WO2014051537A1 - Dispositif et procédé pour l'élimination d'additifs dans les produits sanguins - Google Patents

Dispositif et procédé pour l'élimination d'additifs dans les produits sanguins Download PDF

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Publication number
WO2014051537A1
WO2014051537A1 PCT/US2012/057023 US2012057023W WO2014051537A1 WO 2014051537 A1 WO2014051537 A1 WO 2014051537A1 US 2012057023 W US2012057023 W US 2012057023W WO 2014051537 A1 WO2014051537 A1 WO 2014051537A1
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Prior art keywords
platelet
platelets
photosensitizer
diafiltration
tff
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PCT/US2012/057023
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English (en)
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Lakshman R. Sehgal
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Biovec Transfusion, Llc
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Priority to PCT/US2012/057023 priority Critical patent/WO2014051537A1/fr
Publication of WO2014051537A1 publication Critical patent/WO2014051537A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/02—Blood transfusion apparatus
    • A61M1/0272—Apparatus for treatment of blood or blood constituents prior to or for conservation, e.g. freezing, drying or centrifuging
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2202/00—Aspects relating to methods or apparatus for disinfecting or sterilising materials or objects
    • A61L2202/20—Targets to be treated
    • A61L2202/22—Blood or products thereof

Definitions

  • the present application generally relates to compositions and methods for maintaining platelet functionality and extending the shelf-life of platelets. More particularly, the present invention relates to platelet preservation compositions comprising a
  • photosensitizer and methods for removing the photosensitizer from the composition prior to platelet transfusion.
  • platelets When blood vessels are damaged, cell fragments released from the bone marrow, called platelets, adhere to the walls of blood vessels and form clots to prevent blood loss. It is important to have adequate numbers of normally functioning platelets to maintain effective clotting, or coagulation, of the blood. Occasionally, when the body undergoes trauma, or when the platelets are unable to function properly, it is necessary to replace or transfer platelet components of blood into a patient. Most commonly, platelets are obtained from volunteer donors either as a component of a whole blood unit, or via plateletpheresis (withdrawing only platelets from a donor and re-infusing the remaining of the blood back into the donor). The platelets then are transferred to a patient as needed, a process referred to as "platelet transfusion.”
  • Platelet transfusion is indicated under several different scenarios.
  • an acute blood loss either during an operation or as a result of trauma, can cause the loss of a large amount of platelets in a short period of time.
  • Platelet transfusion is necessary to restore a normal ability to control blood flow, or haemostasis.
  • an individual can develop a condition of decreased number of platelets, a condition known as thrombocytopenia. The condition can occur as a result of chemotherapy, and requires platelet transfusion to restore normal blood clotting.
  • platelets can be stored for a few days. Platelet sterility is difficult to maintain because platelets cannot be stored at low temperatures, for example 4°C to 5°C. A low storage temperature for the platelets initiates an activation process within the platelets that leads to aggregation and cell death. Bacterial growth in the platelet medium at suitable storage temperatures, e.g., room temperature, can lead to an unacceptable occurrence of bacterial contamination in platelets used for transfusion. In fact, bacterial contamination of platelet products has been recognized as the most frequent infectious risk from transfusion, occurring in approximately 1 of 2000 to 1 of 3000 whole blood derived random donor platelets or apheresis derived single donor platelets.
  • bacterial contamination is considered to be the second most common cause of death overall, from transfusion, with mortality rates ranging from 1 :20,000 to 1 :85,000.
  • FDA Food and Drug Administration
  • One aspect of the present application relates to a method for removing a photosensitizer from a platelet preparation.
  • the method comprises passing a platelet preparation comprising a photosenitizer through a tangential flow filtration (TFF) device having a TFF filter with an average pore size of 500 dalton to 5 ⁇ .
  • TFF tangential flow filtration
  • the TFF device has a TFF filter with an average pore size of 500 dalton to 2 ⁇ .
  • the TFF device has a TFF filter with an average pore size of 3000 dalton to 2 ⁇ .
  • the photosensitizer comprises riboflavin.
  • the photosensitizer comprises psoralen.
  • the photosensitizer comprises amotosalen.
  • the photosensitizer comprises methylene blue.
  • the TFF device is a diafiltration device with a diafiltration buffer.
  • the platelet preparation is circulated through the diafiltration device until a 4-6 volume exchange with the diafiltration buffer is reached.
  • the platelet preparation is circulated through the diafiltration device until a 6- 10 volume exchange with the diafiltration buffer is reached.
  • the platelet preparation is circulated through the diafiltration device until a 10-15 volume exchange with the diafiltration buffer is reached.
  • the diafiltration buffer contains no plasma.
  • the TFF filter is a hollow fiber membrane filter.
  • the hollow fiber membrane filter comprises filter membrane tubes with an inner diameter of 0.5 mm or larger.
  • the platelet preparation is passed through the hollow fiber membrane filter at a flow rate in the range of 20-400 ml/minute.
  • the platelet preparation is passed through the hollow fiber membrane filter at a flow rate in the range of 150-400 ml/minute.
  • the platelet preparation further comprises an antiplatelet agent.
  • the platelet preparation further comprises an anticoagulant.
  • Another aspect of the present application relates to a platelet preservation composition
  • a photosensitizer selected from the groups consisting of riboflavin, psoralen and amotosolen
  • one or more platelet preservation agents selected from the groups consisting of platelet activation inhibitors and anticoagulants.
  • Another aspect of the present application relates to a preserved platelet preparation, comprising unactivated platelets, a photosensitizer selected from the group consisting of riboflavin, psoralen or amotosolen; and one or more platelet preservation agents selected from the group consisting of platelet activation inhibitors and anticoagulants.
  • FIG. 1 shows representative chromatograms from samples after diafiltration.
  • the four traces in each chromatogram are: bottom trace - blank control; 2 nd trace from bottom - 5 ⁇ g/mL argatroban control; 3 rd trace from bottom - 0.25 ⁇ g/mL Eptifibatide control (samples 5-7) or 0.25 g/mL tirofiban control (samples 8-10); top trace - diafiltered samples, samples 5-7 were spiked with either 0.1 ⁇ / ⁇ . tirofiban and 8 ⁇ g/mL argatroban before diafiltration, samples 8-10 were spiked with 0.1 ⁇ g/mL tirofiban and 8 ⁇ g/mL argatroban before diafiltration.
  • FIGS. 2A and 2B show the photo-degradation profile of argatroban at 282 nm.
  • FIG. 2A shows HPLC traces of argatroban samples after various exposures to UV (trace A- F), the positive control (unexposed 50 ⁇ g/mL Argatroban) and a the negative control (saline blank).
  • FIG. 2B is a graphical representation of the loss in peak height associated with the exposure to UV 282 , expressed as % relative to the positive control with standard deviations shown.
  • FIGS. 3 A and 3B shows the photo-degradation profile of argatroban at 308 nm.
  • FIG. 3A shows HPLC traces of argatroban samples after various exposures to UV (trace A-F), the positive control (unexposed 50 ⁇ g/mL Argatroban) and a the negative control (saline blank).
  • FIG. 3B is a graphical representation of the loss in peak height associated with the exposure to UV 3 o 8 , expressed as % relative to the positive control with standard deviations shown.
  • FIG. 4 shows HPLC traces reflecting an identical pattern of photodegradation products at UV 282 nm and UV 3 o 8 nm in the two sets of experiments exemplified in FIGS. 2 and 3.
  • FIGS. 5A-5B show the photo-degradation profile of tirofiban at UV 282 nm-
  • FIG. 5A shows HPLC traces of tirofiban samples after various exposures to UV (trace A-F), the positive control (unexposed 50 ⁇ g/mL Argatroban) and a the negative control (saline blank).
  • FIG. 5B is a graphical representation of the loss in peak height associated with the exposure to UV 282) expressed as % relative to the positive control with standard deviations shown. Traces E and F were below the lower limits of quantitation (LLOQ) for the assay.
  • LLOQ lower limits of quantitation
  • FIGS. 6A-6B show the photo-degradation profile of Tirofiban at UV 308 .
  • FIG. 6A shows HPLC traces of tirofiban samples after various exposures to UV (trace A-C), the positive control (unexposed 50 ⁇ g/mL Argatroban) and a the negative control (saline blank).
  • FIG. 6B is a graphical representation of the loss in peak height associated with exposure to UV 3 o8, expressed as % relative to control with standard deviations shown.
  • FIGS. 7A-7B show the photo-degradation profile of Eptifibatide at UV 282 nm .
  • FIG. 7 A shows HPLC traces of Eptifibatide samples after various exposures to UV (trace A- F), the positive control (unexposed 50 ⁇ g/mL Argatroban) and a the negative control (saline blank).
  • FIG. 7B is a graphical representation of the loss in peak height associated with the exposure to UV 30 8, expressed as % relative to control with standard deviations shown.
  • FIGS. 8A-8B show the photo-degradation profile of Eptifibatide at UV 3 o8 nm-
  • FIG. 8 A shows HPLC traces of Eptifibatide samples after various exposures to UV (trace A- F), the positive control (unexposed 50 ⁇ g/mL Argatroban) and the negative control (saline blank).
  • FIG. 8B is a graphical representation of the loss in peak height associated with the exposure to UV 30 8, expressed as % relative to control and with standard deviations shown.
  • the preserved platelet preparation comprises one or more photosensitizers, such as riboflavin, psarelen and amotosalen.
  • the preserved platelet preparation further comprises one or more platelet preservation agents, such as antiplatelet agents and anticoagulants.
  • the preserved platelet preservation agents Prior to the clinical use of the preserved platelets, the
  • the method comprises the step of passing a platelet preparation comprising a photosenitizer through a tangential flow filtration (TFF) device having a TFF filter with an average pore size of 500 dalton to 5 ⁇ .
  • TFF tangential flow filtration
  • the preservation composition comprises a photosensitizer for pathogen inactivation, a platelet activation inhibitor, and an anticoagulant.
  • the photosensitizer is used in a photoradiation pathogen inactivation process to improve pathogen killing and platelet quality.
  • the platelet activation inhibitors and anticoagulants prevent or reduce activation of platelets during the pathogen inactivation process.
  • photosensitizer refers to a compound which absorbs radiation at one or more defined wavelengths and has the ability to utilize the absorbed energy to carry out a chemical process, such as facilitating the formation of phototoxic species sufficient for killing one or more pathogens.
  • a photosensitizer is "sensitive to” or “sensitized by" radiation at a wavelength if it absorbs the radiation at this wavelength.
  • photosensitizer examples include, but are not limited to, riboflavin, psoralen, amotosalen, quinoline, quinolones, nitric oxide, pyrrole derived macrocyclic compounds, naturally occurring or synthetic porphyrins and derivatives thereof naturally occurring or synthetic chlorins and derivatives thereof, naturally occurring or synthetic bacteriochlorins and derivatives thereof, naturally occurring or synthetic isobacteriochlorins and derivatives thereof, naturally occurring or synthetic phthalocyanines and derivatives thereof, naturally occurring or synthetic naphthalocyanines and derivatives thereof, naturally occurring or synthetic porphycenes and derivatives thereof, naturally occurring or synthetic porphycyanines and derivatives thereof, naturally occurring or synthetic pentaphyrins and derivatives thereof, naturally occurring or synthetic sapphyrins and derivatives thereof, naturally occurring or synthetic benzochlorins and derivatives thereof, naturally occurring or synthetic chlorophylls and derivatives thereof, naturally occurring or synthetic azaporphyrins and derivatives thereof,
  • chlorins and bacteriochlorins of the tetra(hydroxyphenyl) porphyrin series phthalocyanines, hematoporphyrin (BP), protoporphyrin, uroporphyrin III, coproporphyrin III, protoporphyrin IX, 5 -amino levulinic acid, pyrromethane boron difluorides, indocyanine green, zinc phthalocyanine, dihematoporphyrin (514 nm), benzoporphyrin derivatives, carotenoporphyrins, hematoporphyrin and porphyrin derivatives, rose bengal (550 nm), bacteriochlorin A (760 nm), epigallocatechin, epicatechin derivatives, hypocrellin B, urocanic acid, indoleacrylic acid, rhodium complexes, etiobenzochlorins, octaethy
  • the photosensitizer is sensitive to ultraviolet (UV) light.
  • the photosensitizer is sensitive to non-UV light, including longer wavelengths ranging from about 600 to about 1200 nm.
  • a combination of photosensitizers may be utilized, wherein at least one is sensitive to UV light and one is sensitive to non-UV light.
  • the photosensitizer is a compound preferentially adsorbing to nucleic acids, such as riboflavin, psoralen andamotosalen, thereby focusing its photodynamic effects upon microorganisms and viruses with little or no effect upon accompanying platelets and other non-nucleated cells or proteins.
  • nucleic acids such as riboflavin, psoralen andamotosalen
  • the photosensitizer may be an endogenous photosensitizer or a non- endogenous photosensitizer.
  • endogenous refers to photosensitizers naturally found in a human or mammalian body, either as a result of synthesis by the body, ingestion (e.g. vitamins), or formation of metabolites and/or byproducts in vivo.
  • Exemplary endogenous photosensitizers include, but are not limited to, alloxazines, such as 7,8- dimethyl- 10-ribityl isoalloxazine (riboflavin or vitamin B2), 7,8,10-trimethylisoalloxazine (lumiflavin), 7,8-dimethylalloxazine (lumichrome), isoalloxazine-adenine dinucleotide (flavine adenine dinucleotide [FAD]), alloxazine mononucleotide (also known as flavine mononucleotide [FMN] and riboflavine-5-phosphate), vitamin Ks, including vitamin Kl, vitamin Kl oxide, vitamin vitamin K5, vitamin K-S (II), vitamin K6, vitamin K7, vitamin L, their metabolites and precursors, and napththoquinones, naphthalenes, naphthols and their derivatives having planar molecular conformations.
  • alloxazines such
  • alloxazine includes isoalloxazines.
  • Endogenously-based derivative photosensitizers include synthetically derived analogs and homologs of endogenous photosensitizers which may have or lack lower (1-5) alkyl or halogen substituents of the photosensitizers from which they are derived, and which preserve the function and substantial non-toxicity thereof.
  • the photosensitizer is riboflavin.
  • riboflavin is used in the concentration range of 1-200 ⁇ , 25-150 ⁇ , or 50- 100 ⁇ .
  • the photosensitizer is psoralen.
  • psoralen is used in the concentration range of 1-200 ⁇ , 25-150 ⁇ , or 50-100 ⁇ .
  • the photosensitizer is amotosalen.
  • amotosalen is used in the concentration range of 1-200 ⁇ , 25-150 ⁇ , or 50-100 ⁇ .
  • the photosensitizer is methylene blue.
  • methylene blue is used in the concentration range of 0.2-50 ⁇ , 1-20 ⁇ , or 2.5-10 ⁇ .
  • the photosensitizer is added in an amount sufficient to produce phototoxic species killing one or more pathogens.
  • the effective concentration varies for each particular photosensitizer. There is a reciprocal relationship between photosensitizer compositions and light dose, thus, determination of effective concentration, suitable wavelength, light intensity, and duration of illumination is within ordinary skill in the art.
  • the photosensitizer is added in an amount sufficient for inactivating one or more blood-borne pathogens, preferably all blood-borne pathogens, but less than a toxic (to humans or other mammals) or insoluble amount.
  • the photosensitizer is used in a concentration of at least about 1 ⁇ up to the solubility of the photosensitizer in the fluid medium.
  • the platelet preservation agents can be any additives that are added to platelet preparation to preserves the activity and/or extends the shelf-life of platelets.
  • platelet preservation agents include, but are not limited to, platelet activation inhibitors, anticoagulants, oxygen carriers, non-steroidal anti-inflammatory drugs, anti-microbial agents, quenchers, and other additives.
  • Platelet activation inhibitors include any agent that reversibly impedes platelet activation and/or aggregation by blocking sites on the platelet surface can be used as the antiplatelet agent in the present invention.
  • Platelet activation inhibitors include, but are not limited to, GPIIb/IIIa antagonists including bifunctional inhibitors of both GPIIb and Ilia, thrombin antagonists, P2Y12 receptor antagonists, and second messenger effectors.
  • the GPIIb/IIIa antagonists are GPIIb/IIIa antagonists that bind GPIIb/IIIa sites in a reversible manner.
  • the term "reversible” or “reversibly” refers to an act, such as binding or associating, that is capable of reverting back to an original condition prior to the act, for example the state of being unbound or
  • GPIIb/IIIa antagonists examples include Eptifibatide (INTEGRILIN ® , Schering-Plough
  • Tirofiban AGGRASTAT ®
  • Abciximab REOPRO ®
  • Lefradafiban Sibrafiban
  • the GPIIb/IIIa antagonists are bifunctional inhibitors of both GPIIb/IIIa as described in U.S. Patent No. 5,242,810, which is incorporated herein by reference.
  • the platelet activation inhibitors include one or more thrombin antagonists. These agents interact with thrombin and block its catalytic activity on fibrinogen, platelets and other substrates. Heparin and its derivatives (low molecular weight heparins and the active pentasaccharide) inhibit thrombin and/or other coagulation serine proteases indirectly via antithrombin, and the warfarin-type drugs interfere with the synthesis of the precursors of the coagulation serine proteases.
  • the direct thrombin inhibitors approved for clinical use at present Lepirudin, Desirudin, Bivalirudin, Argatroban
  • Melagatran/Xirnelagatran Xirnelagatran
  • the platelet activation inhibitors include one or more P2Y12 receptor antagonists.
  • P2Y12 receptor antagonists include, but are not limited to, prasugrel, cungrelor and AZD6140.
  • the platelet activation inhibitors include one or more second messenger effectors.
  • Second messenger effectors include any agent inhibiting a chemical pathway in a platelet so as to reduce platelet activation.
  • second messenger effectors include, but are not limited to, "Thrombosol” (Life Cell Corp), linear or novel cyclic RGD peptide analogs, cyclic peptides, peptidomimetics, non-peptide analogs conjugated to nitric oxide donor, and the like, and mixtures thereof.
  • Second messenger effectors also include calcium sequestering agents, such as calcium channel blockers, a-blockers, ⁇ -adrenergic blockers and mixtures thereof. More specific examples of calcium sequestering agents include, but are not limited to,
  • anticoagulant citrate dextrose solution anticoagulant citrate dextrose solution modified, anticoagulant citrate phosphate dextrose solution, anticoagulant sodium citrate solution, anticoagulant citrate phosphate dextrose adenine solution, potassium oxalate, sodium citrate, sodium oxalate, amlodipine, bepridil hydrochloride, diltiazem hydrochloride, felodipine, isradipine, nicardipine hydrochloride, nifedipine, nimodipine, verapamil hydrochloride, doxazocin mesylate, phenoxybenzamine hydrochloride, phentolamine mesylate, prazosin hydrochloride, terazosin hydrochloride, tolazoline hydrochloride, acebutolol hydrochloride, atenolol, betaxolol hydrochloride, bisoprolol fumarate, carteolo
  • hydrochloride metipranolol hydrochloride, metoprolol tartrate, nadolol, penbutolol sulfate, pindolol, propranolol hydrochloride, terazosin hydrochloride, timolol maleate, guanadrel sulfate, guanethidine monosulfate, metyrosine, reserpine and mixtures thereof.
  • the platelet activation inhibitor has short-to-ultra short half-life.
  • short-to-ultra short half life is meant that the platelet activation inhibitor is cleared from circulation within 15 minutes to 8 hours, preferably within 4 hours or less, after the infusion of the antiplatelet agent into the patient is stopped.
  • the platelet activation inhibitor is an active agent that binds to or associates with the GPIIb/IIIa sites in a reversible manner and has a circulating half-life of inhibition of 4 hours or less.
  • Short to ultra-short acting GPIIb/IIIa antagonist might include Eptifibatide (INTEGRILIN ® ), Tirofiban (AGGRASTAT ® ), Abciximab
  • the preservation composition includes Eptifibatide.
  • the Eptifibatide is present in the composition at a final concentration of about 5-500 ⁇ g per unit of platelets.
  • the platelet activation inhibitor is Eptifibatide at a final concentration of about 50 ⁇ g per unit of platelets.
  • a unit of platelets obtained by the buffy coat method contains about 3 x 10" platelets in approximately 300 ml of plasma or other suitable preservation composition.
  • a unit of platelets collected by apheresis usually contain 5 x 10 9 platelets in 250 ml of plasma or other suitable fluid.
  • the platelet activation inhibitor is present in the composition at a final concentration that is 2-3 times of the therapeutic concentration.
  • therapeutic concentration refers to the inhibitor concentration that is commonly used in the field for platelet preservation.
  • the preservation composition further comprises one or more anticoagulants.
  • anticoagulants include, but are not limited to, heparin, heparin substitutes, prothrombopenic anticoagulants, platelet phosphodiesterase inhibitors, dextrans, thrombin antagonists, and mixtures thereof.
  • heparin and heparin substitutes include, but are not limited to, heparin calcium, such as calciparin; heparin low-molecular weight, such as enoxaparin and lovenox; heparin sodium, such as heparin, lipo-hepin, liquaemin sodium, and panheprin; and heparin sodium dihydroergotamine mesylate.
  • Suitable prothrombopenic anticoagulants are, for example, anisindione, dicumarol, warfarin sodium, and the like. More specific examples of phosphodiesterase inhibitors suitable for use in the invention include, but are not limited to, anagrelide, dipyridamole, pentoxifyllin, and theophylline.
  • dextrans examples include, for example, dextran 70, such as HYSKON ® (CooperSurgical, Inc., Shelton, Connecticut, U.S.A.) and MACRODEX ® (Pharmalink, Inc., Upplands Vasby, Sweden), and dextran 75, such as GENTRAN ® 75 (Baxter Healthcare Corporation, Deerfield, Illinois, U.S.A.).
  • dextran 70 such as HYSKON ® (CooperSurgical, Inc., Shelton, Connecticut, U.S.A.) and MACRODEX ® (Pharmalink, Inc., Upplands Vasby, Sweden
  • dextran 75 such as GENTRAN ® 75 (Baxter Healthcare Corporation, Deerfield, Illinois, U.S.A.).
  • the anticoagulants may also include Xa inhibitors, Ila inhibitors, and mixtures thereof.
  • the anticoagulant is a short-to-ultra short acting
  • the short-to-ultra short acting anticoagulant is a short-to-ultra short acting factor Xa inhibitor with a circulating half-life of less than 4 hours.
  • ultra-short acting factor Xa inhibitors include, but are not limited to, DX-9065a, RPR- 120844, BX-807834 and SEL series Xa inhibitors.
  • DX-9065a is a synthetic, non-peptide, propanoic acid derivative, 571 D selective factor Xa inhibitor (Daichi). It directly inhibits factor Xa in a competitive manner with an inhibition constant in the nanomolar range).
  • RPR- 120844 (Rhone-Poulenc Rorer)
  • Rhone-Poulenc Rorer is one of a series of novel inhibitors which incorporate 3-(S)-amino-2-pyrrolidinone as a central template (Ewing et al., Drugs of Future 24(7):771-787 (1999)).
  • This compound has a Ki of 7 nM with selectivity >150-fold over thrombin, activated protein C, plasmin and t-PA. It prolongs the PT and PTT in a concentration-dependent manner, being more sensitive to the aPTT. It is a fast binding, reversible and competitive inhibitor of factor Xa.
  • BX-807834 has a molecular weight of 527 Da and a Ki of 1 10 ⁇ for factor Xa as compared to 180 ⁇ for TAP and 40 nM for DX-9065a (Baum et al, Circulation. 98 (17), Suppl 1 : 179, (1998)).
  • the SEL series of novel factor Xa inhibitors are pentapeptides based on L-amino acids produced by combinatorial chemistry. They are highly selective for factor Xa and potency in the pM range.
  • the Ki for SEL 271 1 one of the most potent analogues, is 0.003 M for factor Xa and 40 M for thrombin (Ostrem et al., Thromb. Haemost. 73: 1306 (1995); Al-Obeidi and Ostrem., Exp. Opin. Ther. Patents 9(7):931-953 (1999)).
  • the short-to-ultra short acting anticoagulant is a short- to-ultra short acting factor Ila inhibitor.
  • short-to-ultra short acting anticoagulant include, but are not limited to, DUP714, hirulog, hirudin, melgatran and combinations thereof.
  • the anticoagulant is present in the composition at a final concentration that is 2-3 times of the therapeutic concentration.
  • therapeutic concentration refers to the anticoagulant concentration that is commonly used in the field for platelet preservation.
  • the preservation composition may further comprise a pharmaceutically acceptable oxygen carrier.
  • the oxygen carrier can be any suitable red blood cell substitute.
  • the oxygen carrier is a hemoglobin-based oxygen carrier.
  • the oxygen carrier is an acellular hemoglobin-based oxygen carrier substantially free of red cell membrane (stroma) contaminants.
  • a hemoglobin-based oxygen carrier even in small volumes, as part of the platelet preservation composition provides significantly greater concentration of oxygen than amounts currently made available by the use of oxygen-permeable storage bags.
  • an oxygen carrier e.g. , a stroma- free hemoglobin solution
  • platelets can allow for the use of gas impermeable bags, which reduces the high cost associated with using gas permeable bags.
  • the term "pharmaceutically acceptable oxygen carrier” as used herein refers to a substance that has passed the FDA human safety trials at a hemoglobin dosage of 0.5 g/kg body weight or higher.
  • An oxygen carrier suitable for the invention can be hemoglobin, ferroprotoporphyrin, perfluorochemicals (PFCs), and the like.
  • the hemoglobin can be from human or any other suitable mammalian source.
  • the preservation composition has a hemoglobin concentration from the range of 1 to 18 gm/dl and a methemoglobin concentration of less than about 5%.
  • the hemoglobin based oxygen carrier can be chemically modified to mimic the oxygen loading and unloading characteristics of fresh red blood cells. Additionally, the chemical modification can enhance the buffering capacity of the preferred embodiment and preserve normal physiologic pH.
  • the preservation composition may further comprise one or more non-steroidal anti-inflammatory drugs (NSAIDS).
  • NSAIDS suitable for the invention can be salicylate-like or non-salicylate NSAIDS that bind reversibly and inhibit platelet aggregation in vitro, but are cleared rapidly, i.e. quickly eliminated from the body (typically, in less than about 2 hours after infusion).
  • salicylate-like NSAIDS include, but are not limited to, acetaminophen, carprofen, choline salicylate, magnesium salicylate, salicylamide, sodium salicylate, sodium thiosulfate, and mixtures thereof.
  • non-salicylate NSAIDS include, but are not limited to, diclofenac sodium, diflunisal, etodolac, fenoprofen calcium, flurbiprofen, hydroxychloroquin, ibuprofen, indomethacin, ketoprofen, ketorolac tromethamine, meclofenamate sodium, mefenamic acid, nabumetone, naproxen, naproxen sodium, oxyphenbutazone, phenylbutazone, piroxicam, sulfinpyrazone, sulindac, tolmetin sodium, dimethyl sulfoxide and mixtures thereof.
  • the preservation composition may comprise an anti-microbial agent, preferably a short-to-ultra-short acting broad spectrum anti-microbial agent.
  • an anti-microbial agent preferably a short-to-ultra-short acting broad spectrum anti-microbial agent.
  • short or ultra short acting anti-microbial agent is meant that the agent is cleared from circulation within 15 minutes to 8 hours after the infusion of the antimicrobial into the patient is stopped.
  • Such agents include, but are not limited to, penicillin, monobactam,
  • cephalosporin cephalosporin, carbapenems, vancomycin, isoniazid (INH), ethambutol, aminoglycoside, tetracycline, chloramphenicol, macrolide, rifamycin, quinolone, fluoroquinolone,
  • Quenchers may also be added to the preservative composition to make the irradiation process more efficient and selective.
  • quenchers include antioxidants or other agents to prevent damage to desired fluid components or to improve the rate of pathogen inactivation and are exemplified by adenine, histidine, cysteine, tyrosine, tryptophan, ascorbate, N-acetyl-L-cysteine, propyl gallate, glutathione, mercaptopropionylglycine, dithiothreotol, nicotinamide, BHT, BHA, lysine, serine, methionine, glucose, mannitol, vitamin E, trolox, alpha-tocopheral acetate and various derivatives, glycerol, and mixtures thereof.
  • Quenchers may be added to the platelet preservation composition in an amount necessary to prevent damage to the platelets.
  • 2- deoxy-D-glucose may also be used with the platelet preservation composition of this invention.
  • 2- deoxy-D-glucose slows down the rate of glycolysis by competing with glucose for enzymes utilized in the glycolysis pathway.
  • 2-deoxy-D-glucose is phosphorylated by the same enzymes which phosphorylate glucose, but at a slower rate than that of glucose
  • 2- deoxy-D-glucose may be added to the platelet preservation composition at a concentration of about 10 mM.
  • Filtration is a pressure driven separation process that uses membranes (or filters) to separate components in a liquid solution or suspension based on their size differences. Filtration can be broken down into two different operational modes-normal flow filtration (NFF) and tangential flow filtration (TFF). In NFF, fluid is connected directly toward the membrane under an applied pressure. Particulates that are too large to pass through the pores of the membrane accumulate at the membrane surface or in the depth of the filtration media, while smaller molecules pass through to the downstream side. This type of process is also called dead-end filtration.
  • NFF normal flow filtration
  • TMF tangential flow filtration
  • TFF the fluid is pumped tangentially along the surface of the membrane.
  • An applied pressure serves to force a portion of the fluid through the membrane to the filtrate side.
  • particulates and macromolecules that are too large to pass through the membrane pores are retained on the upstream side. However, in this case the retained components do not build up at the surface of the membrane. Instead, they are swept along by the tangential flow.
  • This feature of TFF makes it an ideal process for finer sized-based separations.
  • TFF is also commonly called cross-flow filtration. However, the term
  • the photosensitizers are separated from the platelet preparation by diafiltration, wherein a diafiltration buffer is added to the platelet preparation during circulation to maintain a constant volume of the platelet preparation.
  • the photosensitizers are removed by diafiltration with 4-6 volume exchange with a diafiltration buffer.
  • the buffer can be a physiologic saline solution (0.9% sodium chloride), or any other platelet storage solution, such as Intersol.
  • the photosensitizers, as well as other platelet preservation agents, such as platelet activation inhibitors and anticoagulants are removed by diafiltration with 4-6 volume exchange with a diafiltration buffer that can be a physiologic saline solution (0.9% sodium chloride), or any other suitable platelet storage solution such as Intersol. These diafiltration can contain up to 5% albumin or 20 to 30% plasma.
  • the photosensitizers, as well as other platelet preservation agents, such as platelet activation inhibitors and anticoagulants are removed by diafiltration with 4-6 volume exchange, 6-10 volume exchange, or 10-15 volume exchange with a diafiltration buffer containing no plasma.
  • Diafiltration is a TFF method of "washing” or removing permeable molecules (impurities, salts, solvents, small proteins, etc) from a solution, including antibodies from plasma which are associated with transfusion related acute lung injury. Because it is a significantly faster and scalable method, diafiltration frequently replaces membrane tube dialysis. The success of diafiltration is largely determined by the selection of an appropriate membrane. The membrane pores must be large enough to allow the permeable species to pass through and small enough to retain the larger species. A rule of thumb in selecting the membrane is to choose a membrane whose pore size is rated 2-5 times smaller than anything to be retained, and 2-5 times larger than anything to be removed by the filtration. A large variety of pore sizes are available in the ultrafiltration and micro filtration range for this purpose.
  • an extraction liquid is circulated outside the filtering tube in a counter current manner to facilitate the filtration process.
  • the extraction fluid comprises 0.9% w/v sodium chloride.
  • a typical continuous diafiltration system in which the diafiltration buffer is automatically added to the process reservoir by vacuum suction. It includes a pump, pressure measurement device, flow measurement device, process reservoir, buffer reservoir, and hollow fiber filter module.
  • the pump circulates the process solution from the process reservoir, through the filter and back to the process vessel at a controlled flow and shear rate. Pressure measurements are acquired in this re-circulation loop to control and record the driving force through the membrane. Careful measurement of the permeate flow rate enables accurate process scale up and process optimization. Diafiltration occurs simply by adding the diafiltration buffer to this circulation loop. Working with a hollow fiber module, tubing and an air-tight sealable bottle is a simple means of performing a continuous diafiltration.
  • a vacuum needs to be created in the process vessel. This can be accomplished by submerging the buffer addition tube into a bottle of diafiltration buffer. As permeate flows out of the system, the vacuum in the sealed process reservoir pulls buffer into it at a flow rate equal to the process flux. When the target volume of diafiltration buffer has been collected in the permeate vessel, the process is stopped simply by stopping the permeate flow and breaking the vacuum seal on the feed reservoir.
  • buffer addition can be controlled to match the permeate flow rate through the use of a single- or double-headed secondary pump adding buffer into the feed or process reservoir.
  • buffer addition can be controlled to match the permeate flow rate through the use of a single- or double-headed secondary pump adding buffer into the feed or process reservoir.
  • concentration There is a relationship between the volume of buffer required to remove a permeable species and the product solution volume in the process reservoir. By understanding this relationship, the cost associated with the process time and the volume of buffer can be minimized.
  • the removal of the photosensitizers by TFF involves the use of micro filtration membranes.
  • Microfiltration membrane materials include, but are not limited to, regenerated cellulose, cellulose acetate, polyamide, polyurethane, polypropylene, polysulfone, polyethersulfone, polycarbonate, nylon, polyimide and combinations thereof.
  • the microfiltration membrane is a hollow fiber membrane made of polysulfone or polyethersulfone.
  • the filter membrane tubes has inner diameter of 0.5 mm or greater with the membrane pore size of 0.05 micron or larger.
  • the membrane has a pore size ranging from a molecular weight cut off of 500 daltons to 0.5 micron, from a molecular weight cut off of 500 daltons to 0.2 micron, from a molecular weight cut off of 500 daltons to 0.05 micron, from a molecular weight cut off of 500 daltons to 0.02 micron; or from a molecular weight cut off of 3000 daltons to 0.5 micron, from a molecular weight cut off of 3000 daltons to 0.2 micron, from a molecular weight cut off of 3000 daltons to 0.05 micron, or from a molecular weight cut off of 3000 daltons to 0.02 micron.
  • these membranes can be chemically modified to provide a greater positive or negative charge depending on the specific application thereby selectively binding a solute of interest.
  • the surface chemistry of these membranes can be modified to specifically bind solutes of interest such as the antiplatelet agents or direct thrombin inhibitors.
  • the platelet preparation is passed through the hollow fiber membrane filter at flow rates ranging from 150 ml/minute to 370 ml/minute. Theses flow rates provide acceptable shear forces from 2000-s to 4000-s.
  • An acceptable pump provides a wide range of flow rates and also provides continuous monitoring of inlet, retentate, permeate and transmembrane pressures.
  • the pump is the Kros Flow II pump (Spectrum Labs, Collinso Dominguez, Calif).
  • a replacement fluid suitable for the removal of antiplatelet and anticoagulant agents would be fluids that are used for the storage of platelets. Typically a 10 to 15 volume exchange will result in the removal of better than 99% of the photosensitizer and other added agents.
  • a unit of platelets obtained by the buffy coat method would contain 3 x 10 1 1 platelets in approximately 300 ml of plasma or other suitable preservation composition.
  • a unit of platelets collected by apheresis usually contain 5 x 10 9 platelets in 250 ml of plasma or other suitable fluid.
  • riboflavin riboflavin
  • psoralen dyes such as amotosalen
  • antiplatelet agent such as Eptifibatide
  • anticoagulant such as argabtroban
  • the preserved platelet composition is passed through the hollow fiber filter in a diafiltration device at flow rates ranging from 20 to 400 ml/min, preferably 150 to 400 ml/min.
  • the hollow fiber membrane filters with a pore size ranging from molecular weight cut off of 500 daltons to 0.5 micron are acceptable.
  • the preferred pore size is 0.05 micron.
  • the preferred surface area of the filtration module is 2500 cm 2 . This setting can allow for the complete removal (>99%) of the photosensitizers, platelet activation inhibitors,
  • the diafiltration buffer i.e., replacement fluid
  • the diafiltration buffer can be any solution suitable for platelet storage.
  • the diafiltration buffer is a commercially available platelet storage solution (T-Sol) with 20% plasma.
  • the photosensitizers, platelet activation inhibitors, anticoagulants, and/or plasma components are removed from preserved platelet composition by passing the composition through a porous material that specifically binds to one or more of the undesirable agents.
  • the porous material comprises a nanofiber.
  • nanofiber include, but are not limited to, cellulose nanofibers, biodegradable nanofibers and carbon nanofibers.
  • Cellulose nanofibers may be obtained from various sources such as flax bast fibers, hemp fibers, kraft pulp, and rutabaga, by chemical treatments followed by innovative mechanical techniques.
  • the nanofibers thus obtained have diameters between 5 and 60 nm.
  • the ultrastructure of cellulose nanofibers is investigated by atomic force microscopy and transmission electron microscopy.
  • the cellulose nanofibers are also characterized in terms of crystallinity.
  • the membrane filter is a reinforced composite film comprising 90% polyvinyl alcohol and 10% nanofibers.
  • cellulose fibers can be modified to provide specific binding sites for a given photosensitizer. These fibers can be coated onto the surface of currently available disposable filter platforms like those used for sterilizing small volumes of fluids.
  • Biodegradable polymers such as poly(glycolic acid) (PGA), poly(L-lactic acid) (PLLA) and poly(lactic-co-glycolic acid) (PLGA), can be dissolved individually in the proper solvents and then subjected to electrospinning process to make nanofibrous scaffolds. Their surfaces can then be chemically modified using oxygen plasma treatment and in situ grafting of hydrophilic acrylic acid (AA).
  • the biodegradable nanofibrous scaffold has a fiber thickness in the range of 200-800 nm, a pore size in the range of 2-30 micron, and porosity in the range of 94-96%.
  • the ultimate tensile strength of PGA will be about 2.5 MPa on average and that of PLGA and PLLA will be less than 2 MPa.
  • the elongation-at-break will be 100-130% for the three nanofibrous scaffolds.
  • higher ratios of oxygen to carbon, lower contact angles and the presence of carboxylic (— COOH) groups are identified.
  • the hydrophilic functional groups can be successfully adapted on the surface of electrospun nanofibrous scaffolds.
  • Carbon nanofibers can be synthesized by chemical vapor deposition (CVD).
  • Amino acids such as alanine, aspartic acid, glutamic acid and enzymes such as glucose oxidase (GOx) can be adsorbed on CNF.
  • the properties of CNF are characterized by the pH value, the concentration of acidic/basic sites and by naphthalene adsorption.
  • Preserved platelet composition agents may also be removed from a platelet preparation by centrifugation or chromatography. Briefly, platelets may be precipitated under conditions that do not precipitate the antiplatelet agent and anticoagulant. The precipitated platelets are then washed and resuspended for clinical use. Similarly, chromatographic methods such as column chromatography may also be used to separate the platelets from the antiplatelet agent and anticoagulant. Alternatively, the preserved platelet composition agents may be removed from a platelet preparation by affinity-based removal methods such as magnetic beads coated with antibodies that bind to the preserved platelet composition agents.
  • the preservation composition comprises a photosensitizer and one or more preservative agents.
  • photosensitizers include riboflavin, psoralen and amotosalen.
  • preservative agents include, but are not limited to, platelet activation inhibitors, anticoagulants, oxygen carriers, non-steroidal anti-inflammatory drugs, and anti-microbial agents.
  • the photosensitizer is removed from the platelet preparation by TFF after subjecting the platelet preparation to photoradiation. In other embodiments, the photosensitizer is removed from the platelet preparation by TFF without subjecting the platelet preparation to photoradiation.
  • the preservation composition comprises a photosensitizer, a platelet activation inhibitor and/or an anticoagulant.
  • the photosensitizer is used in a photoradiation pathogen inactivation process to improve pathogen killing and platelet quality.
  • the platelet activation inhibitors and/or anticoagulants prevent or reduce activation of platelets during the pathogen inactivation process.
  • the preservation composition comprises a
  • photosensitizer a platelet activation inhibitor and/or an anticoagulant, and an oxygen carrier.
  • Platelet preservation compositions and preserved platelet compositions of the present invention may be stored in a range of temperatures between about -80°C to about 42°C.
  • room temperature or “ambient temperature” refers to a temperature in the range of 12°C to 30°C
  • body temperature refers to a temperature in the range of 35°C to 42°C
  • refrigeration temperature refers to a temperature in the range of 0°C to 12° C
  • freezing temperature refers to a temperature below 0°C.
  • the term “cold storage” or “storage at low temperature” refers to storage at -20°C to 12°C, preferably 2°C to 12°C, more preferably 4°C to 8°C.
  • the preservation composition of the present invention may be used in an amount from about 60 to about 200 ml for about one unit of platelets (typically about 80 to about 100 ml of platelets).
  • the preservation composition of the present invention may be combined with about one unit of whole blood, typically about one pint, and separated into various components to afford about one-sixth to about one-tenth whole blood unit of treated platelets.
  • the preservation composition contains a photosensitizer and an inhibitor of platelet activation dissolved in about 45 to about 55 ml of an oxygen carrier.
  • the preservation composition comprises Eptifibatide and Argatroban.
  • the inhibitor of platelet activation can also be dissolved in about 45 to about 55 ml of normal saline to preserve the freshness of the platelets without an oxygen carrier.
  • the amount of the preservative agents present in the preservation composition depends on the type of preservative agent.
  • the amount of the platelet activation inhibitor should be sufficient to reversibly inhibit binding to a ligand or site on the platelet in a manner that is sufficient to inhibit platelet function.
  • suitable amounts in the preservation composition may range from about 0.5 mg to about 3 mg for 50 ml of acellular hemoglobin-based oxygen carrier substantially free of red cell membrane (stroma) contaminants.
  • NSAIDs for example, ibuprofen, may be preferably present in the preservation composition in an amount from about 20 mg to about 60 mg for each 50 ml of acellular hemoglobin-based oxygen carrier that is substantially free of red cell membrane contaminants.
  • preservative agents in the preserved platelets are preservative agents in the preserved platelets.
  • pharmaceutically acceptable refers to a substance that complies with the regulations enforced by the FDA regarding the safety of use in a human or animal subject or a substance that has passed FDA human safety trials.
  • pharmaceutically acceptable platelet activation inhibitor refers to an active agent that prevents, inhibits, or suppresses platelet adherence and/or aggregation, and comports with guidelines for pharmaceutical use as set forth by the FDA.
  • platelets can be stored at room temperature or low temperature as further described below. Platelet function also can be better maintained throughout the 5 -day storage period mandated by the FDA, or longer.
  • Another aspect of the present invention relates to a preserved platelet composition, comprising platelets, an effective amount of a photesensitizer, and an effective amount of one or more platelet preservation agents comprising a platelet activation inhibitor and/or an anticoagulant, wherein the preserved platelet composition is sterilized by exposure to a radiation at a wavelength that sensitizes the photosensitizer and wherein the platelet composition is substantially free of red blood cells or other blood nutrients.
  • the term "effective amount,” as used herein, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result, e.g., sufficient to inactivate pathogens in the platelet preparation, or sufficient to inactivate platelets in a platelet preparation or prevent activation of platelets in a platelet preparation.
  • the preserved platelet composition comprises platelets admixed with a preservation composition comprising a photosensitizer and a platelet activation inhibitor.
  • the preserved platelet composition comprises platelets admixed with a preservation composition comprising a photosensitizer and an anticoagulant.
  • the preserved platelet composition comprises platelets admixed with a preservation composition comprising a photosensitizer, a platelet activation inhibitor and an anticoagulant.
  • the preserved platelet composition may include any of the above-described preservation compositions.
  • the photosensitizers and other preservative agents may be removed prior to transfusion in order to reduce any potentially toxic or adverse effects as further described below.
  • the platelets are substantially free of activated platelets both prior to and following treatment of the platelet compositions with the photosensitizers and preservative agents of the present invention. Platelet sources and methods of making the same are further described below.
  • Another aspect of the present invention relates to a method of extending the shelf-life of platelets using the preservation composition described above.
  • the method comprises (a) admixing a platelet composition with an effective amount of a photosensitizer to form a platelet mixture; and (b) irradiating the platelet mixture with light under conditions sufficient to sensitize the photosensitizer and inactivate pathogens in the platelet mixture, wherein an effective amount of a platelet activation inhibitor and/or an effective amount of an anticoagulant are added to the platelet composition either before or immediately after step (b).
  • the photosensitizer is added in an amount sufficient to produce phototoxic species killing or inactivating the reproductive ability of one or more pathogens.
  • the effective concentration varies for each particular photosensitizer.
  • the photosensitizer is used in a concentration of at least about 1 ⁇ up to the solubility of the photosensitizer in the fluid.
  • the photosensitizer is riboflavin and is used at a concentration range between about 1 ⁇ and about 160 ⁇ , preferably about 50 ⁇ .
  • the photosensitizer is added directly to the platelets.
  • the wavelength used will depend on the type of photosensitizer selected such that the light source may provide light of about 270 nm to about 700 nm.
  • the use of ultraviolet radiation, especially in the UVA range is the generally accepted method because of its ability to damage DNA of the pathogens. It however requires a higher dosage and /or longer exposure. This can adversely effect the blood product. This is especially true for platelets and also other cellular components of blood. Therefore, radiations are preferably in the UVB and UVC ranges.
  • the light source may be a simple lamp, or may consist of multiple lamps radiating at different wavelengths.
  • the photoradiation source should be capable of delivering from about 0.01 J/cm 2 to about 120 J/cm 2 .
  • the illumination time varies based upon the type of photosensitizer, but is typically in the range of 30 seconds to 30 minutes.
  • the photoradiation source is a monochromatic radiation source having wavelengths in the range of 250 to 308 nm. Exposure of platelets, plasma or other cellular components of blood, in a highly U.V transmissible container, allows exposure to monochromatic radiation between 3 and 10 Joules/cm , from above and below. This treatment reduces pathogen levels by 4 to 7 logs.
  • Irradiating the preservation composition in the presence of photosensitizers may cause the degradation of preservative agents, including the platelet activation inhibitors and/or anticoagulants, as further described below.
  • the preservative agents may be added at higher concentrations to compensate for this loss in activity, and e.g., retain inhibitory activity in the preservation composition during and after the irradiation (or illumination) step.
  • preservative agents, including platelet activation inhibitors may be additionally supplemented following the irradiation step.
  • platelet activation inhibitor(s) are added at 2-3 times their therapeutic concentration or more.
  • Eptifibatide and Argabotran may be added to platelets at three times their therapeutic concentration (i.e., at about 48 ⁇ g Eptifibatide and 2.4 mg Argabotran in 350 ml of platelets).
  • Inhibitors of platelet activation and anticoagulants may be present in the preservation composition (or added thereto) prior to and/or following the illumination step. Additional preservative agents, including oxygen carriers, NSAID drugs, and/or antimicrobial agents may be similarly present in the preservation composition (or added thereto) prior to and/or following the illumination step.
  • the admixed platelets are substantially free of activated platelets prior to addition of the inhibitor(s) of platelet activation.
  • Preservative agents including inhibitors of platelet activation and
  • anticoagulants may be added to the platelets separately from the photosensitizer or they can be added together.
  • a separate mixing step may optionally be included.
  • the preservation composition including the photosensitizer and the inhibitor(s) of platelet activation are placed in a photopermeable bag container and irradiated in batch mode, preferably while agitating the container to fully distribute the photosensitizer throughout the fluid and expose all the fluid to the radiation.
  • Platelet activation inhibitors may be added to the preservation composition either before irradiation, during irradiation, after irradiation, or combinations thereof.
  • the photopermeable container is a bag (such as a blood bag) made of transparent or semitransparent plastic, and the agitating means preferably includes a mechanism for shaking the bag or container in multiple planes.
  • the container or bag may be oxygen-permeable or oxygen-impermeable.
  • photosensitizers and/or preservative agents including inhibitors of platelet activation and/or anticoagulants may be removed or inactivated, thereby eliminating any concerns of adverse or toxic effects from the photosensitizers, platelet preservative agents, or other plasma components prior to
  • the photosensitizer may be unnecessary to remove the photosensitizer prior to transfusion of the treated platelets.
  • the toxic products may be removed by diafiltration or other suitable removal means, including those as further described below.
  • an additional irradiation step may be employed immediately prior to transfusion to inactivate the preservation agents, such as inhibitors of platelet activation and/or anticoagulants.
  • freshly prepared photosensitizer(s) preferably endogenous photosensitizer(s)
  • this additional irradiation step may provide an alternative to other removal means, including diafiltration as further described below.
  • Platelets may be derived from whole blood or platelet-containing components of whole blood, or they may be further isolated therefrom. Preferably, the platelets are substantially free of red blood cells and other blood nutrients and/or are substantially free of activated platelets.
  • the blood is whole blood isolated from a mammal, for use in the same species.
  • the blood is isolated and separated into the three core components of whole blood, i.e., plasma, cells, and platelets.
  • the whole blood, or only the platelet component of the whole blood can be treated with the preservation composition. If whole blood is treated, a preferred embodiment contemplates the use of only some components of the proposed preservation composition, such as the antiplatelet agent and anticoagulant, for whole blood storage.
  • the blood can then be fractionated and the platelet component can be further mixed with the preservation composition of the present invention for storage.
  • platelets are derived from a non-plasma blood component. More particularly, blood is passed through a filter comprising a filtering membrane to separate plasma in blood from the non-plasma component by tangential flow filtration, wherein a diafiltration solution is added to the non-plasma blood component to replace some or all of the permeate volume.
  • the diafiltration solution can be a plasma- free solution commonly used for the storage of the non-plasma blood component but without any antiplatelet agent and/or anticoagulant. Examples of the diafiltration solution include, but are not limited to, Intersol (Fenwal), T-Sol, PAS II, PAS IIIM, PAS27 (Baxter).
  • the diafiltration buffer is a commercially available platelet storage solution (T- Sol) with 20% to 30% plasma.
  • T- Sol platelet storage solution
  • an extraction liquid is circulated outside the filtering tube in a counter current manner to facilitate the filtration process.
  • the extraction fluid comprises 0.9% w/v sodium chloride.
  • the preserved platelets can be stored at room temperature, at refrigeration temperatures (0° C- 12 °C) or at freezing temperatures (-80 °C- 0 °C) in liquid, frozen, or freeze-dried state to maintain the freshness and functional activity of the platelets. If the platelets will be subsequently frozen or freeze dried, the platelets can be mixed with the preservation composition before freezing.
  • the irradiated mixture is stored at 4 °C to 12 °C. In another embodiment, the irradiated mixture is stored at 4 °C to 8 °C. In yet another embodiment, the platelet mixture is stored at room temperature.
  • the platelets may be stored for a desired period of time.
  • the desired period of time is one, two, three or four weeks, preferably at ambient temperature.
  • Platelet functional activities may be determined by their ability to aggregate in the presence of certain biological agents and their morphology. Platelet function also can be assessed by the maintenance of the pH upon limited storage of a solution containing the platelets and in vivo haemostatic effectiveness using the rabbit kidney injury model described in Krishnamurti et al, Transfusion, 39:967 (1999). Structural integrity of platelets is assessed by in vivo survival following radiolabeling with carbon- 15 or indium- 1 11 and identification of the presence of specific platelet antigens.
  • the platelets may be isolated from the whole blood using methods commonly used in the art.
  • a unit of whole blood is centrifuged using settings that precipitate only the cellular components of the blood (e.g., red blood cells and white blood cells). At these settings, the platelets remain suspended in the plasma.
  • the platelet-rich plasma (PRP) is removed from the precipitated blood cells, then centrifuged at a faster setting to harvest the platelets from the plasma.
  • the whole blood is centrifuged using settings that cause the platelets to become suspended in the "buffy coat” layer, which includes the platelets and the white blood cells.
  • the "buffy coat” is isolated in a sterile bag, suspended in a small amount of red blood cells and plasma, then centrifuged again to separate the platelets and plasma from the red and white blood cells.
  • apheresis platelets are collected using a mechanical device that draws blood from the donor and centrifuges the collected blood to separate out the platelets and other components to be collected. The remaining blood is returned to the donor.
  • an acellular platelet preservation composition for freshly collected platelets can be prepared for improving the functional half- life of platelets.
  • the addition of the platelet preservation composition to freshly collected platelets better maintains the original blood clotting function when infused during the storage period of the platelets.
  • the addition of a platelet preservation composition permits an extended storage of the platelets at refrigeration temperatures and allows the platelets to maintain blood clotting properties without affecting the half-life of the platelets in circulation once transfused. As a result, the platelets stored for an extended period can be used for transfusions while saving a substantial amount of effort and cost.
  • a photosensitizer such as riboflavin, psoralen or methylene blue, in the amount of 1-200 ⁇ (riboflavin or psorale) or 0.2-50 ⁇ (methylene blue ), and
  • a platelet activation inhibitor such as a GPIIb/IIIa antagonist, a thrombin antagonist, a P2Y12 receptor antagonist or a second messenger effector, in an amount of 0.001-5.0 mg.
  • the above platelet preservation composition is added to the platelets and subjected to a sterilization procedure by exposing to radiation at a desired wave-length.
  • An energy source such as glucose or citrate to sustain aerobic metabolism and electrolytes such as Na, CI, and Mg, may be added after the sterilization procedure.
  • TABLE 1 provides the concentration ranges for some commonly used energy sources and electrolytes.
  • TEG The principle of TEG is based on the measurement of the physical viscoelastic characteristics of blood clot. Clot formation was monitored at 37°C in an oscillating plastic cylindrical cuvette ("cup") and a coaxially suspended stationary piston (“pin”) with a 1 mm clearance between the surfaces, using a computerized Thrombelastograph (TEG Model 3000, Haemoscope, Skokie, IL).
  • the cup oscillates in either direction every 4.5 seconds, with a one second mid-cycle stationary period; resulting in a frequency of 0.1 Hz and a maximal shear rate of 0.1 per second.
  • the pin is suspended by a torsion wire that acts as a torque transducer.
  • TEG assesses coagulation by measuring various parameters such as the time latency for the initial initiation of the clot (R), the time to initiation of a fixed clot firmness (k) of about 20 mm amplitude, the kinetic of clot development as measured by the angle (a), and the maximum amplitude of the clot (MA).
  • the parameter A measures the width of the tracing at any point of the MA. Amplitude in mm is a function of clot strength or elasticity.
  • the amplitude on the TEG tracing is a measure of the rigidity of the clot; the peak strength or the shear elastic modulus attained by the clot, G, is a function of clot rigidity and can be calculated from the maximal amplitude (MA) of the TEG tracing.
  • reaction time represents the latent period before the establishment of a 3 -dimensional fibrin gel network (with measurable rigidity of about 2 mm amplitude).
  • Maximum Amplitude (MA, in mm), is the peak rigidity manifested by the clot.
  • G (5000A)/(1 00-A).
  • Blood clot firmness is important function parameters for in vivo thrombosis and hemostasis because the clot must stand the shear stress at the site of vascular injury. TEG can assess the efficacy of different pharmacological interventions on various factors (coagulation activation, thrombin generation, fibrin formation, platelet activation, platelet- fibrin interaction, and fibrin polymerization) involved in clot formation and retraction.
  • Data are expressed as mean + SEM. Data are analyzed by either paired or group analysis using Student's t-test or ANOVA when applicable; differences are considered significant at P ⁇ 0.05 or less.
  • HPLC-based methods were used to define the performance characteristics for assays to detect levels of Tirofiban (AGGRASTAT ® ), Eptifibatide (INTEGRILIN ® ), and Argatroban following their removal via diafiltration or following their degradation by ultraviolet light treatment.
  • Platelet activation inhibitors were accomplished with the use of tangential flow filtration.
  • Platelet concentrates in 100% plasma containing either Eptifibatide and Argatroban, or Tirofiban and Argatroban, were processed through a hollow fiber filter made of polyethyl sulfone with a pore diameter of 0.5 micron and the inner diameter of the lumen of the filter fiber being 1 mm.
  • the diafiltration was conducted as a combination of discontinuous and constant volume exchange. Forty milliliter aliquots were processed at a time. A 4 volume discontinuous exchange was done first, which essentially removed most of the plasma proteins. This was followed by a 6 volume constant volume exchange. The flask was sealed. The loss of the permeate was continuously replaced by isotonic saline.
  • HPLC-based methods were used to define the performance characteristics for assays for detecting Tirofiban, Eptifibatide, and Argatroban following their removal via diafiltration. These methods employed a Beckman-Coulter System Gold HPLC System equipped with a 126 solvent module, a 168 Multiwavelength diode-array detector; and a 508 autosampler module.
  • Analytical standard curve data for Tirofiban, Eptifibatide, and Argatroban were generated, which allowed for a preliminary assessment of standard assay performance characteristics, including: assay range, reproducibility, lower limit of detection (LLOD), lower limits of quantitation (LLOQ)(data not shown).
  • the standard curve data was generated in a non-serial manner and in duplicate with analytical sampling occurring as single runs.
  • Tirofiban and Eptifibatide were each prepared at 0.1, 0.25, 1, 5, 10, 25, and 50 ⁇ g/mL concentrations for assembly of individual standard curves with all dilutions performed in sterile 0.5% saline. Eptifibatide also had 100 and 500 ⁇ g/mL concentrations tested.
  • Argatroban was prepared at 5, 25, 100, 500, and 1,000 ⁇ g/mL concentrations for standard curve assembly with all dilutions performed in sterile 0.5% saline.
  • the HPLC analysis was conducted using a Beckman-Coulter System Gold HPLC System equipped with a 126 solvent module, a 168 Multiwavelength diode-array detector; and a 508 autosampler module.
  • a Shimadzu ShimPac ODS (3.5 ⁇ ; 2.0x30mm) column was employed, wherein a flow rate of 0.2 mL/min. was used with Solvent A consisting of Fisher Optima-grade water w/ 0.1% trifluoroacetic acid (TFA) and Solvent B consisting of Fisher Optima-grade Methanol w/ 0.1% TFA.
  • Solvent A consisting of Fisher Optima-grade water w/ 0.1% trifluoroacetic acid (TFA)
  • Solvent B consisting of Fisher Optima-grade Methanol w/ 0.1% TFA.
  • the gradient sequence is defined in Tables 3 and 4. All data was recorded at 230 and 280 nm and full spectrum (295-700 nm) by the 168 detector module
  • a 5-point parametric fit may be able to extend the effective assay range.
  • FIGS 2 A and 2B The dose-dependent photodegradation of Argatroban upon exposure to UV light at 282 nm is shown in FIGS 2 A and 2B.
  • FIG. 2 A shows HPLC traces of exposures A-F compared to a control (unexposed 50 ⁇ g/mL Argatroban) and a saline blank.
  • FIG. 2B (right) is a graphical representation of the loss in peak height associated with the exposure to UV282 with standard deviations.
  • Relative areas were as follows: Control - 100%; exposure A-84.5%, exposure B-64.7%, exposure C-53.9%, exposure D-38.1%, exposure E-26.7%, and exposure F-23.2%. Although a steady loss in Argatroban was observed at 19.9 minutes (A 2 30 tim), there was no emergence of a secondary peak in the chromatogram of comparable area to account for discrete photo bleached degradation products.
  • FIG. 4 depicts several degradation products associated with the ultraviolet light treatment.
  • FIGS. 3A and 3B depict a similar set of observations for Argatroban exposure to UV308.
  • FIG. 3 A shows HPLC traces of exposures A-C in relation to a control (unexposed 50 ⁇ g/mL Argatroban) and a saline blank.
  • FIG. 3B is a graphical representation of the loss in peak height associated with the exposure of Argatroban to UV308 expressed as % relative to control with standard deviations.
  • FIG. 4 shows representative traces reflecting an identical pattern of photodegradation products at UV 2 8 2 andUV 3 os in the two sets of experiments exemplified in FIGS. 2 and 3.
  • FIGS. 5A and 5B show HPLC traces of exposures A-F in reference to a control (unexposed 5 ⁇ g/mL Tirofiban) and a saline blank.
  • FIG. 5B is a graphical representation of the loss in peak height associated with the exposure to UV3 08 expressed as % relative to a control with standard deviations shown. Traces E and F were below the LLOQ for the assay.
  • FIGS. 6A and 6B show HPLC traces of exposures A-C in reference to a control (unexposed 5 ⁇ g/mL Tirofiban) and a saline blank.
  • FIG. 6B is a graphical representation of the loss in peak height associated with exposure to UV 3 os expressed as % relative to control with standard deviations shown. The degradation profile was as follows: Exposure A-81.2%, B-76.9%, and C-73.0% relative to control.
  • FIGS. 7A and 7B Eptifibatide (INTEGRILIN ® ) exposure to UV 282 nm provided a faster photodegradation profile than the other compounds tested.
  • FIG. 7A shows HPLC traces of exposures A-F in reference to a control (unexposed 5 ⁇ g/mL Eptifibatide) and a saline blank.
  • FIG. 7B is a graphical representation of the loss in peak height associated with the exposure to UV308 expressed as % relative to control. Exposure A was 47.0% relative to control, with the remainder of the exposures being below the LLOD. Notably, an additional species at 17.3 appeared to grow more intense with the initial exposures, but did not increase in intensity at higher exposures.
  • FIGS. 8 A and 8B shows a photodegradation profile of Eptifibatide
  • FIG. 8 A shows HPLC traces of exposures A-C in reference to a control (unexposed 5 ⁇ g/mL Eptifibatide) and a saline blank.
  • FIG. 8B is a graphical representation of the loss in peak height associated with the exposure to UV 3 o 8 expressed as % relative to control and with standard deviations shown. For Eptifibatide, similar degradation products were observed at either wavelength.
  • UV 282 Argatroban > Tirofiban » Eptifibatide
  • UV 308 Tirofiban » Argatroban » Eptifibatide

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Abstract

La présente invention concerne un procédé pour l'élimination d'un photo-sensibilisateur depuis une composition de conservation de plaquettes. Le procédé comprend le passage de la composition de conservation de plaquettes à travers un dispositif de filtration à écoulement tangentiel pour la séparation du photo-sensibilisateur depuis les plaquettes dans la composition de conservation de plaquettes.
PCT/US2012/057023 2012-09-25 2012-09-25 Dispositif et procédé pour l'élimination d'additifs dans les produits sanguins WO2014051537A1 (fr)

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WO2017120545A3 (fr) * 2016-01-07 2017-08-24 Cerus Corporation Systèmes et méthodes pour la préparation de plaquettes
US10822461B2 (en) 2017-10-05 2020-11-03 Fresenius Medical Care Holdings, Inc. Polysulfone-urethane copolymer, membranes and products incorporating same, and methods for making and using same
US11529587B2 (en) 2019-05-03 2022-12-20 Cellphire, Inc. Materials and methods for producing blood products
US11701388B2 (en) 2019-08-16 2023-07-18 Cellphire, Inc. Thrombosomes as an antiplatelet agent reversal agent
US11767511B2 (en) 2018-11-30 2023-09-26 Cellphire, Inc. Platelets as delivery agents
US11903971B2 (en) 2020-02-04 2024-02-20 Cellphire, Inc. Treatment of von Willebrand disease
US11965178B2 (en) 2018-11-30 2024-04-23 Cellphire, Inc. Platelets loaded with anti-cancer agents

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017120545A3 (fr) * 2016-01-07 2017-08-24 Cerus Corporation Systèmes et méthodes pour la préparation de plaquettes
US10822461B2 (en) 2017-10-05 2020-11-03 Fresenius Medical Care Holdings, Inc. Polysulfone-urethane copolymer, membranes and products incorporating same, and methods for making and using same
US11499016B2 (en) 2017-10-05 2022-11-15 Fresenius Medical Care Holdings, Inc. Polysulfone-urethane copolymer, membranes and products incorporating same, and methods for making and using same
US11767511B2 (en) 2018-11-30 2023-09-26 Cellphire, Inc. Platelets as delivery agents
US11965178B2 (en) 2018-11-30 2024-04-23 Cellphire, Inc. Platelets loaded with anti-cancer agents
US11529587B2 (en) 2019-05-03 2022-12-20 Cellphire, Inc. Materials and methods for producing blood products
US11752468B2 (en) 2019-05-03 2023-09-12 Cellphire, Inc. Materials and methods for producing blood products
US11813572B2 (en) 2019-05-03 2023-11-14 Cellphire, Inc. Materials and methods for producing blood products
US11701388B2 (en) 2019-08-16 2023-07-18 Cellphire, Inc. Thrombosomes as an antiplatelet agent reversal agent
US11903971B2 (en) 2020-02-04 2024-02-20 Cellphire, Inc. Treatment of von Willebrand disease

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