be determined primarily based on the severity of the wound or other condition that will be treated. A typical dosage may be 1.5 x 109 microcapsules per kg of body weight. The following examples illustrate the invention. The fibrinogen used in the examples was a full-length product, derived from blood and commercially available, which had been dually inactivated by virus. The HSA microcapsules used in the examples were prepared by spray drying, and then stabilized by heating, as described in WO-A-9615814. The microcapsules were immersed in Tween 80 at 1%, and thoroughly washed with PFPW to remove the Tween 80 and the excipient before use.
PFPW = purified water free of pyrogens. DTNB = 5,5-dithiobis (nitrobenzoic acid). The free thiol content was measured using the Ellman test with DTNB. This reagent participates in a mechanism of exchange of thioi with the free thiols present in the protein under examination, and releases TNB, which can be measured at 412 nm using UV spectrophotometry. IS.
EXAMPLE 1
N-hydroxysuccinimide ester of iodoacetic acid (IAAE) was reacted with tetra-alanine (AAAA) in a mixture of methanol and distilled water for 1 hour at room temperature. EDC was added in a 1.2 molar ratio when compared with AAAA in distilled water for 5 minutes, after which fibrinogen was added, which had been resuspended in distilled water. After stirring for 1 hour at room temperature, the microcapsules were added, and the mixture was stirred at room temperature for another 16 hours. The microcapsules were washed 6 times with distilled water after the reaction to remove all unreacted fibrinogen and separator, and then resuspended in distilled water to give a final concentration of microcapsules of 100 milligrams per milliliter. It was calculated that the amount of fibrinogen used (54 mg) is 0.5 molar equivalent when compared to the microcapsules (20 mg).
This appeared to be the maximum possible charge, given the free thiol content of the microcapsules. The results of the ELISA test revealed that a fibrinogen load of 0.5 mg per 100 mg of HSA had been achieved. A slide test carried out using 5 mg of labeled microcapsules and 0.15 units of thrombin gave a positive result. The aggregation occurred immediately after the addition of thrombin. A control experiment was also carried out using the same amounts of microcapsules and fibrinogen without IAAE, AAAA or EDC. From this sample, the results of the ELISA test showed that a fibrinogen load of 0.06 mg per 100 mg of HSA had been achieved. This had arisen without the use of an interlacer. The sample also gave a positive slide test result, but aggregation did not occur until approximately 12 seconds after the addition of thrombin.
EXAMPLE 2
The procedure of Example 1 was repeated, but to optimize the reaction between IAAE and AAAA, we continued using inverted phase HPLC. For this purpose, it was necessary to investigate the amount of AAAA required to convert the entire IAAE into a product. All unreacted IAAEs could possibly participate in inconvenient side reactions later in the synthesis. Consequently, the amount of AAAA that was reacted increased from 1 molar equivalent to 4, while the amount of IAAE used remained constant.
EXAMPLE 3
The procedure of Example 1 was repeated, but the amount of separator required for a given amount of fibrinogen was investigated using a ratio of IAAE: AAAA of 1: 4 and an excess of EDC of 1.2 molar equivalents with respect to AAAA. The amount of separator used was calculated using the number of moles of IAAE present, since this is the active constituent and the limiting factor of the separator preparation. The IAAE-AAAA-EDC: fibrinogen ratio was thus increased from 1: 1 to 1: 5. The results of the ELISA test revealed that a ratio of fibrinogen: lAAE-AAAA-EDC of 1: 2 reproducibly produced the highest fibrinogen load. The load was calculated to be 0.06 mg per 100 mg of HSA, and the sample was added within 5 seconds using the slide test.
EXAMPLE 4
In a further optimization experiment, Example 1 was repeated, but with a reduced amount of fibrinogen. The ratio of IAAE-AAAA- (EDC) -fibrinogen: microcapsules was varied using 0.5, 0.3, 0.1, 0.05 and equivalents. The results of the ELISA test revealed that all samples except the 1: 0.01 ratio had an acceptable load of fibrinogen. The samples varied between 0.1 and 0.2 mg of fibrinogen per 100 mg of HSA, and the experiment showed that reducing the amount of fibrinogen to only 0.5 molar equivalents produced a load as high as using 0.5 molar equivalents.
EXAMPLE 5
As the results obtained in Example 4 were fairly uniform, two experiments were carried out. Table 1 shows the amounts used to investigate the fibrinogen loads for the first experiment using the following ratios:
IAAE: AAAA (1: 4) lAAE-AAAA-EDC: Fibrinogen (2: 1) IAAE-AAAA- (EDC) -Fibrinogen: Microcapsules (0.3: 1)
Table 2 summarizes the quantities required for the second experiment, in which the same quantities as in the first experiment were used, except that:
IAAE-AAAA- (EDC) -Fibrinogen: Microcapsules (0.05: 1)
TABLE 1 TABLE 2 In both experiments, the microcapsules were reacted with the separator in sodium phosphate at 0.1 M, pH regulator of sodium chloride at 0.15 M at pH 8 and at room temperature for 16 hours. The microcapsules were then thoroughly washed with PFPW, and resuspended to give a final concentration of microcapsules of 100 milligrams per milliliter. The results of the ELISA test obtained for the samples of ratios of 0.3 and 0.05 revealed that 0.38 and 0.42 milligrams of fibrinogen were bound per 100 milligrams of HSA, respectively. The results of the slide test for both samples yielded positive results, with aggregation occurring approximately 2 seconds after the addition of thrombin.
EXAMPLE 6
1 mg / ml tetra-alanine (AAAA) is prepared. 3 mg of tetra-alamin (Sigma) are weighed in a 7 ml container, dissolved in 3 ml of PFPW, and subjected to swirling action. 3 mg / ml of N-hydroxysuccinimide ester of iodoacetic acid (IAAE) are prepared. 3 mg of tetra-alanine (Sigma) are weighed in a 7 ml container and dissolved in 1 ml of methanol. This is subjected to swirling action.
Pipette 70μl of the tetra-alanine in a 7ml container followed by 5.7μl of IAAE. This is subjected to swirling action. The molar ratio of IAAE: tetra-alanine is 1: 4. 2.3 g microcapsules are formulated with glucose. The mixture consists of approximately 80 mg of protein and 1,600 mg of glucose. A concentration of 50 mg / ml of protein in Tween 80 at 1% (v / v) (Sigma) is prepared. The contents are subjected to swirling action, and allowed to stand for approximately 30 minutes at room temperature, to immerse the hollow microcapsules. An aliquot of 400 μl is added to an Eppendorf after subjecting it to swirling action. The sample is centrifuged at 3000 rpm for 2 minutes in a Beckmann GS-15 centrifuge at room temperature (relative centrifugal force, RCF = 1502 radians / second). This is then washed 3 times with 1 ml of PFPW. The pellet is stored at room temperature until required. A vial of human fibrinogen is reconstituted in 20 ml of 0.1 M saline / 0.025 M sodium phosphate buffer (pH 7.2), which produces a theoretical fibrinogen concentration of 60 mg / ml. The fibrinogen solution is converted into PEG 1000 at 5.8% (w / v) weight at room temperature by the addition of 4 milliliters of PEG solution at 35% (w / v) regulated at its pH. The resulting solution is mixed only by inversion, and then cooled on ice for 15 to 20 minutes. The resulting precipitate is centrifuged (4000 rpm / 4 minutes / GS15 Beckman). The supernatant is removed and the volume measured. The pellet is then washed by the addition of 7% PEG (w / v) / 0.1 M saline solution / 0.025M sodium phosphate pH regulator (pH 7.2). Half the volume of the original fibrinogen supply material is used to wash the pellet. The pellet is resuspended in the pH regulator and mixed with the agitator of a test tube. The solution is centrifuged at 4000 rpm for 4 minutes. The supernatant is separated from the washed pellet, and the volume is measured. The pellet is reconstituted in 20 ml of 0.025 M sodium phosphate / 0.1 M saline solution regulated in its pH to pH 7.2. The volume of the purified fibrinogen solution is measured. The total protein concentration is determined by the BCA test. The concentration of fibrinogen is calculated by reading the absorbance of UV light at 280 nm. Since it is known that a solution of fibrinogen at 1% (w / v) has an extinction coefficient of 15.5 to 280 nm, the concentration of fibrinogen can be calculated. (See reference Haemostasis and Thrombosis, volume 1, 3rd edition, page 492, by R. F. Doolittle). 1 mg / ml of fresh EDC is prepared in PFPW. 3 mg of EDC (Sigma) is weighed with a 7 ml flask, and dissolved in 3 ml of PFPW. This is subjected to swirling action. The container containing the separator that was reacted is removed from the agitator. 53 μl of the EDC is added to the reaction mixture. The molar ratio of EDC: tetra-alanine is 1: 1.2. This is subjected to swirling action, and is stirred for approximately 5 minutes. 10.3 mg of fibrinogen are added in 258 μl to 40 mg / ml. The molar ratio of fibrinogen: microcapsules of HSA is 0.1: 1. The molar ratio of lAAE: fibrinogen is 2: 1. The vessel is inverted, and stirred for approximately 60 minutes. 20 mg of the washed microcapsules are resuspended in a pH regulator, ie disodium acid phosphate at 0.1 M / NaCl at 0.15M at pH 8.0. The pH is adjusted with HCl to 12N. 613 μl of the pH regulator in the microcapsules is added. This is added to the reaction mixture of 387 μl. This makes a final volume of 1 mi. This is stirred overnight at room temperature. This reaction lasts approximately 16 hours. The container is removed from the stir plate. Centrifuge at 4500 rpm for 1 minute (RCF = 3379 radians per second). The supernatant is removed to be analyzed by ELISA test. The pellet is washed 6 times in 1 ml of PFPW. The washes are also preserved for ELISA test to determine a mass balance. The pellet is then resuspended in 200 μl of PFPW. A sample is provided for ELISA test (100 μl) and slide test determination (100 μl). The product samples are stored at 4 ° C.
EXAMPLE 7
A bottle of formulated microcapsules is prepared, containing approximately 1 g of protein and 2 g of mannitol. The irradiated flasks are stored at 4 ° C. A concentration of 50 mg / ml of protein in Tween 80 at 1% (v / v) is prepared. The contents are subjected to swirling action, and allowed to stand for approximately 30 minutes at room temperature to immerse the hollow microcapsules. For a 1 g preparation, a 20 ml aliquot is added in a 50 ml Beckman centrifuge tube after swirling. This is centrifuged in the Beckman Avanti J-25 centrifuge at 5000 rpm for 3 minutes at room temperature (Relative Centrifugal Force, RCF = 4648 radians / second). This is then washed 2 times with 20 ml of PFPW with centrifugation at 3300 rpm for 2 minutes at room temperature (RCF = 2025 radians / second). The microcapsules are finally washed with 20 ml of pH buffer of 10 mM phosphate (pH 6.0) with centrifugation at 3300 rpm for 2 minutes at room temperature (RCF = 2025 radians / second). The pellet is resuspended in 20 ml of phosphate pH buffer at 10 mM (pH 6.0), and transferred to a 70 ml Sterilin vessel with a magnetic stirrer. The fibrinogen is reconstituted in WFI to produce a theoretical fibrinogen concentration of about 40 mg / ml. The suspension is mixed gently in a roller mixer for 20 minutes. Fibrinogen that is in excess is instantly frozen in liquid nitrogen in vials of cryogengen, and stored (-20 ° C). For a 1 g preparation, fibrinogen (0.25 ml at 40 mg / ml) is added to the microcapsules, with shaking. The molar ratio of fibrinogen: microcapsules of HSA is 0.002: 1. The mixture is stirred for about 4 hours. The container is removed from the stir plate, and the contents are transferred to a 50 ml Beckman tube. Centrifuge at 3300 rpm for 2 minutes at room temperature (RCF = 2025 radians / second). The supernatant is discarded. The pellet is washed 3 times in 20 ml of WFI. The washes are discarded. The pellet is then resuspended in 10 mi using WFI. A 500 μl sample is provided for ELISA test, determination by slide test, and Couiter count. At the end of the Coulter count, the sample is formulated by adding mannitol supply material (153 mg / ml) and phosphate pH regulator supply material (250 mM) to obtain isotonic mannitol (51 mg / ml) , Phosphate pH regulator at 25 mM (pH 7.0 ± 0.2) and a count of 1500 million microcapsules / ml.
EXAMPLE 8
The microcapsules of HSA (50 mg, 0.757 μmol) were resuspended in pH buffer of 0.1 M sodium borate, pH 8.05 (832 μl), and 2-iminothiolane (520 μg, 3.78 μmol) was added in pH buffer of 0.1 M sodium borate, pH 8.05 (168 μl). The reaction was stirred for 1 hour at room temperature, after which the microcapsules were washed in distilled water (5 x 5 ml). The microcapsules were subjected to a final wash in pH buffer of 0.1 M sodium borate, pH 8.05, and resuspended in the same pH regulator (2 ml).
Control Fibrinogen (20 mg, 0.058 μmol) was resuspended in pH buffer MES at 0.05M, pH 6.03 (1.2 ml), and stirred at room temperature for 2 hours. The HSA microcapsules (50 mg, 0.757 μmol) were added in pH buffer of 0.1 M sodium borate, pH 8.05 (2 ml), and the reaction was stirred for another 2 hours at room temperature.
EXAMPLE
N-iodoacetyl-Gly-Leu-Phe (3 mg, 5.95 μmol) was resuspended in pH buffer MES at 0.05M, pH 6.03 (1.2 ml), and EDC (2 mg, 10.4 μmol) was added, and the reaction was stirred for 5 to 7 minutes at room temperature. Fibrinogen (20 mg, 0.058 μmol) was added, and the mixture was stirred for 2 hours at room temperature. The HSA microcapsules (50 mg, 0.757 μmol) were added in pH buffer of 0.1 M sodium borate, pH 8.05 (2 ml), and the reaction was stirred for another 2 hours at room temperature. A sample of each of the control and experimental reactions was obtained for the determination of free thiol. The remaining sample of experimental and control material was washed with distilled water (2 x 5 ml), and resuspended to give a concentration of HSA microcapsules of 100 mg / ml. In this example, the free thiol content of the HSA microcapsules was increased from 0.211 to 1.73 nmoles of SH per nmole of HSA. No effect was observed on the activity of the control sample (without separator). However, when the modified microcapsules were reacted with the fibrinogen that had been incubated with the separator, an active sample was obtained by slide test. The activity of the slide test increased from 5 to 15 seconds in the HSA microcapsules with a higher content of free thiol. This suggests that the increase in the free thiol content of the HSA microcapsules leads to increased activity of the final product, due to the fact that increased binding of the separator, and thus fibrinogen, can be achieved.
EXAMPLE 9
EDC (69.8: 1 of a 0.5 mg / ml solution in water) was added to
N-iodoacetyl tetraglycine (125.5: 1 of a 0.5 mg / ml solution in water), and the mixture was stirred for 5 minutes at room temperature. Fibrinogen (186: 1 of a 55.5 mg / ml solution) was added, and the reaction was stirred for 1 hour at room temperature. Microcapsules loaded with doxorubicin, containing 0.56 moles of drug per mole of HSA (100 mg), resuspended in 1619 ml of water, were added to the activated fibrinogen solution. The mixture was stirred at room temperature for 16 hours. The microcapsules were collected by centrifugation at 3500 rpm for 2 minutes, and the supernatant was removed and discarded. The microcapsules were washed in water (4 x 5 mL), and resuspended in 1 mL of water to give a final HSA concentration of approximately 100 mg / mL. The sample was tested to determine its activity, and it was found that the addition of fibrinogen did not result in the loss of doxorubicin from the microcapsules. In addition, double-charged microcapsules showed activity with thrombin, indicating that bound fibrinogen has continued to be active despite the presence of doxorubicin. The levels of fibrinogen present were determined using the ELISA test. These results have shown a reasonable load of fibrinogen.
Claims (26)
1. - A pharmaceutically acceptable product, characterized in that it comprises an insoluble carrier to which is attached essentially non-degraded active fibrinogen or a fragment thereof, which has platelet aggregation activity.
2. The product according to claim 1, further characterized in that the bond is non-chemical, for example, by adsorption.
3. The product according to claim 1, further characterized in that the binding is covalent, through a chemical linker, in the absence of surfactant.
4. The product according to claim 1, further characterized in that the binding is covalent, through a chemical linker, of at least 10 nm in length.
5. The product according to any of the preceding claims, further characterized in that the vehicle comprises intertwined protein microparticles.
6. The product according to claim 5, further characterized in that the protein of the microparticles is albumin.
7. - The product according to any of the preceding claims, further characterized in that the fibrinogen is of full length.
8. The product according to claim 7, further characterized in that it comprises from 0.01 to 2.5% by weight of active fibrinogen, and no more than 50% of inactive fibrinogen.
9. A method for preparing a covalently bound conjugate of the formula XSYZ, wherein X-SH is a vehicle component having free thiol groups, Y is a spacer, and Z is an active component, characterized in that it comprises the steps of reacting the active component with a bifunctional reagent of the formula Y1-Y-Y2, wherein Y1 is a group selectively reactive with free thiol groups, and Y2 is a group reactive with the active component, but not with thiol groups; and reacting the resulting Y1-Y-Z with the vehicle component.
10. The method according to claim 9, further characterized in that Y1 is I.
11. The method according to claim 9 or 10, further characterized in that Y2 is COOH.
12. The method according to claim 10, further characterized in that the bifunctional reagent is obtained by reacting a separator component with optionally activated iodoacetic acid.
13. - The method according to any of claims 9 to 12, further characterized in that the separator comprises a peptide or fatty acid chain.
14. The method according to any of claims 9 to 13, further characterized in that the active component has NH2 groups.
15. The method according to any of claims 9 to 14, further characterized in that the separator has 10 to 600 nm in length.
16. The method according to any of claims 9 to 15, further characterized in that the vehicle component is in the form of microparticles.
17. The method according to any of claims 9 to 15, further characterized in that the active component is a protein that includes the sequence of RGDS.
18. The method according to claim 17, further characterized in that the RGD protein is fibrinogen.
19. The method according to any of claims 9 to 18, further characterized in that the vehicle is albumin.
20. The method according to claim 19, further characterized in that the vehicle is human serum albumin.
21. - Albumin conjugated through its thiol groups to an active component, by means of a separator of at least 50 nm in length.
22. The albumin according to claim 21, further characterized in that it is obtained by a method according to claim 18 or 20.
23. The albumin according to claim 21 or 22, further characterized in that it is in the form of microparticles
24. The albumin according to any of claims 20 to 22, further characterized in that the active component is fibrinogen, to be used as a platelet substitute.
25. The albumin according to claim 24, further characterized in that the fibrinogen is as defined in claim 8. 26.- A product according to any of claims 1 to 8 and 21 to 25, further comprising an agent bound cytotoxic such as methotrexate, doxorubicin, cisplatin or 5-fluoro-2'-deoxyuridine.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9702652.0 | 1997-02-10 | ||
GB9621886.2 | 1997-02-10 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA99003677A true MXPA99003677A (en) | 2000-05-01 |
Family
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