WO2018138360A1 - Oxygen carrying blood substitutes and their use as delivery vehicles - Google Patents

Oxygen carrying blood substitutes and their use as delivery vehicles Download PDF

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WO2018138360A1
WO2018138360A1 PCT/EP2018/052168 EP2018052168W WO2018138360A1 WO 2018138360 A1 WO2018138360 A1 WO 2018138360A1 EP 2018052168 W EP2018052168 W EP 2018052168W WO 2018138360 A1 WO2018138360 A1 WO 2018138360A1
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composition
hboc
oxygen carrier
coronary
mmol
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PCT/EP2018/052168
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French (fr)
Inventor
Borja IBÁÑEZ CABEZA
Jose Manuel GARCÍA-RUIZ
Jaume AGÜERO RAMON-LLIN
Rodrigo FERNÁNDEZ-JIMÉNEZ
Valentín FUSTER CARULLA
Enrique Lara-Pezzi
Juan Antonio BERNAL RODRÍQUEZ
Carlos GALÁN-ARRIOLA
Andrés PUN GARCÍA
Enda Joseph CLINTON
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Centro Nacional De Investigaciones Cardiovasculares Carlos Iii (F.S.P.)
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Publication of WO2018138360A1 publication Critical patent/WO2018138360A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0026—Blood substitute; Oxygen transporting formulations; Plasma extender
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein

Definitions

  • the present invention relates to the field of medicine and biotechnology. More specifically it relates to oxygen carrier compositions for the delivery of oxygen to tissues and organs (e.g., to the myocardium) under bloodless situations. It further relates to medical uses and methods of treatment comprising the administration thereof, for instance, as delivery vehicle for gene therapy purposes.
  • Heart failure is a major public health issue with a current prevalence of over 5.8 million in the USA and over 23 million worldwide. Every year in the USA, more than 550,000 individuals are diagnosed with HF for the first time, and there is a lifetime risk of one in five of developing this syndrome. A diagnosis of HF carries substantial risk of morbidity and mortality, which despite advances in management remain unacceptably high. Over 2.4 million patients who are hospitalized have HF as a primary or secondary diagnosis, and nearly 300,000 deaths annually are directly attributable to HF (Bui et al., Nat. Rev. Cardiol. 201 1 , 8(1 ), 30-41 ).
  • Direct gene delivery methods include intrapericardial injection, endocardial injection or intramyocardial injection.
  • Direct injection of the gene therapy construct has the obvious advantage that enables the application of a high concentration of vector directly at the target site. It requires however a fairly aggressive intervention which involves the administration of relatively high volumes of the therapeutic solution into the myocardium and thus this administration route is associated to a higher risk for the patient.
  • An additional drawback is that presence of the transgene is localized around the site of injection and thus results in its heterogeneous distribution along the myocardium.
  • Transvascular gene delivery methods have also been described for cardiac gene therapy. These methods will enable a more diffuse and homogeneous delivery but present other limitations which typically include but are not limited to dilution of the vector in the circulating blood; dissemination into collateral organs; and short contact between vector and cardiomyocytes (Katz et al., J Mol Cell Cardiol 201 1 , 50(5):766-776). Accordingly, issues related to delivery, including vector efficiency, dose, specificity/tropism and safety are areas of concern (Byrne et al., Gene Therapy 2008, 15, 1550-1557).
  • Non-selective (indirect) intracoronary delivery methods are characterized by the cross- clamping of both the pulmonary artery and the aorta, and rely on the creation of a transcoronary myocardial perfusion gradient for vector delivery. This allows perfusion of the virus at relatively low downstream pressure, and the endocardium can be efficiently transfected. Clinical feasibility of this approach may be limited considering the risk of systemic ischemia and acute left ventricle (LV) overload during the aortic cross-clamping and the time of occlusion must be limited (Parsa et al., Semin Thorac Cardiovasc surg. 2003, 15:259-97; Katz et al., J Mol Cell Cardiol 201 1 , 50(5):766-776).
  • LV left ventricle
  • Boekstegers et al. described the advantages of retrograde delivery compared to ante-grade and sustained that blocking the venous outflow can significantly increase viral transfection of the myocardium (Boekstegers P, and Kupatt C, Basic Res Cardiol.
  • Percutaneous retrograde delivery methods involve in vivo isolation of the heart from the systemic circulation and the cardiac recirculation of the gene therapy vector. These typically require a significant degree of instrumentation and consequently are complicated and expensive. Moreover, this high instrumentation may be associated to an increased risk of the gene therapy intervention in patients having a severe heart dysfunction and thus already being at high risk.
  • Catheter-based, percutaneous infusion of the transgene containing vector into the coronary arteries is the most common cardiac gene delivery method. Its perceived advantages are based on its minimal invasiveness, the possibility of transgene delivery to all four myocardial chambers and the delivery of vector genomes using endovascular coronary catheterization— a well-established procedure in the clinical practice. Simple antegrade (arterial-to-venous) intracoronary delivery has been reported to result however in very limited myocardial transduction efficiency (Magovern CJ et al., Ann Thorac Surg. 1996, 62:425-33; Logeart D et al., Hum Gene Ther. 2001 , 12:1601-10; Kaplitt MG et al., Ann Thorac Surg. 1996, 62:1669-76; Logeart D et al., Hum Gene Ther. 2000, 1 1 :1015-22).
  • Reduced transgene transduction efficiency in single pass intracoronary delivery may be associated to the rapid washout of the vector.
  • some strategies have been devised encompassing the interruption of the coronary blood flow.
  • Hayase M et al. discloses a catheter-based percutaneous antegrade intracoronary artery gene delivery method, which was combined with coronary artery occlusion with or without coronary vein occlusion. This method encompasses a 3-minute interruption of the coronary flow prior to the viral vector delivery. The interruption of blood flow for longer periods would be desirable as it would enable prolonging the time of contact of the vector with the myocardial cells, but as a drawback may be associated to an increased the risk of ischemia and ischemia/reperfusion (l/R) injury.
  • l/R ischemia/reperfusion
  • myocardial necrosis typically begins within approximately 15 minutes and, without any intervention, results in irreversible damage over the next 30 to 90 minutes.
  • coronary reperfusion itself induces additional damage to the myocardium, known as ischemia/reperfusion (l/R) injury.
  • Oxygen-carrying blood substitutes (herein after also referred as "oxygen carriers”) were initially developed as an alternative to red blood cells for surgical patients.
  • Oxygen-carrier solutions are characterized by being aqueous compositions which transport oxygen and can be dissolved into a balanced salt buffer.
  • Many oxygen-carrying blood substitutes are based on hemoglobin and are thus named Hemoglobin-based oxygen carriers (HBOCs). Functionally, they allow delivery of more oxygen to hypoxic tissues due to their higher 0 2 affinity, lower viscosity and smaller mean diameter than human erythrocytes.
  • HBOCs are promising candidates to prevent many organs from l/R injury (Caswell, et. al., Am J Physiol Heart Circ Physiol 2005, 288:H1796-H1801 ; George, et. al., Am J Physiol Heart Circ Physiol 2006, 291 :H1 126-H1 137; Burffle, et. al., British Journal of Anaesthesia 2005, 95(6):737-45; Rempf, et. al. British Journal of Anaesthesia 2009, 103(4): 496-504; Lintel, et. al., Am J Physiol Heart Circ Physiol 2010, 298:H1 103-H1 1 13; WO2013/016598).
  • HBOC-201 Hemopure® or hemoglobin glutamer-250
  • Hb glutaraldehyde-polymerized hemoglobin
  • HBOC-201 has been shown to be well-tolerated as an intracoronary perfusate in humans (Meliga, et. al., Eurolntervention 2008, 4:99-107). Moreover, it was under clinical development for the treatment of haemorrhagic shock resulting from traumatic injury, for the treatment of acute coronary syndrome, peripheral arterial disease (PAD)/ peripheral vascular disease (PVD), occlusive coronary artery disease, wounds, and trauma.
  • PAD peripheral arterial disease
  • PVD peripheral vascular disease
  • Meliga, et. al., Eurolntervention 2008, 4:99-107; and US 2010/0209532 A1 provide the results of a Phase II trial which was designed to test the hypothesis that pre-oxygenated HBOC-201 is capable of supporting myocardial metabolism and preserving cardiac function during brief total coronary occlusion in humans. All patients underwent two intra-stent balloon occlusions of 3 minutes of duration with a recovery period of 20 minutes in between. This assay had two arms and subjects were assigned to receive a continuous intracoronary infusion of pre-oxygenated HBOC-201 during the first occlusion period and no infusion (referred as "dry occlusion") during the second occlusion period or vice-versa.
  • US 6 699 231 B1 discloses the treatment of a tissue by occluding an artery and/or a vein and perfusing the tissue with a therapeutic agent and an oxygen carrier in a method further comprising collecting the perfusate and extracorporeal ⁇ pumping the perfusate back and extracorporeally oxygenating the oxygen carrier.
  • US 6 177 403 B1 discloses a composition comprising a gene vector, an oxygen carrier and a vascular permeability- enhancing agent.
  • oxygen carrying blood substitutes' may not only prevent and/or treat cardiac ischemia but also avoid the contact of gene vectors with blood elements which may adversely affect cardiomyocyte transduction. Preventing contact of the viral particles with blood components may be particularly relevant when adeno-associated virus (AAV) are used as gene therapy vectors, as this would enable to solve in particular the problem associated with the presence of neutralizing antibodies against AAVs circulating in the blood of the recipient.
  • AAV adeno-associated virus
  • the inventors have newly devised a cardiac gene delivery method which comprises the occlusion of coronary veins and/or arteries and administration of a vector containing a nucleic acid of interest in an oxygen carrier blood substitute composition.
  • a cardiac gene delivery method which comprises the occlusion of coronary veins and/or arteries and administration of a vector containing a nucleic acid of interest in an oxygen carrier blood substitute composition.
  • an appropriate volume of the oxygen carrier blood substitute may be administered prior to the administration of the vector containing composition.
  • a first pilot assay was conducted in healthy male pigs, see Example 2 and Figure 4, which shows an increased transfer of the green fluorescent protein (GFP)-encoding AAV vector into the myocytes of the left ventricle apex when HBOC-201 is used as vehicle for the anterograde intracoronary administration of the gene therapy vector with respect to the administration of vector particles suspended in saline.
  • GFP green fluorescent protein
  • the first aspect of the invention relates to an oxygen carrier composition for use in a method for the in vivo diagnosis and/or the therapeutic and/or preventive treatment of a myocardial disease; wherein the oxygen carrier composition is administered prior to and/or simultaneously to the administration of a preventive, therapeutic and/or diagnostic agent; wherein the administration of the oxygen carrier composition and said agent is intracoronary; and wherein the method comprises the temporary occlusion of a coronary artery and/or a coronary vein prior to the administration of said oxygen carrier composition.
  • an oxygen carrier composition for use in a method for the in vivo diagnosis and/or treatment of a myocardial disease
  • oxygen carrier composition in ii) is substantially free of the diagnostic, prophylactic and/or therapeutic agent administered in iii);
  • step iii) wherein in step iii) the agent and the oxygen carrier composition are administered simultaneously.
  • it relates to an oxygen carrier composition for use in a method for the in vivo diagnosis and/or the therapeutic and/or preventive treatment of a myocardial disease; wherein said method comprises the following consecutive steps:
  • oxygen carrier composition has been pre-oxygenated; and wherein said agent is a transgene encoding adeno-associated virus (AAV) expression vector.
  • AAV adeno-associated virus
  • Example 1 intracoronary administration of the standard HBOC-201 formulation to an ischemic heart (i.e., after 45 minutes from the interruption of blood flow and prior to blood restoration) significantly increased microvascular obstruction and infarct size.
  • HBOC-pH pH of the solution to 7.35
  • HBOC-pH-Glc-lns glucose, as metabolic substrate, and insulin
  • modified solution is especially safe under ischemic conditions, and thus particularly suitable for myocardial administration after prolonged occlusion periods. Accordingly, bloodless reperfusion of modified oxygen carrier composition has shown to be a promising and safe strategy which may be applied when acute exposure of the ischemic myocardium to therapeutic agents (such as cardioprotective agents) is desired before contact with reperfusing blood.
  • therapeutic agents such as cardioprotective agents
  • the present invention relates to a composition
  • a composition comprising or consisting of:
  • composition is herein also referred as "the modified oxygen carrier composition of the invention".
  • said composition comprises or consists of:
  • the pH of said composition is from 6.8 to 7.4, preferably wherein the pH of said composition is 7.35.
  • the present invention relates to the use of the modified oxygen carrier composition of the invention as a pharmaceutical composition. It also relates to the composition or pharmaceutical composition of the invention for use as a medicament.
  • the invention relates to the composition according to the preceding aspects of the invention, for use in a method of in vivo diagnosis and/or treatment of a tissue or organ which has or is at risk of having ischemia.
  • the invention relates to a method for obtaining a composition according to the preceding aspects of the invention. It may comprise mixing the components of the composition as described herein. It also may comprise adding a metabolic substrate to the oxygen carrier composition and adjusting the pH of the composition as described herein. In a particular embodiment, it comprises:
  • An additional aspect of the invention relates to an oxygen carrier composition as described in the preceding aspects of the invention, for use as delivery vehicle in the administration of a therapeutic and/or diagnostic agent.
  • Figure 1 Study protocols.
  • FigIA Protocol ⁇ - Intracoronary infusion of HBOC-201 in healthy pigs;
  • FigI B Protocol 2.- Effect of intracoronary infusion of pre-oxygenated oxygen carrier infusion at the end of prolonged ischemia (before blood flow restoration).
  • Figure 2. Effect of HBOC-201 intracoronary infusion rate + duration combinations on left ventricular ejection fraction (Protocol 1 ) Tukey boxplots show median values, interquartile range and extreme values. LVEF, left ventricular ejection fraction. * p-value ⁇ 0.05 compared with baseline; #p-value ⁇ 0.05 compared with the 12 min infusion group.
  • FIG. 4 CMR-derived parameters from HBOC-pH and HBOC-pH-Glc-lns infusion after myocardial infarction (Protocol 2). Tukey boxplots show median values, interquartile range and extreme values. LVEF, left ventricular ejection fraction. * p-value ⁇ 0.05 compared to baseline.
  • GFP Green Fluorescent Protein
  • a solution or "aqueous composition” may mean a fluid (liquid) preparation that contains water, optionally in combination with other mutually miscible solvents (e.g. water-soluble organic solvents), and one or more chemical substances dissolved therein.
  • compositions may refer to a formulation(s) that it is suitable for injection and/or administration into an individual in need thereof.
  • a “composition” may also be referred to as a “pharmaceutical composition.”
  • the compositions provided herein are substantially sterile and do not contain any agents that are unduly toxic or infectious to the recipient.
  • balanced refers to an osmotically balanced salt solution to prevent acute cell or tissue damage.
  • This term as used herein may mean that the osmolality is close to the physiological osmolality in the human body, thus leading to more suitable compositions to be used in parenteral administration.
  • Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm/L, preferably about 290 mOsm/L. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example.
  • diagnosis refers to determining the presence or absence of a disease when a subject shows signs or symptoms of the disease. It also encompasses early detection or screening of a disease in asymptomatic individuals who may have the disease.
  • disease as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disorder” and “condition” (as in medical condition), in that all reflect an abnormal condition of the body or of one of its parts that impairs normal functioning and is typically manifested by distinguishing signs and symptoms.
  • treatment refers to the prophylactic and/or therapeutic treatment.
  • therapeutic treatment refers to bringing a body from a pathological state or disease back to its normal, healthy state. Specifically, unless otherwise indicated, includes the amelioration, cure, and/or maintenance of a cure (i.e., the prevention or delay of relapse) of a disease or disorder. Treatment after a disorder has started aims to reduce, alleviate, ameliorate or altogether eliminate the disorder, and/or its associated symptoms, to prevent it from becoming worse, to slow the rate of progression, or to prevent the disorder from re-occurring once it has been initially eliminated (i.e., to prevent a relapse). It is noted that, this term as used herein is not understood to include the term “prophylactic treatment” as defined herein.
  • prophylactic treatment or “preventive treatment” as used herein refers to preventing a pathological state. It is noted that, this term as used herein is not understood to include the term “therapeutic treatment” as defined herein.
  • effective amount refers to an amount that is effective, upon single or multiple dose administration to a subject (such as a human patient) in the prophylactic and/or therapeutic treatment of a disease, disorder or pathological condition.
  • subject refers to a mammalian subject.
  • it is selected from a human, companion animal, non-domestic livestock or zoo animal.
  • the subject may be selected from a human, mouse, rat, dog, cat, cow, pig, sheep, horse, bear, and so on.
  • said mammalian subject is a human subject.
  • pharmaceutically acceptable carrier and/or excipient is intended to include formulation used to stabilize, solubilize and otherwise be mixed with active ingredients to be administered to living animals, including humans. This includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.
  • nucleic acid sequence and “nucleotide sequence” may be used interchangeably to refer to any molecule composed of or comprising monomeric nucleotides.
  • a nucleic acid may be an oligonucleotide or a polynucleotide.
  • a nucleotide sequence may be a DNA or RNA (e.g., microRNAs).
  • a nucleotide sequence may be chemically modified or artificial. Nucleotide sequences include peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acid (TNA).
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • GAA glycol nucleic acids
  • TPA threose nucleic acid
  • Each of these sequences is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule.
  • phosphorothioate nucleotides may be used.
  • Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoramidates and oligoribonucleotide phosphorothioates and their 2'-0-allyl analogs and 2'-0-methylribonucleotide methylphosphonates which may be used.
  • nucleic acid construct refers to a man-made nucleic acid molecule resulting from the use of recombinant DNA technology.
  • a nucleic acid construct is a nucleic acid molecule, either single- or double-stranded, which has been modified to contain segments of nucleic acids sequences, which are combined and juxtaposed in a manner, which would not otherwise exist in nature.
  • a nucleic acid construct usually is a "vector", i.e. a nucleic acid molecule which is used to deliver exogenously created DNA into a host cell.
  • a "coding sequence” or a sequence which "encodes” a gene product as used herein refers to a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA), in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • expression vector or “vector” as used herein refers to a recombinant nucleotide sequence that is capable of effecting expression of a gene (transgene) in host cells or host organisms compatible with such sequences. In particular, this term encompasses a plasmid, phage, transposon, cosmid, chromosome, virus, etc.
  • expression vectors typically include at least suitable transcription regulatory sequences and optionally, 3' transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements able to respond to a precise inductive signal (endogenous or chimeric transcription factors) or specific for certain cells, organs or tissues.
  • DNA “regulatory sequences”, “control sequences” and “control elements” as used herein, refer collectively to promoter sequences (e.g., an eukaryotic promoter), polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences/elements need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
  • promoter sequences e.g., an eukaryotic promoter
  • polyadenylation signals e.g., polyadenylation signals
  • transcription termination sequences e.g., upstream regulatory domains
  • upstream regulatory domains e.g., origins of replication
  • origins of replication e.g., origins of replication
  • IRS internal ribosome entry sites
  • eukaryotic promoter refers to a DNA sequence region that initiates transcription of a particular gene, or one or more coding sequences, in eukaryotic cells.
  • a promoter can work in concert with other regulatory regions or elements to direct the level of transcription of the gene or coding sequence/s. These regulatory elements include, without limitation, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter, including e.g. attenuators, enhancers, and silencers.
  • the promoter is located near the transcription start site of the gene or coding sequence to which is operably linked, on the same strand and upstream of the DNA sequence (towards the 5' region of the sense strand).
  • a promoter can be about 100-1000 base pairs long. Positions in a promoter are designated relative to the transcriptional start site for a particular gene (i.e., positions upstream are negative numbers counting back from -1 , for example -100 is a position 100 base pairs upstream).
  • operably linked refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous; where it is necessary to join two protein encoding regions, they are contiguous and in reading frame.
  • polyadenylation signal or "poly(A) signal” refers to a specific recognition sequence within 3' untranslated region (3' UTR) of the gene, which is transcribed into precursor mRNA molecule and guides the termination of the gene transcription.
  • Poly(A) signal acts as a signal for the endonucleolytic cleavage of the newly formed precursor mRNA at its 3'-end, and for the addition to this 3'-end of a RNA stretch consisting only of adenine bases (polyadenylation process; poly(A) tail).
  • Poly(A) tail is important for the nuclear export, translation, and stability of mRNA.
  • the polyadenylation signal is a recognition sequence that can direct polyadenylation of mammalian genes and/or viral genes, in mammalian cells.
  • Poly(A) signals typically consist of a) a consensus sequence AAUAAA, which has been shown to be required for both 3'-end cleavage and polyadenylation of pre-messenger RNA (pre-mRNA) as well as to promote downstream transcriptional termination, and b) additional elements upstream and downstream of AAUAAA that control the efficiency of utilization of AAUAAA as a poly(A) signal.
  • pre-mRNA pre-messenger RNA
  • packaging cells refers to a cell or cell line which may be transfected with a helper vector or virus or a nucleic acid construct, and provides in trans all the missing functions which are required for the complete replication and packaging of a viral vector.
  • the packaging cells express in a constitutive or inducible manner one or more of said missing viral functions.
  • substantially free may refer to a composition containing less than 0.033%, less than 0.001 %, less than 0.0005%, less than 0.0003%, or less than 0.0001 % of the referred component of the composition.
  • the composition does not comprise said component.
  • the invention relates to an oxygen carrier composition for use in a method for the in vivo diagnosis and/or the therapeutic and/or preventive treatment of a myocardial disease; wherein the oxygen carrier composition is administered prior to and/or simultaneously to the administration of a preventive, therapeutic and/or diagnostic agent; wherein the administration of the oxygen carrier composition and said agent is intracoronary; and wherein the method comprises the temporary occlusion of a coronary artery and/or a coronary vein prior to the administration of said oxygen carrier composition.
  • the invention refers to a method for the cardiac delivery of a preventive, therapeutic and/or diagnostic agent wherein said method comprises the administration of an oxygen carrier composition prior to and/or simultaneously to the administration of said preventive, therapeutic and/or diagnostic agent; wherein the administration of the oxygen carrier composition and said agent is intracoronary; and wherein the method comprises the temporary occlusion of a coronary artery and/or a coronary vein prior to the administration of said oxygen carrier composition.
  • coronary artery also includes “coronary arteries” and may refer to one or more of the left anterior descending artery, the left circumflex artery and the right coronary artery. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, said coronary artery is the left anterior descending artery.
  • coronary vein also includes “coronary veins” and may refer to one or more of the coronary sinus, the anterior cardiac vein, the right atrial veins and the right ventricular veins.
  • oxygen carrier composition or "oxygen-carrying blood substitutes” are used herein interchangeably and refer to compositions, typically aqueous compositions, which can transport oxygen in excess of what can be dissolved into a balanced salt solution and which are isosmotic with blood.
  • An ideal blood substitute should have an oxygen carrying capacity as good as that of the natural hemoglobin molecule within the red blood cells (RBCs), be less antigenic, have a long shelf life (preferably at room temperature), have a long intravascular half-life, and be free of toxicity and side effects.
  • oxygen-carrying blood substitutes are based on hemoglobin and are named Hemoglobin-based oxygen carriers (HBOCs).
  • HBOCs Hemoglobin-based oxygen carriers
  • said oxygen carrier composition is a hemoglobin-based oxygen carrier.
  • HBOCs are physiological solutions that contain hemoglobin from purified human, animal, or recombinant sources.
  • purified hemoglobins typically have undergone multiple chemical modifications, including crosslinking and/or polymerization to change their physiochemical characteristics.
  • Hemoglobin can be cross-linked and polymerized for instance with glutaraldehyde and/or o- raffinose to increase its ability to deliver oxygen and increase its duration of action in circulation (Levy J H, May 2009, American Council on Science and Health).
  • hemoglobin solutions can also include one or more pharmaceutically acceptable carriers and/or excipients.
  • pharmaceutically acceptable carriers include water (including water for injection), isotonic solutions, such as saline solution (e.g., 0.9% NaCI), buffered saline, glucosalin solution (e.g., 5% glucose and 0.9% NaCI), lactated Ringer's solution (e.g., 102 mmol/L sodium chloride; 28 mmol/L sodium lactate; 4 mmol/L of potassium chloride, and 1 .5 mmol/L of calcium chloride), plasma-lyte solution (lactated Ringer-like mixture with presence of magnesium, acetate and gluconate ions) and the like.
  • water including water for injection
  • isotonic solutions such as saline solution (e.g., 0.9% NaCI), buffered saline, glucosalin solution (e.g., 5% glucose and 0.9% NaCI), lactated Ringer'
  • excipients include an agent for regulating the osmotic pressure and/or the pH (e.g., an acid/salt buffer system, a salt, a monosaccharide, a disaccharide or a polyol), a surfactant, a preservative agent, as well as combinations thereof.
  • said HBOC composition comprises one or more, preferably all, the excipients selected from the group consisting of sodium chloride, potassium chloride, dihydrated calcium chloride, sodium hydroxide, sodium lactate and N- acetyl-L-cysteine.
  • said composition comprises the following excipients and the carrier is water for injection:
  • N-acetyl-L-cysteine 10.0-15.0 mmol/L (preferably, 12.3 mmol/L)
  • suitable oxygenated hemoglobin solutions employed by the method of the invention are prepared in vitro by oxygenating hemoglobin solutions that include hemoglobin to convert at least about 80%, more preferably at least about 90%, by weight of the hemoglobin to oxyhemoglobin.
  • about 18% by weight, or less, of the hemoglobin that is included in the hemoglobin solutions to be oxygenated has a molecular weight of over 500,000 Daltons; about 5% by weight, or less, of the hemoglobin that is included in the hemoglobin solutions to be oxygenated has a molecular weight equal to or less than 65,000 Daltons; and/or an the endotoxin content of the hemoglobin solution that is included in the hemoglobin solutions to be oxygenated is less than about 0.5 endotoxin units per milliliter, preferably less than about 0.05 endotoxin units per milliliter.
  • a P 50 of the polymerized hemoglobin is preferably in a range of between about 24 and about 46 mm Hg, preferably between about 34 and about 46 mm Hg.
  • Said oxygenated hemoglobin solution may include from about 10 grams to about 250 grams of chemically modified (e.g., polymerized) hemoglobin per liter of solution.
  • said HBOC composition is characterized by one or more, preferably all of the following features:
  • the hemoglobin tetramer has been cross-linked with glutaraldehyde to stabilize the molecule and minimize existence of dimer subunits;
  • Hemoglobin tetramers have been polymerized to increase average MW to 250 kD to increase retention in vascular compartment, thereby also decreasing vasoconstriction activity and a 3-yr shelf life at 2-30 degrees C.
  • the largest polymers are still much smaller than an RBC (1/1000 diameter of an erythrocyte), facilitating access to tissues not available to RBCs, particularly in pathological conditions (i.e., micro-occluded vasculature as in no-reflow phenomenon during ischemia-reperfusion injury);
  • Residual tetramer is fractionated out to ⁇ 3% of total Hb to further to increase retention in vascular compartment, thereby also decreasing vasoconstriction activity;
  • the HBOC composition comprises N-acetyl-L-cysteine to maintain Hb in reduced state during storage in original packaging;
  • the HBOC composition comprises a modified Ringers solution to achieve physiological osmolality and maintain normal ion gradients between plasma, cells and tissues;
  • the HBOC composition has a physiological oncotic pressure to prevent net fluid exchange with blood cells and tissues;
  • the HBOC composition has a viscosity lower than blood, facilitating fluid mechanics, particularly in microvasculature;
  • the HBOC composition has an elevated 0 2 p50 (40 mmHg), facilitating 02 unloading and diffusive 02 delivery to tissues;
  • the HBOC composition is ready for immediate use without thawing or reformulating.
  • the oxygenated carrier composition is a polymerized hemoglobin solution as described in US 2010/0209532. More preferably, said oxygenated carrier composition comprises a purified hemoglobin intra/intermolecular crosslinked with glutaradehyde; even more preferably, it comprises a purified hemoglobin intra/intermolecular crosslinked with glutaradehyde, and further comprises NaCI from 100 to 130 mmol/L (preferably, 1 14 mmol/L), KCI from 2.0 to 6.0 mmol/L (preferably, 4.0 mmol/L), CaCI 2 -2H 2 0 from 0.5 to 2.5 mmol/L (preferably, 1 .4 mmol/L), NaOH from 10.0 to 15.0 mmol/L (preferably, 12.5 mmol/L), sodium lactate from 20.0 to 35.0 mmol/L (preferably, 27.1 mmol/L), N-acetyl-L-cysteine from 10.0 to 20.0 mmol/L (preferably, 12.3 mmol/L)
  • said oxygen carrier composition is HBOC-201 .
  • HBOC-201 also referred as Hemopure® or hemoglobin glutamer-250; Hb02 Therapeutics, LLC
  • HBOC-201 is a polymerized, iso-oncotic, high-molecular weight, bovine haemoglobin-based oxygen carrier suitable for intravenous infusion.
  • HBOC-201 comprises purified bovine hemoglobin intra/intermolecular cross-linked with glutaraldehyde (shown below) formulated in a balanced salt solution, more specifically in a modified Ringer's lactate solution at pH of 7.6 to 7.9. This cross-linked hemoglobin has been described to have a half- life of about 24 hours.
  • HBOC-201 is a sterile solution with no risk of disease transmission.
  • this cross-linked hemoglobin carries the same amount of oxygen as the hemoglobin in red blood cells.
  • the hemoglobin molecules in HBOC-201 have the advantage that are smaller, have lower viscosity and more readily release oxygen to tissues than red blood cells. Consequently, they can carry oxygen at low blood pressure and through constricted or partially blocked blood vessels to areas of the body that red blood cells cannot reach due to their larger size.
  • Hemolink® Hemosol, Inc., Missiassauga, Canada
  • Hemolink® Hemosol, Inc., Missiassauga, Canada
  • PolyHeme® (Northfield Laboratories, Inc.) which comprises a human cross-linked hemoglobin polymer purified from outdated erythrocytes. More specifically, it is intramolecular cross-linked with pyridoxal phosphate (PLP) (A) and intermolecular cross- linked with glutaraldehyde (B).
  • PRP pyridoxal phosphate
  • B glutaraldehyde
  • Hemospan® (Sangart, Inc., San Diego, CA) which comprises a maleimide-polyethylene glycol-modified human hemoglobin.
  • Diaspirin cross-linked hemoglobin (DCLHb, HemAssist®; Baxter Healthcare Corp), which comprise a intramolecular cross-linked with bis (3,5-dibromosalicyl)-fumarate).
  • VitalHeme® (Caged Nitric Oxide (cNO) Labeled Polynitroxylated Pegylated Hemoglobin (PNPH); SynZyme Technologies, LLC) is under development for the treatment of traumatic brain injury complicated by hemorrhagic shock, stroke and sickle cell disease.
  • VitalHeme® comprises a multifunctional neuroprotective cNO labeled polynitroxylated pegylated hemoglobin nano-particle.
  • Polynitroxylated pegylated hemoglobin as a multifunctional therapeutic which takes advantage of the ability of hemoglobin (Hb) to transport oxygen, the antioxidative stress activities from the redox coupling of nitroxide and heme iron and the hypercolloid properties of pegylation.
  • VitalHeme® acts by reducing oxidative stress in hemorrhagic shock and sickle cell disease.
  • Sanguinate® (Pegylated Hemoglobin, PEG-Hb, Pegylated Carboxyhemoglobin Bovine) from Prolong Pharmaceuticals.
  • Sanguinate® comprises a PEGylated hemoglobin which facilitates the transfer of red blood cells to the tissue and begins the re-oxygenation process. This process helps to release other molecules which protect the vasculature and surrounding tissue during the transfer. It reduces the oxygen debt and protects from the ischemic reperfusion injury.
  • the drug candidate is under development for the treatment of sickle cell disease (SCD), delayed cerebral ischemia after acute aneurysmal subarachnoid hemorrhage, end-stage renal disease and ischemia reperfusion injury in myocardial infarction.
  • SCD sickle cell disease
  • delayed cerebral ischemia after acute aneurysmal subarachnoid hemorrhage end-stage renal disease and ischemia reperfusion injury in myocardial infarction.
  • PFC Perfluorocarbon
  • said oxygen carrier composition is a composition comprising PFCs, such as PFC emulsions.
  • the viscosity of the oxygen carrier composition is generally between 1 and 3 centipoises at 37°C, preferably between 1.5 and 2.5 centipoises at 37°C, more preferably has a viscosity about 2.2 centipoises at 37°C.
  • said oxygen carrier composition is a modified oxygen carrier composition of the invention as described herein.
  • Said preventive, therapeutic and/or diagnostic agent can be any agent suitable for the treatment and/or diagnosis of myocardial diseases.
  • myocardial disease is used to encompass numerous conditions affecting the heart, and the heart valves, and encompasses diseases and conditions including, but not limited to arteriosclerosis, atherosclerosis, myocardial infarction, acute coronary syndrome, angina, heart failure, atrial fibrillation, systolic dysfunction, diastolic dysfunction, myocarditis, atrial tachycardia, ventricular fibrillation, endocarditis, acute coronary syndrome, acute ischemic attack, and sudden cardiac death.
  • This agent may be any type of therapeutic, prophylactic and/or diagnostic agent, such as traditional chemical agents or biological agents, which includes but is not limited to peptides, proteins (e.g., antibodies, growth factors and cytokines), cell therapy and gene therapy, and combinations thereof.
  • traditional chemical agents or biological agents which includes but is not limited to peptides, proteins (e.g., antibodies, growth factors and cytokines), cell therapy and gene therapy, and combinations thereof.
  • peptides e.g., antibodies, growth factors and cytokines
  • cell therapy and gene therapy e.g., cell therapy and gene therapy, and combinations thereof.
  • a person skilled in the art will know the most appropriate agent according to the disease to be treated or diagnosed.
  • said agent is a cardioprotective agent.
  • Cardioprotective agents are typically used in the treatment of ischemia/reperfusion injury, and include but are not limited to Glucagon-like peptide-1 (GLP-1 ) and analogues thereof; Cyclosporin-A and Metop
  • said agent is a nucleic acid sequence.
  • said nucleic acid sequence is a nucleic acid construct.
  • said agent comprises or consists of a transgene encoding expression vector and may be used for gene therapy purposes.
  • a person skilled in the art will select the nucleic acid sequence to be transduced into the cardiomyocytes according to the myocardial disease to be treated. These typically include but are not limited to growth factors and cytokines.
  • Non-limiting examples of nucleic acid sequences are SUM01 , IGF-1 , VEGF, calcineurin A beta 1 , Niemann-Pick type C gene, FGF, HGF, PDGF, HIF1 a, SERCA2a, SDF-1 , ADCY6, pARK-ct-carboxy terminal peptide from GRK2, S100A1 , PVALB, KCNH2-G628S, SCN4A, connexin 32, connexin 40, connexin 43, ADCY1 and Kir2.1.
  • genes used for gene therapy purposes in coronary heart disease, heart failure and arrhythmia are as specified in tables 2, 3 and 4 of Wolfram and Donahue (Journal of the American Heart Association 2013, e0001 19), which is hereby incorporated by reference.
  • Non-viral transfection methods include both chemical and physical systems.
  • chemical-based systems synthetic or naturally occurring compounds such as calcium phosphate (CaP), DEAE-dextran, cationic lipids, and cationic polymers may facilitate the transfer of a nucleic acid construct through the cell membrane.
  • CaP calcium phosphate
  • DEAE-dextran DEAE-dextran
  • cationic lipids and cationic polymers
  • the efficacy of chemical non-viral gene delivery methods and their safety for cells is dependent on various factors such as the type of method, ratio of nucleic acid construct to reagents, charge and size of complexes, time of exposure, type of target cell, and correct cell density.
  • nucleic acid construct is a viral vector.
  • Viruses have been reported to be highly efficient at nucleic acid delivery to specific cell types while avoiding immunosurveillance by an infected host. These properties make viruses attractive gene-delivery vehicles, or vectors, for gene therapy.
  • Recombinant viruses used for gene therapy purposes include but are not limited to retrovirus, adenovirus, adeno- associated virus (AAV), and herpesvirus.
  • the nucleic acid construct further comprises a 5'ITR and a 3'ITR of a virus.
  • inverted terminal repeat refers to a nucleotide sequence located at the 5'-end (5'ITR) and a nucleotide sequence located at the 3'-end (3'ITR) of a virus, that contain palindromic sequences and that can fold over to form T-shaped hairpin structures that function as primers during initiation of DNA replication. They are also needed for viral genome integration into host genome; for the rescue from the host genome; and for the encapsidation of viral nucleic acid into mature virions. The ITRs are required in c/ ' s for the vector genome replication and its packaging into the viral particles.
  • the nucleic acid construct comprises a 5'ITR, a ⁇ packaging signal, and a 3'ITR of a virus
  • ⁇ packaging signal is a cis-acting nucleotide sequence of the virus genome, which in some viruses (e.g. adenoviruses, lentiviruses ...) is essential for the process of packaging the virus genome into the viral capsid during replication.
  • the nucleic acid construct comprises a 5'ITR and a 3'ITR of a virus selected from the group consisting of parvoviruses (in particular adeno- associated viruses), adenoviruses, alphaviruses, retroviruses (in particular gamma retroviruses, and lentiviruses), herpesviruses, and SV40.
  • the virus is an adeno-associated virus (AAV).
  • the nucleic acid construct comprises a 5'ITR and a 3'ITR of an AAV.
  • the AAV genome is composed of a linear, single-stranded DNA molecule which contains 4681 bases (Berns and Bohenzky, (1987) Advances in Virus Research (Academic Press, Inc.) 32:243-307).
  • the genome includes inverted terminal repeats (ITRs) at each end which function in c/ ' s as origins of DNA replication and as packaging signals for the virus.
  • ITRs are approximately 145 bp in length.
  • the internal non-repeated portion of the genome includes two large open reading frames, known as the AAV rep and cap genes, respectively. These genes code for the viral proteins involved in replication and packaging of the virion.
  • At least four viral proteins are synthesized from the AAV rep gene, Rep 78, Rep 68, Rep 52 and Rep 40, named according to their apparent molecular weight.
  • the AAV cap gene encodes at least three proteins, VP1 , VP2 and VP3.
  • the construction of recombinant AAV virions is generally known in the art and has been described for instance in US 5,173,414 and US5.139.941 ; WO 92/01070, WO 93/03769, (Lebkowski et al. (1988) Molec.
  • the ITRs are the only AAV viral elements which are required in c/ ' s for the AAV genome replication and its packaging into the viral particles.
  • the ITRs may be synthetic or have been derived from viruses of different serotypes. All other viral genes required for viral vector replication can be provided in trans within the virus-producing cells (packaging cells).
  • the nucleic acid construct may comprise ITRs of any AAV serotype, including AAV1 , AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1 , AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV serotype now known or later discovered.
  • the ITRs are from a human AAV serotype.
  • the ITRs are from the AAV2 serotype.
  • the ITRs are selected from the group consisting of AAV1 , AAV6, AAV8 and AAV9 serotypes.
  • control sequences and “control elements” refer, among others, to promoter and termination sequences.
  • Useful promoter sequences include those derived from sequences encoding vertebrate cardiac-specific expression genes.
  • the nucleic acid construct comprises a coding sequence operably linked to a vertebrate cardiac-specific gene expression promoter and a transcription termination sequence located 3' to the coding sequence, wherein the coding sequence operably linked to the control sequences is in turn flanked by the AAV ITRs and wherein the preferred AAV ITRs are derived from the AAV-2 serotype.
  • vertebrate cardiac-specific gene expression promoters include, but are not limited to, the chicken cardiac troponin T (cTnT/TNNT2) promoter, the human cTnT promoter, the mouse cTnT promoter, the human alpha-myosin heavy chain promoter, the mouse alpha-myosin heavy chain promoter, rat alpha-myosin heavy chain promoter, human myosin light chain 2v promoter, 5 mouse myosin light chain 2v promoter, rat myosin light chain 2v promoter, frog myosin light chain 2v promoter, human cardiomyocyte-specific Na + - Ca 2+ exchange promoter, human cardiac alpha-actinin promoter, mouse alpha-actinin promoter, human cardiac troponin I (TNNI3) promoter.
  • cTnT/TNNT2 the chicken cardiac troponin T
  • human cTnT/TNNT2 promoter the human cTnT promoter
  • the vertebrate cardiac-specific gene expression promoter is the chicken cardiac troponin T (cTnT) promoter.
  • the "Chicken cardiac troponin T (cTnT) promoter” refers to a regulatory sequence that controls transcription of the cardiac troponin T gene in chicken.
  • the nucleic acid construct comprises a coding sequence operably linked to the chicken cardiac troponin T (cTnT) promoter and a polyA transcription termination sequence located 3' to the coding sequence, wherein the coding sequence operably linked to the control sequences is in turn flanked by the AAV ITRs and wherein the preferred AAV ITRs are derived from the AAV-2 serotype.
  • cTnT chicken cardiac troponin T
  • transcription termination sequences include, but are not limited to, polyA, SV40 polyA, human growth hormone polyA and bovine growth hormone polyA.
  • said transcription termination sequence is a SV40 polyA transcription termination sequence.
  • SV40 pA transcription termination sequence refers to a regulatory sequence that controls termination of transcription in the Simian Virus 40 and includes the typical polyadenylation core sequence AATAAA.
  • the nucleic acid construct comprises a coding sequence operably linked to the chicken cardiac troponin T (cTnT) promoter and a SV40 polyA transcription termination sequence located 3' to the coding sequence, wherein the coding sequence operably linked to the control sequences is in turn flanked by AAV ITRs derived from the AAV-2 serotype.
  • cTnT chicken cardiac troponin T
  • SV40 polyA transcription termination sequence located 3' to the coding sequence
  • AAV ITRs derived from the AAV-2 serotype.
  • an AAV virion may comprise capsid proteins from any AAV serotype.
  • the AAV viral particle comprises capsid proteins from a serotype selected from the group consisting of AAV1 , AAV2, AAV6, AAV8, and AAV9 which have been described as more suitable for delivery to the myocardial cells (Prasad et al., 201 1 , Gene Therapy, 18(l):43-52), preferably capsid proteins are from a serotype selected from the group consisting of AAV8 and AAV9, more preferably the capsid proteins are from the AAV9 serotype.
  • the viral particle comprises a nucleic acid construct of invention wherein the 5'ITR and 3'ITR sequences of the nucleic acid construct are of an AAV2 serotype and the capsid proteins are of an AAV9 serotype, referred as AAV2/9 vector.
  • nucleic acid construct and expression vector may be obtained by conventional methods known to those skilled in the art: Sambrook and Russell (Molecular Cloning: a Laboratory Manual; Third Edition; 2001 Cold Spring Harbor Laboratory Press); and Green and Sambrook (Molecular Cloning: a Laboratory Manual; Fourth Edition; 2012 Cold Spring Harbor Laboratory Press).
  • the invention relates to an oxygen carrier composition (e.g. an HBOC composition) for use in a method for the in vivo diagnosis and/or the therapeutic and/or preventive treatment of a myocardial disease; wherein said method comprises the following consecutive steps:
  • an oxygen carrier composition e.g. an HBOC composition
  • AAV adeno- associated virus
  • the method of the invention as described herein results in an increase of the transgene transduction in comparison with the administration of the diagnostic, prophylactic and/or therapeutic transgene encoding expression vector in the absence of an oxygen carrier composition of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 1 10%, at least 120%, at least 130%, at least 140% or at least 150%.
  • Gene transduction is generally higher in those areas of the myocardium directly irrigated by the coronary vessel wherein administration of the expression vector has been conducted.
  • gene transduction is achieved at least in the left ventricle.
  • gene transduction is achieved in all the myocardium.
  • Methods to determine efficiency of gene transduction include but are not limited to determining the number of cells positive for the transgene in the target tissue, and determining the total amount of the transgene and/or its expression levels in the target tissue, for instance by means of determining the expression of the therapeutic, prophylactic and/or diagnostic gene or of a reporter gene comprised within the expression vector.
  • Reporter genes that may be used to determine transfection efficiency include but are not limited to chloramphenicol acetyl transferase (CAT), ⁇ -galactosidase, Photinus pyralis luciferase, and Renilla reniformis green fluorescent protein (GFP).
  • the determination of the expression of the transgene or reporter gene may be carried out at protein level.
  • methods for the quantification of peptides and proteins well known to one skilled in the art, including but not limited to enzymatic assays and immunoassays.
  • immunoassays are known to one skilled in the art for the quantitation of proteins of interest, either in solution or using a solid phase assay. These methods are based on the use of affinity reagents, which may be any antibody or ligand specifically binding to the target protein, which is preferably labeled.
  • affinity reagents which may be any antibody or ligand specifically binding to the target protein, which is preferably labeled.
  • western blotting or immunoblotting allows comparison abundances separate proteins by an electrophoretic gel, eg. SDS-PAGE.
  • a sheet of polymeric material generally nitrocellulose, nylon, or polyvinylidene difluoride
  • proteins are detected directly in cells of a tissue.
  • target proteins are revealed by using a solution containing a specific antibody (staining).
  • the antibody can be conjugated directly with a radioactive, fluorescent or enzymatic (direct detection method) or may be used a secondary antibody that recognizes the primary antibody and thus amplifies the signal (indirect method of detection or sandwich assay).
  • a specific capture antibody for the target protein in a polymer or plastic surface immobilized and added to the support a solution containing the protein of interest (for example, cell lysate). Finally, the sample is incubated on the support for a time to allow antigen-antibody complexes are formed. Then, usually perform one or more washes to remove the solution and the target protein is detected with a second antibody that recognizes an epitope different from that recognized by the capture antibody protein. As in the case of Western blotting, this detection antibody may be labeled directly or can be recognized with a secondary antibody.
  • An immunoassay commonly used for protein quantification is the test enzyme-linked immunosorbent assay (ELISA) in which the detection antibody carries an enzyme that converts a commonly colorless substrate into a colored compound or a non-fluorescent substrate to a fluorescent compound. Also in other solid phase immunoassays, the antibody may be labeled with a radioactive isotope or fluorescence.
  • MS mass spectrometry
  • LC / MS liquid chromatography coupled to mass spectrometry
  • Gene transduction may also be measured by the transgene quantification or the mRNA expression levels thereof.
  • Molecular biology methods for measuring quantities of target nucleic acid sequences are well known in the art. These methods include but are not limited to end point PCR, competitive PCR, reverse transcriptase-PCR (RT-PCR), quantitative PCR (qPCR), reverse transcriptase qPCR (RT-qPCR), PCR-pyrosequencing, PCR-ELISA, DNA microarrays, in situ hybridization assays such as dot-blot or Fluorescence In Situ Hybridization assay (FISH), branched DNA (Nolte, Adv. Clin. Chem.
  • Such methods may also include the pre-conversion of mRNA into cDNA by the reaction with a reverse transcriptase (RT), for example the PCR or qPCR reaction is usually preceded by conversion of mRNA into cDNA and referred to as RT-PCR or RT-qPCR, respectively.
  • RT reverse transcriptase
  • said molecular method for gene quantification is selected from the group consisting of quantitative Polymerase Chain Reaction (qPCR), PCR-pyrosequencing, fluorescence in-situ hybridization (FISH), DNA microarrays, and PCR-ELISA.
  • qPCR quantitative Polymerase Chain Reaction
  • FISH fluorescence in-situ hybridization
  • DNA microarrays DNA microarrays
  • PCR-ELISA quantitative Polymerase Chain Reaction
  • gene transduction efficiency is determined by measuring the transgene mRNA expression levels by reverse transcriptase quantitative PCR (RT-qPCR).
  • Expression levels may be absolute or relative. It is generally preferred that expression levels are normalized. Normalization can be performed with respect to different measures in the sample. These procedures are well known to one skilled in the art. Typically, expression levels are normalized with respect to an "Endogenous control".
  • An “Endogenous control” as used herein relates to a gene expression product whose expression levels do not change or change only in limited amounts.
  • the "endogenous control” is the expression product from housekeeping genes and which code for proteins which are constitutively expressed and carry out essential cellular functions.
  • housekeeping genes for use in the present invention include ⁇ -2- microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, GAPDH, actin and HPRT.
  • the oxygen carrier composition for use in a method as described herein is characterized by said method comprising the following consecutive steps:
  • the oxygen carrier composition in ii) and iv) is substantially free of the diagnostic, prophylactic and/or therapeutic agent administered in iii). More preferably, the oxygen carrier composition in ii) and iv) is substantially free of any diagnostic, prophylactic and/or therapeutic agent.
  • the intracoronary administration of the agent and the oxygen carrier composition in step iii) occurs simultaneously.
  • the agent and the oxygen carrier composition may be administered in the same composition or in a separate composition.
  • the agent and the oxygen carrier composition are formulated in a single composition. This formulation can be extemporaneous (i.e., prepared immediately before the administration to the patient).
  • occlusion is performed by the closed chest model, i.e., comprises inflating a catheter occluding balloon.
  • the method of the invention comprises temporary occluding a coronary artery and/or a coronary vein, as defined above.
  • the aim of this step is mainly isolating the vessel from the blood circulatory system of the subject prior to administering the oxygen carrier composition vessel (optionally comprising the prophylactic, therapeutic or diagnostic agent).
  • Administration may be antegrade (e.g. through the arteries in the direction of blood flow), in a retrograde manner (through veins, such as the coronary sinus, in opposition to the normal blood flow direction), or in a combination of retrograde and antegrade administration.
  • administration is antegrade and administration downstream of the site of occlusion.
  • administration is retrograde and administration upstream of the site of occlusion.
  • said method comprises in step i) the temporary occlusion of the left anterior descending (LAD) artery, preferably a mid-LAD artery occlusion, wherein preferably occlusion is conducted by inflating a catheter occluding balloon.
  • LAD left anterior descending
  • Occlusion time of the coronary artery and/or the coronary vein may be from a few seconds to several minutes and will be determined in order to maximize the time of contact of the agent with the target site in the absence of deleterious effects.
  • Possible occlusion times include occlusion times typically used for gene therapy purposes which are generally of less than 10 minutes, preferably of less than 5 minutes, to prevent ischemia-related injury, preferably from 1 to 10 minutes, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes, including fractions thereof.
  • the method of the invention which comprises the administration of an oxygenated oxygen carrier composition also encompasses longer occlusion times, such as 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 35, 40, 45 or more minutes.
  • the occlusion time is of between 1 and 15 minutes, preferably of 12 or 13 minutes. In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the occlusion time is of between 1 and 5 minutes, preferably of 2 or 3 minutes.
  • the volume of the oxygen carrier composition to be perfused without removal of blood is physiologically limited.
  • the total volume of the oxygen carrier injected is generally recommended to be below 20%, preferably about 17%, of the subject total circulatory volume (12 ml/kg).
  • the volume which may be perfused would be of 600 mL, and for an individual of 60 kg (typically the average weight of a human subject) this volume would be of 720 mL.
  • a maximum total volume of 12 mL/kg is administered.
  • Said oxygen carrier composition may be perfused at a maximum infusion rate of 1 ml/kg/min (equating to normal coronary flow).
  • a maximum infusion rate of 1 ml/kg/min (equating to normal coronary flow).
  • preferred infusion rates are from 0.4 ml/kg/min to 1 ml/kg/min, such as an infusion rate of about 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 ml/kg/min.
  • the perfusion rate is of 1 ml/kg/min.
  • the perfusion rate is of 1 ml/kg/min and the a perfusion time is of 12 minutes (corresponding to an administered volume of 12 ml/kg and a total volume of 600 mL in a 50 kg individual).
  • the volume of the oxygen carrier composition to be perfused prior to the agent administration may be from 100 mL to 500 mL, preferably from 200 to 400 mL, such as of about 250, 300 or 350 mL.
  • administration is of 250 mL (corresponding to 5 minutes at an infusion rate of 1 ml/kg/min for an individual of 50 kg).
  • a person skilled in the art will determine the appropriate perfusion time according to the selected volume to be perfused and the perfusion rate.
  • the volume of the oxygen carrier composition comprising the agent may be from 100 mL to 500 mL, preferably from 200 to 400 mL, such as of about 250, 300 or 350 mL.
  • administration is of 350 mL (corresponding to 7 minutes at an infusion rate of 1 ml/kg/min for an individual of 50 kg).
  • the oxygen carrier composition is administered during 5 minutes at a rate of 1 ml/kg/min (corresponding to 250 mL for a 50 kg individual), subsequently the composition comprising the oxygen carrier and the agent is administered during 7 minutes at a rate of 1 ml/kg/min (corresponding to 350 mL for a 50 kg individual).
  • the oxygen carrier composition may be administered for an additional time period, e.g. 1 minute at an infusion rate of 1 ml/kg/min, prior to blood flow restoration.
  • the present invention relates to a composition comprising or consisting of:
  • composition is herein also referred as "the modified oxygen carrier composition of the invention".
  • said composition comprises or consists of:
  • a metabolic substrate may be any potential source of ATP, including fatty acids, glucose, ketone bodies, pyruvate, lactate, amino acids and even constituent proteins, as well as combinations thereof.
  • Amino acids are not particularly limited, and can be selected for instance from the group consisting of alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, taurine, threonine, tryptophan, tyrosine, valine and combinations thereof.
  • amino acids are selected from the group consisting of glutamate, glutamine, asparagine and alanine, and combinations thereof.
  • said metabolic substrate is glucose.
  • said metabolic substrate is glucose and said composition further comprises insulin.
  • the pH of the composition might be from pH 4.0 to pH 7.5, preferably from pH 6.8 to pH 7.4, more preferably from pH 7.0 to pH 7.4, for instance any pH selected from 7.0, 7.05, 7.1 , 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.
  • the pH of said composition is about 7.35.
  • a pH modulator or buffering agent may be an acid/salt buffer system.
  • the acid component in the buffer mixtures can be used inorganic acids (e.g., phosphate, carbonate, hydrogencarbonate), polyvalent carboxylic acids (e.g., succinic acid, maleic acid, benzoic acid), hydroxycarboxylic acids (such as glycolic acid, citric acid, malic acid or lactic acid), keto acids (eg, a-ketoglutaric acid) or sulfonic acids (e.g., 2- [ 4- (2- hydroxyethyl) -1 -piperazino] -ethanesulfonic acid (HEPES)), and amino acids (e.g., glycine, histidine, aspartic acid, phenylalanine, lysine, arginine, cysteine; including the natural L- amino acids as well as derivatives thereof), and combinations thereof.
  • the buffer substances can either be present in the free acid form and/or in the form of
  • said pH modulator agent is n- acetylcysteine.
  • said pH modulator agent is n-acetylcysteine and the pH of said composition is adjusted to 7.35.
  • said composition comprises or consists of:
  • composition is from 6.8 to 7.4, preferably wherein the pH of said composition is 7.35.
  • Other pharmaceutically acceptable excipients which may be included in said composition include but are not limited to monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, trehalose, maltose and sucrose; trisaccharides such as raffinose, tetrasaccharides such as stachyose; polysaccharides such as dextran; and sugar alcohols such as mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur- containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thiogly
  • the modified oxygen carrier composition of the invention may be suitable for in vitro or in vivo use.
  • said composition is a pharmaceutical composition.
  • the present invention relates to the use of the modified oxygen carrier composition of the invention as a pharmaceutical composition. It also relates to the composition or pharmaceutical composition of the invention for use as a medicament.
  • a "pharmaceutical composition” refers to a composition that is pharmaceutically acceptable.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.
  • Said pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • Methods to accomplish the administration are known to those of ordinary skill in the art. This includes, for example, injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical.
  • the pharmaceutical composition of the invention is administered to a subject by intravascular administration.
  • the administration is intracoronary.
  • the composition may be injected or infused using a syringe, as well as other devices known in the art.
  • the pharmaceutical composition may be administered via the central lumen of the catheter which may have been introduced percutaneously.
  • the invention relates to the composition according to the preceding aspects of the invention, for use in a method of in vivo diagnosis and/or treatment of a tissue or organ which has or is at risk of having ischemia, this includes for instance those organs which may be transplanted, such as the heart, liver or kidney.
  • a tissue or organ which has or is at risk of having ischemia is the myocardium.
  • a related aspect is directed to methods for in vivo diagnosis and/or treating a tissue or organ which has or is at risk of having ischemia with a composition or pharmaceutical composition of the present invention.
  • the method to in vivo diagnosing or treating said ischemia comprises administering to a subject a composition or pharmaceutical composition of the present invention in an amount effective to treat it.
  • the invention refers to the modified composition of the invention for use in a method for the preventive and/or therapeutic treatment of ischemia/reperfusion injury, for instance after acute myocardial infarction.
  • Said method comprises administration of said modified composition before and/or after interruption of the blood flow. Preferably, it is administered after interruption of the blood flow and before blood flow restoration.
  • composition may be particularly suitable for administration to an ischemic organ, such as an ischemic myocardium.
  • the modified composition of the invention is administered after 15 minutes from oxygen deprivation, for instance after 20, 25, 30, 35, 40 or 45 minutes.
  • administration of the composition is performed after 45 minutes or more from oxygen deprivation. Oxygen deprivation may occur for instance by interruption of the blood flow.
  • said oxygen carrier composition is perfused at an infusion rate from 0.4 to 1 mL/kg/min, preferably at an infusion rate of 1 mL/kg/min.
  • the invention relates to a method for obtaining a composition according to the preceding aspects of the invention. It may comprise mixing the components of the composition as described herein. It also may comprise adding a metabolic substrate to the oxygen carrier composition and adjusting the pH of the composition as described herein. In a particular embodiment, it comprises:
  • composition of the present invention has been found to be particularly suitable for bloodless drug delivery. Accordingly, in a further aspect, the invention relates to a delivery vehicle comprising a pharmaceutical composition as described herein.
  • a delivery vehicle comprising a pharmaceutical composition as described herein.
  • An additional aspect of the invention relates to an oxygen carrier composition as described in the preceding aspects of the invention, for use as delivery vehicle in the administration of a therapeutic and/or diagnostic agent, preferably in a method as described herein under the first aspect of the invention.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • the term “comprises” also encompasses and expressly discloses the terms “consists of” and “consists essentially of”.
  • the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim except for, e.g., impurities ordinarily associated with the element or limitation.
  • words of approximation such as, without limitation, "about”, “around”, “approximately” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature.
  • a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by ⁇ 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15%. Accordingly, the term “about” may mean the indicated value ⁇ 5% of its value, preferably the indicated value ⁇ 2% of its value, most preferably the term "about” means exactly the indicated value ( ⁇ 0%).
  • Example 1 Pre-oxygenated HBOC -201 intracoronary infusion strategies 1.1. Material and Methods
  • Protocol 1 preparation and procedures Intracoronary infusion of HBOC-201 in healthy pigs
  • pigs were monitored by continuous electrocardiography, pulse-oximetry, and invasive measurement of systemic and pulmonary artery pressures (using a Swan-Ganz catheter).
  • a conventional 0.014-inch guidewire was advanced into the LAD coronary artery.
  • a short, highly compliant over-the-wire balloon (Helios Occlusion Balloon Catheter, LightLab) was then placed in the LAD artery proximal to the origin of the first diagonal branch and connected to the infusion pump (PHD ULTRA Series Syringe Pump, Harvard Apparatus). The guidewire was then removed and the balloon catheter was inflated (coronary occlusion).
  • the HBOC-201 solution was warmed using an inline clinical fluid warmer (Astotherm® plus, Model AP220S, Futuremed America) positioned immediately proximal to the intracoronary Helios balloon catheter.
  • the HBOC-201 solution was contained within the sterile, high-pressure infusion line wrapped around the warmer's heating coil. Balloon location and state of inflation were monitored regularly by angiography. After balloon deflation, a coronary angiogram was recorded to confirm patency of the coronary artery. In the event of ventricular fibrillation, a biphasic defibrillator was used to deliver non-synchronized shocks as needed. Postoperative animal recovery and care were carried out by CNIC veterinarians and technicians.
  • Protocol 2 preparation and procedures Myocardial infarction induction protocol followed by bloodless or regular reperfusion
  • pigs were sedated by intramuscular injection of ketamine (20 mg/kg), xylazine (2 mg/kg), and midazolam (0.5 mg/kg).
  • Central venous and arterial lines were placed and a single bolus of unfractionated heparin (300 lU/kg) was administered immediately before catheter introduction.
  • pigs were monitored by continuous electrocardiography, pulse-oximetry, and invasive measurement of systemic and pulmonary artery pressures (using a Swan-Ganz catheter).
  • Femoral artery was percutaneously accessed and a sheath was placed.
  • a guiding catheter was placed in the origin of the left coronary artery.
  • a conventional 0.014-inch guidewire was advanced into the LAD coronary artery.
  • a short, highly compliant over-the-wire balloon (Helios Occlusion Balloon Catheter, LightLab) was then placed in the LAD artery distal to the origin of the first diagonal branch and connected to the infusion pump (PHD ULTRA Series Syringe Pump, Harvard Apparatus). The guidewire was then removed and the balloon catheter was inflated (coronary occlusion).
  • the oxygen carrier used in these experiments was HBOC-201 (Hb02 Therapeutics; (Burkhoff D, Lefer DJ, Am Heart J 2015, 149:573-579 ; Cabrales P, Intaglietta M, ASAIO J 2013, 59:337-354; Street JS, Moallempour M, Lim JC , Expert Opin Biol Ther 2008, 8:1425- 1433 ; Te Lintel Hekkert M et al., Am J Physiol Heart Circ Physiol 2010, 298:1-11 103-1 1 13).
  • HBOC-201 was pre-oxygenated under sterile conditions 2 hours before each experiment. More specifically, HBOC-201 250ml bags were pre-chilled to 4°C overnight. Then, 80ml_ of 100% oxygen were injected into the HBOC-201 bag using an oxygenation tube set (Advanced Scientifics Inc., product no. B105885-1 ). Oxygenated bags were equilibrated by rocking for 90 minutes in a refrigerator at 4°C.
  • the total volume of the oxygen carrier injected was 17% of the animal's total circulatory volume (12 ml/kg).
  • Two different rates of infusion of the fixed oxygen carrier solution were tested (1 ml/Kg/min (physiological rate, resulting in 12 min of infusion) and 0.7 ml/Kg/min (reduced rate, resulting in 17 min of infusion).
  • Pre-oxygenated warm (37°C) oxygen carrier was infused via the central lumen of the catheter.
  • the oxygen carrier solution was warmed using an in-line clinical fluid warmer (Astotherm® plus, Model AP220S, Futuremed America) positioned immediately proximal to the intracoronary Helios balloon catheter.
  • the HBOC-201 solution was contained within the sterile, high-pressure infusion line wrapped around the warmer's heating coil.
  • CMR studies were performed at several time points to assess IS and LV performance. Pigs were anesthetized as described above, and anesthesia was maintained by continuous intravenous infusion of midazolam. CMR studies were performed using a Philips 3-Tesla Achieva Tx whole body scanner (Philips Medical Systems, Best, the Netherlands) equipped with a 32-element cardiac phased-array surface coil. Images were acquired with the use of ECG gating by operators blinded to the study arm.
  • Segmented cine steady-state free precession was performed to acquire 1 1 -13 contiguous short-axis slices covering the heart from the base to the apex in order to evaluate global and regional LV motion (FOV, 280 x 280 mm; slice thickness, 8 mm without gap; TR, 2.8 ms; TE, 1 .4 ms, flip angle, 45°; cardiac phases, 25; voxel size, 1 .8 x 1 .8 mm; number of excitations [NEX], 3).
  • FOV global and regional LV motion
  • T2W-STIR T2-weighted, triple inversion-recovery fast spin-echo sequence
  • Late gadolinium enhancement imaging was performed 15 minutes after administration of 0.2 mmol/kg gadopentate dimeglumine, using an inversion-recovery fast gradient-echo sequence to determine IS (FOV, 280 x 280 mm; 1 1 -13 short-axis slices, thickness 8 mm without gap; TR, 5.6 ms; TE, 2.8 ms; voxel size, 1 .6 x 1 .6 mm; time interval optimized to null normal myocardium; trigger delay, longest; bandwidth, 304 Hz per pixel; NEX, 2).
  • AAR myocardium at risk
  • AAR The area of myocardium at risk (AAR) was defined as the extent of the LV showing high signal intensity on T2W-STIR images.
  • AAR and necrosis were identified as hyperintense regions defined as regions exceeding 50% of the peak myocardial signal intensities (full width half maximum) with manual adjustment when needed. If present, a central core of hypointense signal within the area of increased signal was included in the T2W-STIR or late gadolinium enhancement analysis. IS was expressed both as a percentage of LV mass and normalized to AAR.
  • Protocol 1 Optimization of intracoronary HBOC-201 infusion rate and duration in healthy animals.
  • This protocol was undertaken in animals without infarction to assess the ability of healthy myocardium to tolerate prolonged coronary perfusion with pre-oxygenated HBOC-201 instead of blood.
  • the first goal was to identify conditions for HBOC-201 intracoronary infusion during coronary occlusion that does not cause permanent myocardial damage (scar formation or persistent LVEF deterioration).
  • We hypothesized that maintaining bloodless reperfusion for as long as possible without inducing myocardial damage (the goal of protocol 2) would maximize the opportunity to control factors implicated in reperfusion-related injury.
  • the HBOC-201 solution is colloidal, the maximum top-load (no blood extraction) infusion volume is limited to the volume that can be administered without risking circulatory volume overload.
  • animals were randomized to HBOC-201 infusion at the following rates of infusion: 1 , 0.7, 0.4 or 0.2 ml/Kg/min, corresponding to infusion durations of 12, 17, 30, and 60 min.
  • rates of infusion 1 , 0.7, 0.4 or 0.2 ml/Kg/min, corresponding to infusion durations of 12, 17, 30, and 60 min.
  • Hemodynamic data were obtained at baseline and 5 min before finishing the 600 mL HBOC- 201 infusion.
  • Hemodynamic changes in the 1 , 0.7, and 0.4 ml/Kg/min (12, 17, and 30 min) infusion groups were characterized by increases in systemic arterial pressure, pulmonary arterial pressure, and filling pressures (right atrial pressure and pulmonary capillary wedge pressure), without significant changes in heart rate or cardiac output (Table 1 ).
  • Pigs allocated to the 0.2 ml/Kg/min (60 min) infusion group had a much worse hemodynamic response, characterized by increased filling pressures and reduced cardiac output.
  • Coronary infusion groups correspond to infusion rates as follows: 12 min, 1 ml/kg/min; 17 min, 0.7 ml/kg/min; 30 min, 0.4 ml/kg/min; 60 min, 0.2 ml/kg/min.
  • HR heart rate
  • mPAP mean pulmonary arterial pressure
  • mSAP mean systemic arterial pressure
  • mRAP mean right atrial pressure
  • mPCWP mean pulmonary capillary wedge pressure
  • CO cardiac output. *p-value ⁇ 0.05 compared with baseline. #p ⁇ 0.05 compared with 12min infusion group.
  • AIVR Accelerated idioventricular rhythm
  • PAC Premature atrial complex
  • PVC Premature ventricular complex
  • PSVT Paroxistic supraventricular tachycardia
  • NSVT nonsustained ventricular tachycardia
  • Intracoronary infusion of pre-oxygenated HBOC-201 results in dynamic changes in LV performance depending on the infusion strategy.
  • all groups showed a significant reduction in LVEF compared to baseline levels (12 min infusion (median of differences [IQR] absolute points reduction in EF]: -5.8 [-8.5 to -3.8]; 17 min infusion: -6.9 [-12.1 to -4.4]; 30 min infusion: -13.7 [-14.1 to -6.3], and 60 min infusion: - 34.7 [-37.6 to -31.9]; p-value ⁇ 0.05).
  • Myocardial edema (% pigs) 0 0 0 100 #
  • Protocol 2 Study of the effect on infarct size of pre-oxygenated oxygen carrier
  • Fernandez-Jimenez R et al., J Am Coll Cardiol 2015, 65:315-323; Garcia-Prieto J et al., Basic Res Cardiol 2014, 109:422; Garcia-Ruiz JM et al., J Am Coll Cardiol 2016, 67:2093- 2104) was used to test whether the intracoronary perfusion of an oxygen carrier after a prolonged ischemic period (and before blood flow restoration) resulted in infarct size reduction compared with regular reperfusion (i.e. blood flow restoration) at the end of the index ischemia duration (i.e., 45 minutes).
  • Castrated male -30 kg
  • Large-White pigs were subjected to myocardial infarction induced by 45 minutes of mid left anterior descending (LAD) coronary artery occlusion.
  • pigs After 45 minutes of mid LAD artery occlusion, pigs were randomly assigned to regular reperfusion (direct blood flow restoration) or intracoronary infusion of pre-oxygenated oxygen carrier (at 2 different infusion rates) followed by ulterior blood flow restoration, see the treatment protocol in Figure 1 B.
  • the injection of the pre-oxygenated oxygen carrier at the end of the 45 min ischemia time results in a prolongation of the no-blood flow period.
  • the pH of the oxygenated HBOC-201 was 7.6 to 7.9.
  • NAC dose-pH curves were generated and the stability of the pH, %0 2 Hb and %metHb were checked over 24 hours,
  • the HBOC-201 solution was enriched with glucose (140 mg/dl) and insulin (Actrapid, 150 ⁇ /ml) by adding these components into the HBOC-201 bag before oxygenation.
  • the pH, glucose and insulin modified HBOC-201 solution is referred herein as "HBOC-pH-Glc-lns".
  • Pigs were randomized to control (regular reperfusion immediately after 45 min of ischemia) or to one of the two strategies for intracoronary oxygen carrier infusion (12 or 17 min infusion after 45 min of ischemia and before restoration of coronary blood flow), see Figure 1 B. Due to deaths before completion of the 7-day CMR, final group numbers were 8 control pigs and 9 and 13 pigs in the 12 min and 17 min HBOC-pH-Glc-lns infusion groups, respectively.
  • mSAP (mmHg) -26 [-31.8 to -22.3] -7 [-28.3 to 1] -21.5 [-35.8 to -11] mRAP (mmHg) 3.5 [0 to 5] 6 [2 to 7] 7 [5.5 to 8] * mPCWP (mmHg) 7.5 [4.8 to 9] 7 [6 to 12.5] 8 [5.5 to 16]
  • HR heart rate
  • mPAP mean pulmonary arterial pressure
  • mSAP mean systemic arterial pressure
  • mRAP mean right atrial pressure
  • mPCWP mean pulmonary capillary wedge pressure
  • CO cardiac output. *p-value ⁇ 0.05 compared with the control group (blood-only reperfusion).
  • Infarct size (%LV) 27.1 [24.2 to 34] 30.5 [29.4 to 37.5] 25.9 [22.6 to 36.6]
  • Infarct size (%Edema) 98.4 [77.6 to 100] 100 [87.5 to 100] 88.4 [68.9 to 100]
  • Data are shown as median [Q1 to Q3]; CMR, cardiac magnetic resonance; LVEF, left ventricular ejection fraction; LVEDVi: left ventricle end-diastolic volume index; LVESVi: left ventricle end-systolic volume index; *p-value ⁇ 0.05 compared with control group (blood-only reperfusion).
  • AAV adeno associated virus
  • the authors further investigated whether administration of HBOC-201 prior to and together with a GFP-encoding AVV vector enabled to prevent the vector contact with the remaining blood after coronary occlusion and resulted in an improvement of the gene transduction efficiency into the cardiomyocytes.
  • Green Fluorescent Protein (GFP)-expressing AAV vector AAV serotype 9 particles were produced in HEK293 cells. More specifically, an AAV shuttle vector (pAcTnTGFP) was used. It is derived from the pAcTnT vector (kindly provided by Dr B.A. French; Prasad et al., Gene Therapy 201 1 , 18(l):43-52) which contains AAV2-ITRs and carries the green fluorescent protein (GFP) reporter gene under the control of the chicken troponin promoter.
  • the nucleic acid construct was packaged into AAV9 capsids by using the triple transfection method as previously described (Prasad et al., Gene Therapy 201 1 , 18(l):43-52; Gao et al., PNAS 2002, 99 (18), 1 1854-1 1859).
  • Healthy pigs i.e. not in the context of infarction were instrumented as detailed in Example 1 above.
  • 2 castrated male (-30 kg) Large-White pigs were anesthetized, intubated and mechanically ventilated.
  • Femoral artery was percutaneously accessed and a sheath was placed. Through the sheath, a guiding catheter was placed in the origin of the left coronary artery.
  • control pig In the control pig (IC), the AAV solution was administered through the guiding catheter without stopping blood flow.
  • pre-oxygenated oxygen carrier pig a conventional 0.014-inch guidewire was advanced into the LAD coronary artery through the guiding catheter.
  • a short, highly compliant over-the-wire balloon Helios Occlusion Balloon Catheter, LightLab
  • Preoxygenated HBOC-201 with the modifications detailed in Example 1.3 above (i.e., HBOC-pH-Glc-lns) was mixed with AAV solution.
  • the modified pre-oxygenated oxygen was administered for 5 minutes at an infusion rate of of 1 ml/kg/min (the normal coronary blood flow).
  • the oxygen carrier containing the AAV solution was injected distal to occlusion site through the balloon for 7 minutes at an infusion rate of of 1 ml/kg/min.
  • the balloon was deflated and blood flow was restored. Total occlusion time was 12 minutes.
  • Myocardial tissue stored at -80°C was crushed using a pestle and mortar on dry ice. Powdered tissue was added to 1 ml of Trizol® reagent (Invitrogen). Tissue was homogenized (Qiagen tissue homogenizer) and incubated at room temperature for 5 minutes. 200 ⁇ of chloroform was added to each sample and samples were inverted vigorously and incubated for 10 minutes at room temperature. Samples were then spun at 12,000g for 15 minutes at 4°C and the aqueous phase was placed in a clean tube. A total of 1 ml of 75% ethanol was added to the samples which were then shaken and centrifuged at 7500 g for 5 minutes at 4°C.
  • cDNA was synthesized using 100 ng of isolated total RNA per sample and the High Capability cDNA Reverse Transcription kit (Applied Biosystems). Samples were run at 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 minutes and held at 4°C until removed from thethermocycler.
  • Quantitative Real Time PCR was carried out on Applied Biosystems 7900.
  • Fast real time PCR kit (Applied Biosystems) was used to prepare the samples with eGFP primers: EGFP Fw (SEQ ID N01 : CCAGGAGCGCACCATCTTCTT) and EGFP Rv (SEQ ID N02: GTAGTGGTTGTGGGCAGCAG).
  • EGFP Fw SEQ ID N01 : CCAGGAGCGCACCATCTTCTT
  • EGFP Rv SEQ ID N02: GTAGTGGTTGTGGGCAGCAG
  • LinRegPCR Relative Units of expression were normalized with a porcine GAPDH probe (Ss03374854_g1 , Applied Biosystems).
  • One animal received the AAV solution via regular intracoronary administration (IC, control). More specifically, a total of 10 13 particles of an AAV9 vector carrying the GFP reporter gene under the control of the troponin promoter were administered in normal saline via intracoronary injection without obstructing native coronary artery flow, by infusing the AAV solution through the catheter placed at the origin of the coronary artery. The AAV solution is thus mixed with flowing blood.
  • IC intracoronary administration
  • the modified pre-oxygenated solution has shown to be safe in healthy individuals and even after a prolonged ischemia.

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Abstract

The present invention relates to the field of medicine and biotechnology. More specifically it relates to oxygen carrier compositions for the delivery of oxygen to tissues and organs (e.g., to the myocardium) under bloodless situations. It further relates to medical uses and methods of treatment comprising the administration thereof, for instance, as delivery vehicle for gene therapy purposes. In a particular embodiment, it relates to an oxygen carrier composition for use in a method for the in vivo diagnosis and/or treatment of a myocardial disease; wherein said method comprises the following consecutive steps: i. temporary occluding a coronary artery, and optionally, temporary occluding a coronary vein; ii. administering into the coronary artery downstream to the site of occlusion the oxygen carrier composition, wherein said oxygen carrier composition has been pre-oxygenated; and iii. administering into the coronary artery downstream to the site of occlusion a diagnostic, prophylactic and/or therapeutic agent and the oxygen carrier composition; wherein the oxygen carrier composition in ii) is substantially free of the diagnostic, prophylactic and/or therapeutic agent administered in iii); and wherein in step iii) the agent and the oxygen carrier composition are administered simultaneously.

Description

OXYGEN CARRYING BLOOD SUBSTITUTES AND THEIR USE AS DELIVERY
VEHICLES
FIELD OF INVENTION
The present invention relates to the field of medicine and biotechnology. More specifically it relates to oxygen carrier compositions for the delivery of oxygen to tissues and organs (e.g., to the myocardium) under bloodless situations. It further relates to medical uses and methods of treatment comprising the administration thereof, for instance, as delivery vehicle for gene therapy purposes.
BACKGROUND OF THE INVENTION
Heart failure (HF) is a major public health issue with a current prevalence of over 5.8 million in the USA and over 23 million worldwide. Every year in the USA, more than 550,000 individuals are diagnosed with HF for the first time, and there is a lifetime risk of one in five of developing this syndrome. A diagnosis of HF carries substantial risk of morbidity and mortality, which despite advances in management remain unacceptably high. Over 2.4 million patients who are hospitalized have HF as a primary or secondary diagnosis, and nearly 300,000 deaths annually are directly attributable to HF (Bui et al., Nat. Rev. Cardiol. 201 1 , 8(1 ), 30-41 ).
Gene therapy and cell therapy approaches have attracted particular attention in the recent years, as well as methods for delivering thereof which may significantly impact on the outcome of such therapies (Dib et al., JACC: cardiovascular intervention 2010, 3(3), 265- 275; Katz et al., J Mol Cell Cardiol 201 1 , 50(5)766-776). In particular, with respect to gene therapy, cardiac delivery methods have been described aiming to maximize the amount of vector which is efficiently transfected into the cardiomyocytes. Myocardial gene delivery techniques may be broadly divided as: i) direct gene delivery; ii) transvascular gene delivery; and iii) ex vivo gene delivery. The third group is perceived as clinically relevant particularly in the setting of heart transplantation.
Direct gene delivery methods include intrapericardial injection, endocardial injection or intramyocardial injection. Direct injection of the gene therapy construct has the obvious advantage that enables the application of a high concentration of vector directly at the target site. It requires however a fairly aggressive intervention which involves the administration of relatively high volumes of the therapeutic solution into the myocardium and thus this administration route is associated to a higher risk for the patient. An additional drawback is that presence of the transgene is localized around the site of injection and thus results in its heterogeneous distribution along the myocardium.
Transvascular gene delivery methods (including injection in the peripheral veins, coronary veins, coronary sinus and coronary arteries) have also been described for cardiac gene therapy. These methods will enable a more diffuse and homogeneous delivery but present other limitations which typically include but are not limited to dilution of the vector in the circulating blood; dissemination into collateral organs; and short contact between vector and cardiomyocytes (Katz et al., J Mol Cell Cardiol 201 1 , 50(5):766-776). Accordingly, issues related to delivery, including vector efficiency, dose, specificity/tropism and safety are areas of concern (Byrne et al., Gene Therapy 2008, 15, 1550-1557). In the recent years there has been a significant effort in the research community aiming to overcome said problems by developing vectors (e.g., adeno-associated vectors) with cardiac tropism in order to deliver genes specifically and efficiently to the cardiomyocytes (Prasad et al., 201 1 , Gene Therapy, 18(l):43-52).
As an alternative approach to solve these issues, methods involving intracoronary delivery of a gene therapy vector, which may comprise a temporary interruption of blood flow, have been described. By using this approach the safety concerns associated to the specificity of the specificity/tropism of the vector as well as those associated to the dilution into the blood flow may be substantially reduced.
Non-selective (indirect) intracoronary delivery methods are characterized by the cross- clamping of both the pulmonary artery and the aorta, and rely on the creation of a transcoronary myocardial perfusion gradient for vector delivery. This allows perfusion of the virus at relatively low downstream pressure, and the endocardium can be efficiently transfected. Clinical feasibility of this approach may be limited considering the risk of systemic ischemia and acute left ventricle (LV) overload during the aortic cross-clamping and the time of occlusion must be limited (Parsa et al., Semin Thorac Cardiovasc surg. 2003, 15:259-97; Katz et al., J Mol Cell Cardiol 201 1 , 50(5):766-776).
The feasibility and efficacy of percutaneous retrograde (venous-to-arterial) gene delivery by selective pressure regulated retroinfusion of the coronary veins has been reported by various authors (Boekstegers P, and Kupatt C, Basic Res Cardiol. 2004; 99:373-81 ; and White et al., Gene Therapy 201 1 , 18, 546-552). By using their constructed apparatus, consisting of a pump unit, extracorporeal circuit, retroinfusion catheter and suction device, Boekstegers et al. described the advantages of retrograde delivery compared to ante-grade and sustained that blocking the venous outflow can significantly increase viral transfection of the myocardium (Boekstegers P, and Kupatt C, Basic Res Cardiol. 2004; 99:373-81 ; Boekstegers P, et al., Gene Ther. 2000,7:232-40; Katz et al., J Mol Cell Cardiol 201 1 , 50(5):766-776). Moreover, selective coronary retroinfusion was reported by the authors to prolong adhesion time of the vector and increase endothelial permeability, although it did not enable to reduce transduction of extracardiac organs like liver and lung (Raake PW et al., Gene Ther. 2008, 15:12-7; Katz et al., J Mol Cell Cardiol 201 1 , 50(5):766-776).
Percutaneous retrograde delivery methods involve in vivo isolation of the heart from the systemic circulation and the cardiac recirculation of the gene therapy vector. These typically require a significant degree of instrumentation and consequently are complicated and expensive. Moreover, this high instrumentation may be associated to an increased risk of the gene therapy intervention in patients having a severe heart dysfunction and thus already being at high risk.
Catheter-based, percutaneous infusion of the transgene containing vector into the coronary arteries is the most common cardiac gene delivery method. Its perceived advantages are based on its minimal invasiveness, the possibility of transgene delivery to all four myocardial chambers and the delivery of vector genomes using endovascular coronary catheterization— a well-established procedure in the clinical practice. Simple antegrade (arterial-to-venous) intracoronary delivery has been reported to result however in very limited myocardial transduction efficiency (Magovern CJ et al., Ann Thorac Surg. 1996, 62:425-33; Logeart D et al., Hum Gene Ther. 2001 , 12:1601-10; Kaplitt MG et al., Ann Thorac Surg. 1996, 62:1669-76; Logeart D et al., Hum Gene Ther. 2000, 1 1 :1015-22).
Reduced transgene transduction efficiency in single pass intracoronary delivery may be associated to the rapid washout of the vector. To prolong the exposure of the vector in the target site and increase gene transfer efficiency, some strategies have been devised encompassing the interruption of the coronary blood flow.
Logeart D et al. (Hum Gene Ther. 2000, 1 1 :1015— 22) describes the antegrade delivery of an adenoviral vector via the coronary arteries into isolated perfused rat hearts. This document reports that 1 -min no-flow period after adenovirus delivery enabled to increase gene transfer efficiency into the cardiac myocytes.
Hayase M et al. (Am J Physiol Heart Circ Physiol. 2005, 288:H2995-3000) discloses a catheter-based percutaneous antegrade intracoronary artery gene delivery method, which was combined with coronary artery occlusion with or without coronary vein occlusion. This method encompasses a 3-minute interruption of the coronary flow prior to the viral vector delivery. The interruption of blood flow for longer periods would be desirable as it would enable prolonging the time of contact of the vector with the myocardial cells, but as a drawback may be associated to an increased the risk of ischemia and ischemia/reperfusion (l/R) injury. Following coronary occlusion and oxygen deprivation, myocardial necrosis typically begins within approximately 15 minutes and, without any intervention, results in irreversible damage over the next 30 to 90 minutes. Despite being a pre-requisite for myocardial salvage, it is generally accepted that coronary reperfusion itself induces additional damage to the myocardium, known as ischemia/reperfusion (l/R) injury.
An additional problem associated to efficacy of viral vectors, in particular AAVs, is that a significant proportion of humans and nonhuman primates have antibodies in their blood that react to some of the existing serotypes of AAV (Blacklow, N. Ret al., J. Natl. Cancer Inst. 1968, 40:319-327; Chirmule, N et al., Gene Ther. 1999, 6:1574-1583).
In view of the above, methods for cardiac gene delivery that are safe and result in homogeneous and long-term expression of the gene in the cardiomyocytes in a therapeutically relevant amount, free of immune responses and which do not require complicated surgical procedures need still to be developed.
Oxygen-carrying blood substitutes (herein after also referred as "oxygen carriers") were initially developed as an alternative to red blood cells for surgical patients. Oxygen-carrier solutions are characterized by being aqueous compositions which transport oxygen and can be dissolved into a balanced salt buffer. Many oxygen-carrying blood substitutes are based on hemoglobin and are thus named Hemoglobin-based oxygen carriers (HBOCs). Functionally, they allow delivery of more oxygen to hypoxic tissues due to their higher 02 affinity, lower viscosity and smaller mean diameter than human erythrocytes.
HBOCs are promising candidates to prevent many organs from l/R injury (Caswell, et. al., Am J Physiol Heart Circ Physiol 2005, 288:H1796-H1801 ; George, et. al., Am J Physiol Heart Circ Physiol 2006, 291 :H1 126-H1 137; Burmeister, et. al., British Journal of Anaesthesia 2005, 95(6):737-45; Rempf, et. al. British Journal of Anaesthesia 2009, 103(4): 496-504; Lintel, et. al., Am J Physiol Heart Circ Physiol 2010, 298:H1 103-H1 1 13; WO2013/016598). However, for each of these HBOC compositions safety and tolerability needs to be stablished (Chen J-Y, et al. Clinics 2009; 64(8):803-13; Alayash A. I., Trends Biotechnol. 2014, 32(4): 177-185; Yang, et. al., Oxidative Medicine and Cellular Longevity 2015, ID125106). HBOC-201 (Hemopure® or hemoglobin glutamer-250) is a hemoglobin based oxygen carrier comprising crosslinked and glutaraldehyde-polymerized hemoglobin (Hb) extracted from isolated bovine red blood cells. HBOC-201 has been shown to be well-tolerated as an intracoronary perfusate in humans (Meliga, et. al., Eurolntervention 2008, 4:99-107). Moreover, it was under clinical development for the treatment of haemorrhagic shock resulting from traumatic injury, for the treatment of acute coronary syndrome, peripheral arterial disease (PAD)/ peripheral vascular disease (PVD), occlusive coronary artery disease, wounds, and trauma.
Meliga, et. al., Eurolntervention 2008, 4:99-107; and US 2010/0209532 A1 (Example 4, pages 12-15) provide the results of a Phase II trial which was designed to test the hypothesis that pre-oxygenated HBOC-201 is capable of supporting myocardial metabolism and preserving cardiac function during brief total coronary occlusion in humans. All patients underwent two intra-stent balloon occlusions of 3 minutes of duration with a recovery period of 20 minutes in between. This assay had two arms and subjects were assigned to receive a continuous intracoronary infusion of pre-oxygenated HBOC-201 during the first occlusion period and no infusion (referred as "dry occlusion") during the second occlusion period or vice-versa. Accordingly, intracoronary administration of HBOC-201 was performed after a brief coronary artery occlusion and not after prolonged ischemia. US 6 699 231 B1 discloses the treatment of a tissue by occluding an artery and/or a vein and perfusing the tissue with a therapeutic agent and an oxygen carrier in a method further comprising collecting the perfusate and extracorporeal^ pumping the perfusate back and extracorporeally oxygenating the oxygen carrier. US 6 177 403 B1 discloses a composition comprising a gene vector, an oxygen carrier and a vascular permeability- enhancing agent. It also discloses a method of delivering a gene vector to an extravascular tissue, when the vasodilating agent and the gene vector are administered prior to providing an oxygen- transporting agent. These documents do not disclose however a treatment and/or diagnostic method comprising the co-administration in the myocardium of an oxygen carrier composition and a transgene expressing adeno-associated vector. Moreover, these documents are also silent on the particular administration sequence wherein the oxygen carrier composition is further administered immediately before the co-administration step. SUMMARY OF THE INVENTION
The inventors have developed the new hypothesis that oxygen carrying blood substitutes' may not only prevent and/or treat cardiac ischemia but also avoid the contact of gene vectors with blood elements which may adversely affect cardiomyocyte transduction. Preventing contact of the viral particles with blood components may be particularly relevant when adeno-associated virus (AAV) are used as gene therapy vectors, as this would enable to solve in particular the problem associated with the presence of neutralizing antibodies against AAVs circulating in the blood of the recipient.
On this basis, the inventors have newly devised a cardiac gene delivery method which comprises the occlusion of coronary veins and/or arteries and administration of a vector containing a nucleic acid of interest in an oxygen carrier blood substitute composition. To maximize the protective effect, an appropriate volume of the oxygen carrier blood substitute may be administered prior to the administration of the vector containing composition.
A first pilot assay was conducted in healthy male pigs, see Example 2 and Figure 4, which shows an increased transfer of the green fluorescent protein (GFP)-encoding AAV vector into the myocytes of the left ventricle apex when HBOC-201 is used as vehicle for the anterograde intracoronary administration of the gene therapy vector with respect to the administration of vector particles suspended in saline.
The positive results obtained with intracoronary administration of an oxygen carrier composition further to the coronary blockade, prior and concomitant to the administration of gene therapy vectors, has shown bloodless reperfusion of an oxygen carrier composition to be a promising delivery strategy which could be applied to other prophylactic, therapeutic and/or diagnostic agents. For instance, in addition to gene therapy purposes, this strategy may be used as a myocardial delivery system for cardioprotective agents or for other agents where the absence of blood during transfer might result in increased delivery efficiency (e.g. cell therapy) in different clinical scenarios. This strategy may be applied to acute-ischemia and non-acute ischemia scenarios.
The first aspect of the invention relates to an oxygen carrier composition for use in a method for the in vivo diagnosis and/or the therapeutic and/or preventive treatment of a myocardial disease; wherein the oxygen carrier composition is administered prior to and/or simultaneously to the administration of a preventive, therapeutic and/or diagnostic agent; wherein the administration of the oxygen carrier composition and said agent is intracoronary; and wherein the method comprises the temporary occlusion of a coronary artery and/or a coronary vein prior to the administration of said oxygen carrier composition.
In a particular embodiment, it relates to an oxygen carrier composition for use in a method for the in vivo diagnosis and/or treatment of a myocardial disease;
wherein said method comprises the following consecutive steps:
i. temporary occluding a coronary artery, and optionally, temporary occluding a coronary vein;
ii. administering into the coronary artery downstream to the site of occlusion the oxygen carrier composition, wherein said oxygen carrier composition has been pre-oxygenated; and
iii. administering into the coronary artery downstream to the site of occlusion a diagnostic, prophylactic and/or therapeutic agent and the oxygen carrier composition;
wherein the oxygen carrier composition in ii) is substantially free of the diagnostic, prophylactic and/or therapeutic agent administered in iii); and
wherein in step iii) the agent and the oxygen carrier composition are administered simultaneously. In another particular embodiment, it relates to an oxygen carrier composition for use in a method for the in vivo diagnosis and/or the therapeutic and/or preventive treatment of a myocardial disease; wherein said method comprises the following consecutive steps:
i. temporary occluding a coronary artery and/or a coronary vein;
ii. intracoronary administering the oxygen carrier composition prior to and/or simultaneously to the administration of a preventive, therapeutic and/or diagnostic agent;
wherein said oxygen carrier composition has been pre-oxygenated; and wherein said agent is a transgene encoding adeno-associated virus (AAV) expression vector.
Moreover, the inventors have developed an oxygen carrier formulation which is particularly suitable for administration under bloodless conditions, in particular into an ischemic myocardium under bloodless conditions. In Example 1 intracoronary administration of the standard HBOC-201 formulation to an ischemic heart (i.e., after 45 minutes from the interruption of blood flow and prior to blood restoration) significantly increased microvascular obstruction and infarct size. With the aim to avoid the observed deleterious effects the inventors modified the pH of the solution to 7.35 (referred herein as "HBOC-pH") and further incorporated glucose, as metabolic substrate, and insulin, to enhance glucose in-take by the cardiomyocytes (referred herein as "HBOC-pH-Glc-lns").
Therefore, it was shown that the modified solution is especially safe under ischemic conditions, and thus particularly suitable for myocardial administration after prolonged occlusion periods. Accordingly, bloodless reperfusion of modified oxygen carrier composition has shown to be a promising and safe strategy which may be applied when acute exposure of the ischemic myocardium to therapeutic agents (such as cardioprotective agents) is desired before contact with reperfusing blood.
In a second aspect, the present invention relates to a composition comprising or consisting of:
- an oxygen carrier composition;
- a metabolic substrate; and/or
- a pH modulator agent.
Said composition is herein also referred as "the modified oxygen carrier composition of the invention". In a particular embodiment, said composition comprises or consists of:
- HBOC-201 ;
- glucose;
- insulin; and
- N-acetylcysteine;
wherein the pH of said composition is from 6.8 to 7.4, preferably wherein the pH of said composition is 7.35.
In a third aspect, the present invention relates to the use of the modified oxygen carrier composition of the invention as a pharmaceutical composition. It also relates to the composition or pharmaceutical composition of the invention for use as a medicament.
In a fourth aspect, the invention relates to the composition according to the preceding aspects of the invention, for use in a method of in vivo diagnosis and/or treatment of a tissue or organ which has or is at risk of having ischemia. In a further aspect, the invention relates to a method for obtaining a composition according to the preceding aspects of the invention. It may comprise mixing the components of the composition as described herein. It also may comprise adding a metabolic substrate to the oxygen carrier composition and adjusting the pH of the composition as described herein. In a particular embodiment, it comprises:
- adding a metabolic substrate to the oxygen carrier composition; and
- adjusting the pH with a pH modulator agent to a pH from 6.8 to 7.4, preferably to a pH of 7.35. An additional aspect of the invention relates to an oxygen carrier composition as described in the preceding aspects of the invention, for use as delivery vehicle in the administration of a therapeutic and/or diagnostic agent.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 . Study protocols. FigIA: Protocol \ - Intracoronary infusion of HBOC-201 in healthy pigs; FigI B: Protocol 2.- Effect of intracoronary infusion of pre-oxygenated oxygen carrier infusion at the end of prolonged ischemia (before blood flow restoration). Figure 2. Effect of HBOC-201 intracoronary infusion rate + duration combinations on left ventricular ejection fraction (Protocol 1 ) Tukey boxplots show median values, interquartile range and extreme values. LVEF, left ventricular ejection fraction. *p-value <0.05 compared with baseline; #p-value <0.05 compared with the 12 min infusion group. Figure 3. Effect of different flow rates of post-myocardial-infarction modified oxygen carrier injection on infarct size (IS) as evaluated by cardiac magnetic resonance (CMR) 7 days after infarction (primary endpoint) measured as % LV (Fig.3a) and % edema (Fig.3b) and hemodynamic parameters (LVEF%; 3c and 3d). Tukey boxplots show median values, interquartile range (IQR) and extreme values.
Figure 4. CMR-derived parameters from HBOC-pH and HBOC-pH-Glc-lns infusion after myocardial infarction (Protocol 2). Tukey boxplots show median values, interquartile range and extreme values. LVEF, left ventricular ejection fraction. *p-value <0.05 compared to baseline. Figure 5: Green Fluorescent Protein (GFP) mRNA expression normalized to that of GAPDH analyzed by qRT-PCR from total RNA extracted from left ventricular myocardial samples isolated from the apex . **p<0.01 HBOC vs intracoronary injection, 2-way ANOVA followed by Bonferroni's post-test. n=2.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used herein, a solution or "aqueous composition" may mean a fluid (liquid) preparation that contains water, optionally in combination with other mutually miscible solvents (e.g. water-soluble organic solvents), and one or more chemical substances dissolved therein.
As used herein, the term "composition" or "compositions" may refer to a formulation(s) that it is suitable for injection and/or administration into an individual in need thereof. A "composition" may also be referred to as a "pharmaceutical composition." In certain embodiments, the compositions provided herein are substantially sterile and do not contain any agents that are unduly toxic or infectious to the recipient.
The term "balanced", "physiological" or "isotonic" salt solution or buffer refers to an osmotically balanced salt solution to prevent acute cell or tissue damage. This term as used herein may mean that the osmolality is close to the physiological osmolality in the human body, thus leading to more suitable compositions to be used in parenteral administration. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm/L, preferably about 290 mOsm/L. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example.
The term "diagnosis" as used herein refers to determining the presence or absence of a disease when a subject shows signs or symptoms of the disease. It also encompasses early detection or screening of a disease in asymptomatic individuals who may have the disease. The term "disease" as used herein is intended to be generally synonymous, and is used interchangeably with, the terms "disorder" and "condition" (as in medical condition), in that all reflect an abnormal condition of the body or of one of its parts that impairs normal functioning and is typically manifested by distinguishing signs and symptoms. The term "treatment" as used herein refers to the prophylactic and/or therapeutic treatment. The term "therapeutic treatment" as used herein refers to bringing a body from a pathological state or disease back to its normal, healthy state. Specifically, unless otherwise indicated, includes the amelioration, cure, and/or maintenance of a cure (i.e., the prevention or delay of relapse) of a disease or disorder. Treatment after a disorder has started aims to reduce, alleviate, ameliorate or altogether eliminate the disorder, and/or its associated symptoms, to prevent it from becoming worse, to slow the rate of progression, or to prevent the disorder from re-occurring once it has been initially eliminated (i.e., to prevent a relapse). It is noted that, this term as used herein is not understood to include the term "prophylactic treatment" as defined herein.
The term "prophylactic treatment" or "preventive treatment" as used herein refers to preventing a pathological state. It is noted that, this term as used herein is not understood to include the term "therapeutic treatment" as defined herein. The term "effective amount" as used herein refers to an amount that is effective, upon single or multiple dose administration to a subject (such as a human patient) in the prophylactic and/or therapeutic treatment of a disease, disorder or pathological condition.
The term "subject" as used herein refers to a mammalian subject. Preferably, it is selected from a human, companion animal, non-domestic livestock or zoo animal. For example, the subject may be selected from a human, mouse, rat, dog, cat, cow, pig, sheep, horse, bear, and so on. In a preferred embodiment, said mammalian subject is a human subject.
The term "pharmaceutically acceptable carrier and/or excipient" is intended to include formulation used to stabilize, solubilize and otherwise be mixed with active ingredients to be administered to living animals, including humans. This includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.
The terms "nucleic acid sequence" and "nucleotide sequence" may be used interchangeably to refer to any molecule composed of or comprising monomeric nucleotides. A nucleic acid may be an oligonucleotide or a polynucleotide. A nucleotide sequence may be a DNA or RNA (e.g., microRNAs). A nucleotide sequence may be chemically modified or artificial. Nucleotide sequences include peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acid (TNA). Each of these sequences is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule. Also, phosphorothioate nucleotides may be used. Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoramidates and oligoribonucleotide phosphorothioates and their 2'-0-allyl analogs and 2'-0-methylribonucleotide methylphosphonates which may be used.
The term "nucleic acid construct" as used herein refers to a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. A nucleic acid construct is a nucleic acid molecule, either single- or double-stranded, which has been modified to contain segments of nucleic acids sequences, which are combined and juxtaposed in a manner, which would not otherwise exist in nature. A nucleic acid construct usually is a "vector", i.e. a nucleic acid molecule which is used to deliver exogenously created DNA into a host cell. A "coding sequence" or a sequence which "encodes" a gene product as used herein, refers to a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA), in vitro or in vivo when placed under the control of appropriate regulatory sequences. The term "expression vector" or "vector" as used herein refers to a recombinant nucleotide sequence that is capable of effecting expression of a gene (transgene) in host cells or host organisms compatible with such sequences. In particular, this term encompasses a plasmid, phage, transposon, cosmid, chromosome, virus, etc. Together with the transgene, expression vectors typically include at least suitable transcription regulatory sequences and optionally, 3' transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements able to respond to a precise inductive signal (endogenous or chimeric transcription factors) or specific for certain cells, organs or tissues. The terms DNA "regulatory sequences", "control sequences" and "control elements" as used herein, refer collectively to promoter sequences (e.g., an eukaryotic promoter), polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences/elements need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. As used herein, the term "eukaryotic promoter" refers to a DNA sequence region that initiates transcription of a particular gene, or one or more coding sequences, in eukaryotic cells. A promoter can work in concert with other regulatory regions or elements to direct the level of transcription of the gene or coding sequence/s. These regulatory elements include, without limitation, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter, including e.g. attenuators, enhancers, and silencers. The promoter is located near the transcription start site of the gene or coding sequence to which is operably linked, on the same strand and upstream of the DNA sequence (towards the 5' region of the sense strand). A promoter can be about 100-1000 base pairs long. Positions in a promoter are designated relative to the transcriptional start site for a particular gene (i.e., positions upstream are negative numbers counting back from -1 , for example -100 is a position 100 base pairs upstream).
As used herein, the term "operably linked" refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous; where it is necessary to join two protein encoding regions, they are contiguous and in reading frame.
As used herein, the term "polyadenylation signal" or "poly(A) signal" refers to a specific recognition sequence within 3' untranslated region (3' UTR) of the gene, which is transcribed into precursor mRNA molecule and guides the termination of the gene transcription. Poly(A) signal acts as a signal for the endonucleolytic cleavage of the newly formed precursor mRNA at its 3'-end, and for the addition to this 3'-end of a RNA stretch consisting only of adenine bases (polyadenylation process; poly(A) tail). Poly(A) tail is important for the nuclear export, translation, and stability of mRNA. In the context of the invention, the polyadenylation signal is a recognition sequence that can direct polyadenylation of mammalian genes and/or viral genes, in mammalian cells. Poly(A) signals typically consist of a) a consensus sequence AAUAAA, which has been shown to be required for both 3'-end cleavage and polyadenylation of pre-messenger RNA (pre-mRNA) as well as to promote downstream transcriptional termination, and b) additional elements upstream and downstream of AAUAAA that control the efficiency of utilization of AAUAAA as a poly(A) signal. There is considerable variability in these motifs in mammalian genes. The term "packaging cells" as used herein, refers to a cell or cell line which may be transfected with a helper vector or virus or a nucleic acid construct, and provides in trans all the missing functions which are required for the complete replication and packaging of a viral vector. Typically, the packaging cells express in a constitutive or inducible manner one or more of said missing viral functions.
The term "substantially free" as used herein may refer to a composition containing less than 0.033%, less than 0.001 %, less than 0.0005%, less than 0.0003%, or less than 0.0001 % of the referred component of the composition. For example, the composition does not comprise said component.
Detailed description
In a first aspect, the invention relates to an oxygen carrier composition for use in a method for the in vivo diagnosis and/or the therapeutic and/or preventive treatment of a myocardial disease; wherein the oxygen carrier composition is administered prior to and/or simultaneously to the administration of a preventive, therapeutic and/or diagnostic agent; wherein the administration of the oxygen carrier composition and said agent is intracoronary; and wherein the method comprises the temporary occlusion of a coronary artery and/or a coronary vein prior to the administration of said oxygen carrier composition.
In a related aspect, the invention refers to a method for the cardiac delivery of a preventive, therapeutic and/or diagnostic agent wherein said method comprises the administration of an oxygen carrier composition prior to and/or simultaneously to the administration of said preventive, therapeutic and/or diagnostic agent; wherein the administration of the oxygen carrier composition and said agent is intracoronary; and wherein the method comprises the temporary occlusion of a coronary artery and/or a coronary vein prior to the administration of said oxygen carrier composition.
The term "coronary artery" also includes "coronary arteries" and may refer to one or more of the left anterior descending artery, the left circumflex artery and the right coronary artery. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, said coronary artery is the left anterior descending artery. Similarly, the term "coronary vein" also includes "coronary veins" and may refer to one or more of the coronary sinus, the anterior cardiac vein, the right atrial veins and the right ventricular veins.
The term "oxygen carrier composition" or "oxygen-carrying blood substitutes" are used herein interchangeably and refer to compositions, typically aqueous compositions, which can transport oxygen in excess of what can be dissolved into a balanced salt solution and which are isosmotic with blood. An ideal blood substitute should have an oxygen carrying capacity as good as that of the natural hemoglobin molecule within the red blood cells (RBCs), be less antigenic, have a long shelf life (preferably at room temperature), have a long intravascular half-life, and be free of toxicity and side effects.
Many oxygen-carrying blood substitutes are based on hemoglobin and are named Hemoglobin-based oxygen carriers (HBOCs). In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, said oxygen carrier composition is a hemoglobin-based oxygen carrier.
HBOCs (also referred herein as "hemoglobin solutions") are physiological solutions that contain hemoglobin from purified human, animal, or recombinant sources. For therapeutic use, purified hemoglobins typically have undergone multiple chemical modifications, including crosslinking and/or polymerization to change their physiochemical characteristics. Hemoglobin can be cross-linked and polymerized for instance with glutaraldehyde and/or o- raffinose to increase its ability to deliver oxygen and increase its duration of action in circulation (Levy J H, May 2009, American Council on Science and Health). Other modifications to improve natural hemoglobin's 02 transfer efficiency and/or decrease its toxicity, such as polynitroxylation or pegylation, are well known by a person skilled in the art and are also encompassed herein. Toxicity issues identified with clinically assayed hemoglobin solutions as well as possible strategies for overcoming these are discussed for instance in Alayash A. I., (Trends Biotechnol. 2014, 32(4): 177-185) and Chen J-Y, et al. (Clinics 2009; 64(8):803-13) which are incorporated herein by reference.
These hemoglobin solutions can also include one or more pharmaceutically acceptable carriers and/or excipients. Examples of such carriers include water (including water for injection), isotonic solutions, such as saline solution (e.g., 0.9% NaCI), buffered saline, glucosalin solution (e.g., 5% glucose and 0.9% NaCI), lactated Ringer's solution (e.g., 102 mmol/L sodium chloride; 28 mmol/L sodium lactate; 4 mmol/L of potassium chloride, and 1 .5 mmol/L of calcium chloride), plasma-lyte solution (lactated Ringer-like mixture with presence of magnesium, acetate and gluconate ions) and the like. Non-limiting examples of excipients include an agent for regulating the osmotic pressure and/or the pH (e.g., an acid/salt buffer system, a salt, a monosaccharide, a disaccharide or a polyol), a surfactant, a preservative agent, as well as combinations thereof. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, said HBOC composition comprises one or more, preferably all, the excipients selected from the group consisting of sodium chloride, potassium chloride, dihydrated calcium chloride, sodium hydroxide, sodium lactate and N- acetyl-L-cysteine. Preferably, said composition comprises the following excipients and the carrier is water for injection:
NaCI 100-130 mmol/L (preferably, 1 14 mmol/L)
KCI 2.0-6.0 mmol/L (preferably, 4.0 mmol/L)
CaCI2-2H20 0.5-2.5 mmol/L (preferably, 1.4 mmol/L)
NaOH 10.0-15.0 mmol/L (preferably, 12.5 mmol/L)
Sodium lactate 20.0-35.0 mmol/L (preferably, 27.1 mmol/L)
N-acetyl-L-cysteine 10.0-15.0 mmol/L (preferably, 12.3 mmol/L)
Generally suitable oxygenated hemoglobin solutions employed by the method of the invention are prepared in vitro by oxygenating hemoglobin solutions that include hemoglobin to convert at least about 80%, more preferably at least about 90%, by weight of the hemoglobin to oxyhemoglobin. In some embodiments, about 18% by weight, or less, of the hemoglobin that is included in the hemoglobin solutions to be oxygenated has a molecular weight of over 500,000 Daltons; about 5% by weight, or less, of the hemoglobin that is included in the hemoglobin solutions to be oxygenated has a molecular weight equal to or less than 65,000 Daltons; and/or an the endotoxin content of the hemoglobin solution that is included in the hemoglobin solutions to be oxygenated is less than about 0.5 endotoxin units per milliliter, preferably less than about 0.05 endotoxin units per milliliter. Also, a P50 of the polymerized hemoglobin is preferably in a range of between about 24 and about 46 mm Hg, preferably between about 34 and about 46 mm Hg. Said oxygenated hemoglobin solution may include from about 10 grams to about 250 grams of chemically modified (e.g., polymerized) hemoglobin per liter of solution. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, said HBOC composition is characterized by one or more, preferably all of the following features:
1 . During the hemoglobin purification process erythrocyte membrane and all cellular content except Hb has been eliminated, thereby eliminating toxicity otherwise associated with RBC fragments and making the product universally acceptable to all patients;
2. During the hemoglobin purification process all contaminants are eliminated, including in particular, endotoxin, viruses and prion proteins;
3. The hemoglobin tetramer has been cross-linked with glutaraldehyde to stabilize the molecule and minimize existence of dimer subunits;
4. Hemoglobin tetramers have been polymerized to increase average MW to 250 kD to increase retention in vascular compartment, thereby also decreasing vasoconstriction activity and a 3-yr shelf life at 2-30 degrees C. The largest polymers are still much smaller than an RBC (1/1000 diameter of an erythrocyte), facilitating access to tissues not available to RBCs, particularly in pathological conditions (i.e., micro-occluded vasculature as in no-reflow phenomenon during ischemia-reperfusion injury);
5. Residual tetramer is fractionated out to < 3% of total Hb to further to increase retention in vascular compartment, thereby also decreasing vasoconstriction activity;
6. The HBOC composition comprises N-acetyl-L-cysteine to maintain Hb in reduced state during storage in original packaging;
7. The HBOC composition comprises a modified Ringers solution to achieve physiological osmolality and maintain normal ion gradients between plasma, cells and tissues;
8. The HBOC composition has a physiological oncotic pressure to prevent net fluid exchange with blood cells and tissues;
9. The HBOC composition has a viscosity lower than blood, facilitating fluid mechanics, particularly in microvasculature;
10. The HBOC composition has an elevated 02 p50 (40 mmHg), facilitating 02 unloading and diffusive 02 delivery to tissues; and
1 1 . The HBOC composition is ready for immediate use without thawing or reformulating.
Preferably the oxygenated carrier composition is a polymerized hemoglobin solution as described in US 2010/0209532. More preferably, said oxygenated carrier composition comprises a purified hemoglobin intra/intermolecular crosslinked with glutaradehyde; even more preferably, it comprises a purified hemoglobin intra/intermolecular crosslinked with glutaradehyde, and further comprises NaCI from 100 to 130 mmol/L (preferably, 1 14 mmol/L), KCI from 2.0 to 6.0 mmol/L (preferably, 4.0 mmol/L), CaCI2-2H20 from 0.5 to 2.5 mmol/L (preferably, 1 .4 mmol/L), NaOH from 10.0 to 15.0 mmol/L (preferably, 12.5 mmol/L), sodium lactate from 20.0 to 35.0 mmol/L (preferably, 27.1 mmol/L), N-acetyl-L-cysteine from 10.0 to 20.0 mmol/L (preferably, 12.3 mmol/L) and water (preferably, water for injection), such as the commercially available composition HBOC-201. In a particularly preferred embodiment, said oxygen carrier composition is HBOC-201 . HBOC-201 (also referred as Hemopure® or hemoglobin glutamer-250; Hb02 Therapeutics, LLC) is a polymerized, iso-oncotic, high-molecular weight, bovine haemoglobin-based oxygen carrier suitable for intravenous infusion. More specifically, HBOC-201 comprises purified bovine hemoglobin intra/intermolecular cross-linked with glutaraldehyde (shown below) formulated in a balanced salt solution, more specifically in a modified Ringer's lactate solution at pH of 7.6 to 7.9. This cross-linked hemoglobin has been described to have a half- life of about 24 hours. Furthermore, HBOC-201 is a sterile solution with no risk of disease transmission.
Figure imgf000019_0001
M, Wt, = 1.30-500 kDa;
The qualitative and quantitative chemical composition as well as physicochemical characteristics of HBOC-201 and the method of obtaining thereof are described by Dube G.P. et al. (Eurolntervention J. 2008, 4 161 -165), in particular see Table 1 shown below. HBOC-201 was also described in US 6133425 A.
Table 1 : Characteristics of HBOC-201
Average molecular weight 250 kDa
MW > 500 kD < 15%
MW < 65 kD < 2.5%
Relative size vs. a red blood cell 1/100,000,000 X the volume of a RBC
Storage properties Room temperature (2°C to 30°C) for 3 years"
Osmolality 290 to 310 milliosmole/kg
Oncotic pressure 25-27 mm Hg
Viscosity (37°C) 2.2 centipoise at 13 g/dL, 37° C
Reconstitution None required
Administration i.v., via peripheral or central vein
Figure imgf000020_0001
a kD = kiloDalton
b Data also supports stability at 40°C for 18 months
c Slightly less than human RBC Hb which binds 1.34 ml 02/g Hb
On a gram-for-gram basis, this cross-linked hemoglobin carries the same amount of oxygen as the hemoglobin in red blood cells. The hemoglobin molecules in HBOC-201 have the advantage that are smaller, have lower viscosity and more readily release oxygen to tissues than red blood cells. Consequently, they can carry oxygen at low blood pressure and through constricted or partially blocked blood vessels to areas of the body that red blood cells cannot reach due to their larger size.
In 2001 , HBOC-201 was approved in South Africa for treatment of adult surgical patients who are acutely anemic and for eliminating, reducing, or delaying the need for allogeneic RBC transfusion in these patients (Greenburg AG, Kim HW, Crit Care 2004, 8 Suppl 2: S61 - 4). Other non-liming examples of H BOCs which have been assayed in clinical trials (Alayash A. I ., Trends Biotechnol. 2014, 32(4): 177-185) and may be used as oxygen carrier compositions in the method of the invention are: Hemolink® (Hemosol, Inc., Missiassauga, Canada) which comprises a cross-linked o- raffinose polymerized human hemoglobin with a half-life of about 20 hours.
Figure imgf000021_0001
M.Wt, = 64kDa;
p5o = 34mraHg
PolyHeme® (Northfield Laboratories, Inc.) which comprises a human cross-linked hemoglobin polymer purified from outdated erythrocytes.. More specifically, it is intramolecular cross-linked with pyridoxal phosphate (PLP) (A) and intermolecular cross- linked with glutaraldehyde (B).
Figure imgf000021_0002
B
Figure imgf000021_0003
M. Wt. = 130-250 kDa;
Pso = 28-30 mml lg
Hemospan® (Sangart, Inc., San Diego, CA) which comprises a maleimide-polyethylene glycol-modified human hemoglobin.
Figure imgf000022_0001
H -Cy»^*" Maleimide ΡΕΘ
M, Wt. = 90 kDa
Diaspirin cross-linked hemoglobin (DCLHb, HemAssist®; Baxter Healthcare Corp), which comprise a intramolecular cross-linked with bis (3,5-dibromosalicyl)-fumarate).
Figure imgf000022_0002
M. Wt. = 64 kDa;
P50 = 30mmHg
It was studied in clinical trials for coronary artery surgery. However, side effects detected in other studies ended further investigation of this agent (Levy JH, Anesthesiology 2000, 92: 639-41 ).
VitalHeme® (Caged Nitric Oxide (cNO) Labeled Polynitroxylated Pegylated Hemoglobin (PNPH); SynZyme Technologies, LLC) is under development for the treatment of traumatic brain injury complicated by hemorrhagic shock, stroke and sickle cell disease. VitalHeme® comprises a multifunctional neuroprotective cNO labeled polynitroxylated pegylated hemoglobin nano-particle. Polynitroxylated pegylated hemoglobin as a multifunctional therapeutic which takes advantage of the ability of hemoglobin (Hb) to transport oxygen, the antioxidative stress activities from the redox coupling of nitroxide and heme iron and the hypercolloid properties of pegylation. VitalHeme® acts by reducing oxidative stress in hemorrhagic shock and sickle cell disease.
Sanguinate® (Pegylated Hemoglobin, PEG-Hb, Pegylated Carboxyhemoglobin Bovine) from Prolong Pharmaceuticals. Sanguinate® comprises a PEGylated hemoglobin which facilitates the transfer of red blood cells to the tissue and begins the re-oxygenation process. This process helps to release other molecules which protect the vasculature and surrounding tissue during the transfer. It reduces the oxygen debt and protects from the ischemic reperfusion injury. The drug candidate is under development for the treatment of sickle cell disease (SCD), delayed cerebral ischemia after acute aneurysmal subarachnoid hemorrhage, end-stage renal disease and ischemia reperfusion injury in myocardial infarction.
Perfluorocarbon (PFC) emulsions have also been studied as oxygen-carrying blood substitutes. These contain halogen-substituted hydrocarbons that augment oxygen solubility(Jahr JS et al., Am J Ther 2002, 9: 437-43).
In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, said oxygen carrier composition is a composition comprising PFCs, such as PFC emulsions.
The viscosity of the oxygen carrier composition is generally between 1 and 3 centipoises at 37°C, preferably between 1.5 and 2.5 centipoises at 37°C, more preferably has a viscosity about 2.2 centipoises at 37°C.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, said oxygen carrier composition is a modified oxygen carrier composition of the invention as described herein. Said preventive, therapeutic and/or diagnostic agent can be any agent suitable for the treatment and/or diagnosis of myocardial diseases. As used herein, the term "myocardial disease" is used to encompass numerous conditions affecting the heart, and the heart valves, and encompasses diseases and conditions including, but not limited to arteriosclerosis, atherosclerosis, myocardial infarction, acute coronary syndrome, angina, heart failure, atrial fibrillation, systolic dysfunction, diastolic dysfunction, myocarditis, atrial tachycardia, ventricular fibrillation, endocarditis, acute coronary syndrome, acute ischemic attack, and sudden cardiac death.
This agent may be any type of therapeutic, prophylactic and/or diagnostic agent, such as traditional chemical agents or biological agents, which includes but is not limited to peptides, proteins (e.g., antibodies, growth factors and cytokines), cell therapy and gene therapy, and combinations thereof. A person skilled in the art will know the most appropriate agent according to the disease to be treated or diagnosed. In a particular embodiment, said agent is a cardioprotective agent. Cardioprotective agents are typically used in the treatment of ischemia/reperfusion injury, and include but are not limited to Glucagon-like peptide-1 (GLP-1 ) and analogues thereof; Cyclosporin-A and Metoprolol (Hausenloy et al., Basic Res Cardiol 2016, 1 1 1 :70).
In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, said agent is a nucleic acid sequence. In a preferred embodiment, said nucleic acid sequence is a nucleic acid construct. Preferably, said agent comprises or consists of a transgene encoding expression vector and may be used for gene therapy purposes. A person skilled in the art will select the nucleic acid sequence to be transduced into the cardiomyocytes according to the myocardial disease to be treated. These typically include but are not limited to growth factors and cytokines. Non-limiting examples of nucleic acid sequences are SUM01 , IGF-1 , VEGF, calcineurin A beta 1 , Niemann-Pick type C gene, FGF, HGF, PDGF, HIF1 a, SERCA2a, SDF-1 , ADCY6, pARK-ct-carboxy terminal peptide from GRK2, S100A1 , PVALB, KCNH2-G628S, SCN4A, connexin 32, connexin 40, connexin 43, ADCY1 and Kir2.1. More specifically, examples of genes used for gene therapy purposes in coronary heart disease, heart failure and arrhythmia are as specified in tables 2, 3 and 4 of Wolfram and Donahue (Journal of the American Heart Association 2013, e0001 19), which is hereby incorporated by reference.
Viral and non-viral transfection methods may be used. Non-viral transfection methods include both chemical and physical systems. In chemical-based systems, synthetic or naturally occurring compounds such as calcium phosphate (CaP), DEAE-dextran, cationic lipids, and cationic polymers may facilitate the transfer of a nucleic acid construct through the cell membrane. The efficacy of chemical non-viral gene delivery methods and their safety for cells is dependent on various factors such as the type of method, ratio of nucleic acid construct to reagents, charge and size of complexes, time of exposure, type of target cell, and correct cell density. Physical methods such as microinjection, gene gun and electroporation are carrier-free gene delivery techniques that employ the use of a physical force to permeate the cell membrane and facilitate intracellular transfer of naked nucleic acid construct. Factors to be considered for optimal gene transfer using electroporation are the electrical field strength and pulse duration, ionic strength of the electroporation buffer, nucleic acid concentration, cell density and viability. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, said nucleic acid construct is a viral vector. Viruses have been reported to be highly efficient at nucleic acid delivery to specific cell types while avoiding immunosurveillance by an infected host. These properties make viruses attractive gene-delivery vehicles, or vectors, for gene therapy. Recombinant viruses used for gene therapy purposes, include but are not limited to retrovirus, adenovirus, adeno- associated virus (AAV), and herpesvirus.
Thus, in one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the nucleic acid construct further comprises a 5'ITR and a 3'ITR of a virus.
As used herein the term "inverted terminal repeat (ITR)" refers to a nucleotide sequence located at the 5'-end (5'ITR) and a nucleotide sequence located at the 3'-end (3'ITR) of a virus, that contain palindromic sequences and that can fold over to form T-shaped hairpin structures that function as primers during initiation of DNA replication. They are also needed for viral genome integration into host genome; for the rescue from the host genome; and for the encapsidation of viral nucleic acid into mature virions. The ITRs are required in c/'s for the vector genome replication and its packaging into the viral particles.
In one embodiment, the nucleic acid construct comprises a 5'ITR, a ψ packaging signal, and a 3'ITR of a virus, "ψ packaging signal" is a cis-acting nucleotide sequence of the virus genome, which in some viruses (e.g. adenoviruses, lentiviruses ...) is essential for the process of packaging the virus genome into the viral capsid during replication.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the nucleic acid construct comprises a 5'ITR and a 3'ITR of a virus selected from the group consisting of parvoviruses (in particular adeno- associated viruses), adenoviruses, alphaviruses, retroviruses (in particular gamma retroviruses, and lentiviruses), herpesviruses, and SV40. In a preferred embodiment the virus is an adeno-associated virus (AAV). In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the nucleic acid construct comprises a 5'ITR and a 3'ITR of an AAV.
The AAV genome is composed of a linear, single-stranded DNA molecule which contains 4681 bases (Berns and Bohenzky, (1987) Advances in Virus Research (Academic Press, Inc.) 32:243-307). The genome includes inverted terminal repeats (ITRs) at each end which function in c/'s as origins of DNA replication and as packaging signals for the virus. The ITRs are approximately 145 bp in length. The internal non-repeated portion of the genome includes two large open reading frames, known as the AAV rep and cap genes, respectively. These genes code for the viral proteins involved in replication and packaging of the virion. In particular, at least four viral proteins are synthesized from the AAV rep gene, Rep 78, Rep 68, Rep 52 and Rep 40, named according to their apparent molecular weight. The AAV cap gene encodes at least three proteins, VP1 , VP2 and VP3. For a detailed description of the AAV genome, see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol, and Immunol. 158:97-129. The construction of recombinant AAV virions is generally known in the art and has been described for instance in US 5,173,414 and US5.139.941 ; WO 92/01070, WO 93/03769, (Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol, and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801 .
The ITRs are the only AAV viral elements which are required in c/'s for the AAV genome replication and its packaging into the viral particles. The ITRs may be synthetic or have been derived from viruses of different serotypes. All other viral genes required for viral vector replication can be provided in trans within the virus-producing cells (packaging cells).
The nucleic acid construct may comprise ITRs of any AAV serotype, including AAV1 , AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1 , AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV serotype now known or later discovered. Preferably, the ITRs are from a human AAV serotype. In a particular embodiment, the ITRs are from the AAV2 serotype. In another particular embodiment, the ITRs are selected from the group consisting of AAV1 , AAV6, AAV8 and AAV9 serotypes. As already stated "control sequences" and "control elements" refer, among others, to promoter and termination sequences. Useful promoter sequences include those derived from sequences encoding vertebrate cardiac-specific expression genes.
Therefore, in a preferred embodiment, optionally in combination with one or more features of the various embodiments described above or below, the nucleic acid construct comprises a coding sequence operably linked to a vertebrate cardiac-specific gene expression promoter and a transcription termination sequence located 3' to the coding sequence, wherein the coding sequence operably linked to the control sequences is in turn flanked by the AAV ITRs and wherein the preferred AAV ITRs are derived from the AAV-2 serotype.
Examples of vertebrate cardiac-specific gene expression promoters include, but are not limited to, the chicken cardiac troponin T (cTnT/TNNT2) promoter, the human cTnT promoter, the mouse cTnT promoter, the human alpha-myosin heavy chain promoter, the mouse alpha-myosin heavy chain promoter, rat alpha-myosin heavy chain promoter, human myosin light chain 2v promoter, 5 mouse myosin light chain 2v promoter, rat myosin light chain 2v promoter, frog myosin light chain 2v promoter, human cardiomyocyte-specific Na+- Ca2+ exchange promoter, human cardiac alpha-actinin promoter, mouse alpha-actinin promoter, human cardiac troponin I (TNNI3) promoter.
Preferably, the vertebrate cardiac-specific gene expression promoter is the chicken cardiac troponin T (cTnT) promoter. The "Chicken cardiac troponin T (cTnT) promoter" refers to a regulatory sequence that controls transcription of the cardiac troponin T gene in chicken. E.g.: Robust cardiomyocyte-specific gene expression following systemic injection of AAV: in vivo gene delivery follows a Poisson distribution. Prasad et al., 201 1 , Gene Therapy, 18(l):43-52.
Thus, in another preferred embodiment, optionally in combination with one or more features of the various embodiments described above or below, the nucleic acid construct comprises a coding sequence operably linked to the chicken cardiac troponin T (cTnT) promoter and a polyA transcription termination sequence located 3' to the coding sequence, wherein the coding sequence operably linked to the control sequences is in turn flanked by the AAV ITRs and wherein the preferred AAV ITRs are derived from the AAV-2 serotype.
Examples of transcription termination sequences include, but are not limited to, polyA, SV40 polyA, human growth hormone polyA and bovine growth hormone polyA. Preferably, said transcription termination sequence is a SV40 polyA transcription termination sequence. "SV40 pA transcription termination sequence" refers to a regulatory sequence that controls termination of transcription in the Simian Virus 40 and includes the typical polyadenylation core sequence AATAAA. In another preferred embodiment, optionally in combination with one or more features of the various embodiments described above or below, the nucleic acid construct comprises a coding sequence operably linked to the chicken cardiac troponin T (cTnT) promoter and a SV40 polyA transcription termination sequence located 3' to the coding sequence, wherein the coding sequence operably linked to the control sequences is in turn flanked by AAV ITRs derived from the AAV-2 serotype. The terms "viral particle", and "virion" are used herein interchangeably and relate to an infectious and typically replication-defective virus particle comprising the viral genome (i.e. the nucleic acid construct of the expression viral vector) packaged within a capsid and, as the case may be, a lipidic envelope surrounding the capsid. The skilled person will appreciate that an AAV virion may comprise capsid proteins from any AAV serotype. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the AAV viral particle comprises capsid proteins from a serotype selected from the group consisting of AAV1 , AAV2, AAV6, AAV8, and AAV9 which have been described as more suitable for delivery to the myocardial cells (Prasad et al., 201 1 , Gene Therapy, 18(l):43-52), preferably capsid proteins are from a serotype selected from the group consisting of AAV8 and AAV9, more preferably the capsid proteins are from the AAV9 serotype. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the viral particle comprises a nucleic acid construct of invention wherein the 5'ITR and 3'ITR sequences of the nucleic acid construct are of an AAV2 serotype and the capsid proteins are of an AAV9 serotype, referred as AAV2/9 vector.
The nucleic acid construct and expression vector may be obtained by conventional methods known to those skilled in the art: Sambrook and Russell (Molecular Cloning: a Laboratory Manual; Third Edition; 2001 Cold Spring Harbor Laboratory Press); and Green and Sambrook (Molecular Cloning: a Laboratory Manual; Fourth Edition; 2012 Cold Spring Harbor Laboratory Press).
In a preferred embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to an oxygen carrier composition (e.g. an HBOC composition) for use in a method for the in vivo diagnosis and/or the therapeutic and/or preventive treatment of a myocardial disease; wherein said method comprises the following consecutive steps:
i. temporary occluding a coronary artery and/or a coronary vein;
ii. intracoronary administering the oxygen carrier composition prior to and/or simultaneously to the administration of a preventive, therapeutic and/or diagnostic agent; wherein said oxygen carrier composition has been pre-oxygenated; and wherein said agent is a transgene encoding expression vector, preferably an adeno- associated virus (AAV) expression vector. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the method of the invention as described herein results in an increase of the transgene transduction in comparison with the administration of the diagnostic, prophylactic and/or therapeutic transgene encoding expression vector in the absence of an oxygen carrier composition of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 1 10%, at least 120%, at least 130%, at least 140% or at least 150%. Gene transduction is generally higher in those areas of the myocardium directly irrigated by the coronary vessel wherein administration of the expression vector has been conducted. In a particular embodiment, gene transduction is achieved at least in the left ventricle. Preferably, gene transduction is achieved in all the myocardium.
Methods to determine efficiency of gene transduction are well known in the art and include but are not limited to determining the number of cells positive for the transgene in the target tissue, and determining the total amount of the transgene and/or its expression levels in the target tissue, for instance by means of determining the expression of the therapeutic, prophylactic and/or diagnostic gene or of a reporter gene comprised within the expression vector. Reporter genes that may be used to determine transfection efficiency include but are not limited to chloramphenicol acetyl transferase (CAT), β-galactosidase, Photinus pyralis luciferase, and Renilla reniformis green fluorescent protein (GFP).
The determination of the expression of the transgene or reporter gene may be carried out at protein level. There are several methods for the quantification of peptides and proteins well known to one skilled in the art, including but not limited to enzymatic assays and immunoassays. Various types of immunoassays are known to one skilled in the art for the quantitation of proteins of interest, either in solution or using a solid phase assay. These methods are based on the use of affinity reagents, which may be any antibody or ligand specifically binding to the target protein, which is preferably labeled. For example, western blotting or immunoblotting allows comparison abundances separate proteins by an electrophoretic gel, eg. SDS-PAGE. In this technique, separated by gel electrophoresis proteins are transferred onto a sheet of polymeric material (generally nitrocellulose, nylon, or polyvinylidene difluoride), which are immobilized. In immunohistochemistry assays, proteins are detected directly in cells of a tissue. In these immunoassays, target proteins are revealed by using a solution containing a specific antibody (staining). The antibody can be conjugated directly with a radioactive, fluorescent or enzymatic (direct detection method) or may be used a secondary antibody that recognizes the primary antibody and thus amplifies the signal (indirect method of detection or sandwich assay).
Traditionally, the specific identification of proteins in solution has been carried out by immunoassays on a solid support. Typically, a specific capture antibody for the target protein in a polymer or plastic surface immobilized and added to the support a solution containing the protein of interest (for example, cell lysate). Finally, the sample is incubated on the support for a time to allow antigen-antibody complexes are formed. Then, usually perform one or more washes to remove the solution and the target protein is detected with a second antibody that recognizes an epitope different from that recognized by the capture antibody protein. As in the case of Western blotting, this detection antibody may be labeled directly or can be recognized with a secondary antibody. An immunoassay commonly used for protein quantification is the test enzyme-linked immunosorbent assay (ELISA) in which the detection antibody carries an enzyme that converts a commonly colorless substrate into a colored compound or a non-fluorescent substrate to a fluorescent compound. Also in other solid phase immunoassays, the antibody may be labeled with a radioactive isotope or fluorescence. Other methods that can be used for quantification of proteins are techniques based on mass spectrometry (MS) such as liquid chromatography coupled to mass spectrometry (LC / MS), described for example in US2010 / 0173786, or tandem LC-MS / MS (WO2012 / 155019, US201 1 / 0039287, M. Rauh, J Chromatogr B Analyt Technol Biomed Life Sci 2012 February 1 , 883-884. 59-67) and the use of arrays of peptides, proteins or antibodies and multiplex versions of the above techniques, as well as the next generation of such techniques and combinations thereof.
Gene transduction may also be measured by the transgene quantification or the mRNA expression levels thereof. Molecular biology methods for measuring quantities of target nucleic acid sequences are well known in the art. These methods include but are not limited to end point PCR, competitive PCR, reverse transcriptase-PCR (RT-PCR), quantitative PCR (qPCR), reverse transcriptase qPCR (RT-qPCR), PCR-pyrosequencing, PCR-ELISA, DNA microarrays, in situ hybridization assays such as dot-blot or Fluorescence In Situ Hybridization assay (FISH), branched DNA (Nolte, Adv. Clin. Chem. 1998,33:201 -235) and to multiplex versions of said methods (see for instance, Andoh et al., Current Pharmaceutical Design, 2009;15,2066-2073) and the next generation of any of the techniques listed and combinations thereof, all of which are within the scope of the present invention. Such methods may also include the pre-conversion of mRNA into cDNA by the reaction with a reverse transcriptase (RT), for example the PCR or qPCR reaction is usually preceded by conversion of mRNA into cDNA and referred to as RT-PCR or RT-qPCR, respectively. Preferably, said molecular method for gene quantification is selected from the group consisting of quantitative Polymerase Chain Reaction (qPCR), PCR-pyrosequencing, fluorescence in-situ hybridization (FISH), DNA microarrays, and PCR-ELISA.
In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, gene transduction efficiency is determined by measuring the transgene mRNA expression levels by reverse transcriptase quantitative PCR (RT-qPCR).
Expression levels may be absolute or relative. It is generally preferred that expression levels are normalized. Normalization can be performed with respect to different measures in the sample. These procedures are well known to one skilled in the art. Typically, expression levels are normalized with respect to an "Endogenous control". An "Endogenous control" as used herein relates to a gene expression product whose expression levels do not change or change only in limited amounts. Preferably, the "endogenous control" is the expression product from housekeeping genes and which code for proteins which are constitutively expressed and carry out essential cellular functions. Preferred housekeeping genes for use in the present invention include β-2- microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, GAPDH, actin and HPRT.
In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the oxygen carrier composition for use in a method as described herein, is characterized by said method comprising the following consecutive steps:
i. temporary occluding a coronary artery, and optionally, temporary occluding a coronary vein;
ii. administering into the coronary artery downstream to the site of occlusion an oxygen carrier composition, wherein said oxygen carrier composition has been pre-oxygenated;
iii. administering into the coronary artery downstream to the site of occlusion a diagnostic, prophylactic and/or therapeutic agent and the oxygen carrier composition; and
iv. optionally, prior to restoring the coronary flow administering an additional volume of the oxygen carrier composition. Preferably, the oxygen carrier composition in ii) and iv) is substantially free of the diagnostic, prophylactic and/or therapeutic agent administered in iii). More preferably, the oxygen carrier composition in ii) and iv) is substantially free of any diagnostic, prophylactic and/or therapeutic agent.
Preferably, the intracoronary administration of the agent and the oxygen carrier composition in step iii) occurs simultaneously. The agent and the oxygen carrier composition may be administered in the same composition or in a separate composition. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the agent and the oxygen carrier composition are formulated in a single composition. This formulation can be extemporaneous (i.e., prepared immediately before the administration to the patient).
Several methodologies for the occlusion of veins and/or arteries have been described and are well known in the art, non-limiting examples include the Open Chest Model (surgical ligation method) and the Closed Chest Model (Balloon Inflation Model) which is the most commonly used model (Budhani MK _et al., Regulatory Toxicology and Pharmacology 2016, 2016), doi: 10.1016/j.yrtph). Preferably, said occlusion is performed by the closed chest model, i.e., comprises inflating a catheter occluding balloon. In step i) the method of the invention comprises temporary occluding a coronary artery and/or a coronary vein, as defined above. The aim of this step is mainly isolating the vessel from the blood circulatory system of the subject prior to administering the oxygen carrier composition vessel (optionally comprising the prophylactic, therapeutic or diagnostic agent). Administration may be antegrade (e.g. through the arteries in the direction of blood flow), in a retrograde manner (through veins, such as the coronary sinus, in opposition to the normal blood flow direction), or in a combination of retrograde and antegrade administration. In a particular embodiment, administration is antegrade and administration downstream of the site of occlusion. In another particular embodiment, administration is retrograde and administration upstream of the site of occlusion. In a preferred embodiment, it comprises the occlusion of a coronary artery and the administration of the oxygen carrier composition downstream of the site of occlusion, wherein a coronary vein is optionally occluded. In a particularly preferred embodiment, optionally in combination with one or more features of the various embodiments described above or below, said method comprises in step i) the temporary occlusion of the left anterior descending (LAD) artery, preferably a mid-LAD artery occlusion, wherein preferably occlusion is conducted by inflating a catheter occluding balloon. Occlusion time of the coronary artery and/or the coronary vein may be from a few seconds to several minutes and will be determined in order to maximize the time of contact of the agent with the target site in the absence of deleterious effects. Possible occlusion times include occlusion times typically used for gene therapy purposes which are generally of less than 10 minutes, preferably of less than 5 minutes, to prevent ischemia-related injury, preferably from 1 to 10 minutes, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes, including fractions thereof. The method of the invention which comprises the administration of an oxygenated oxygen carrier composition also encompasses longer occlusion times, such as 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 35, 40, 45 or more minutes. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the occlusion time is of between 1 and 15 minutes, preferably of 12 or 13 minutes. In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the occlusion time is of between 1 and 5 minutes, preferably of 2 or 3 minutes.
The volume of the oxygen carrier composition to be perfused without removal of blood is physiologically limited. To avoid circulatory volume overload, the total volume of the oxygen carrier injected is generally recommended to be below 20%, preferably about 17%, of the subject total circulatory volume (12 ml/kg). For instance for an individual of 50 kg the volume which may be perfused would be of 600 mL, and for an individual of 60 kg (typically the average weight of a human subject) this volume would be of 720 mL. Accordingly, in a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, a maximum total volume of 12 mL/kg is administered.
Said oxygen carrier composition may be perfused at a maximum infusion rate of 1 ml/kg/min (equating to normal coronary flow). In the Examples, the administration of 600 mL to pigs of an average weight of 50 kg at infusion rates of 0.4 ml/kg/min did not damage the healthy myocardium. Accordingly, preferred infusion rates are from 0.4 ml/kg/min to 1 ml/kg/min, such as an infusion rate of about 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 ml/kg/min. Preferably, the perfusion rate is of 1 ml/kg/min.
In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the perfusion rate is of 1 ml/kg/min and the a perfusion time is of 12 minutes (corresponding to an administered volume of 12 ml/kg and a total volume of 600 mL in a 50 kg individual). The volume of the oxygen carrier composition to be perfused prior to the agent administration may be from 100 mL to 500 mL, preferably from 200 to 400 mL, such as of about 250, 300 or 350 mL. For instance administration is of 250 mL (corresponding to 5 minutes at an infusion rate of 1 ml/kg/min for an individual of 50 kg). A person skilled in the art will determine the appropriate perfusion time according to the selected volume to be perfused and the perfusion rate.
Similarly, the volume of the oxygen carrier composition comprising the agent may be from 100 mL to 500 mL, preferably from 200 to 400 mL, such as of about 250, 300 or 350 mL. For instance administration is of 350 mL (corresponding to 7 minutes at an infusion rate of 1 ml/kg/min for an individual of 50 kg).
In a preferred embodiment, the oxygen carrier composition is administered during 5 minutes at a rate of 1 ml/kg/min (corresponding to 250 mL for a 50 kg individual), subsequently the composition comprising the oxygen carrier and the agent is administered during 7 minutes at a rate of 1 ml/kg/min (corresponding to 350 mL for a 50 kg individual). Optionally, the oxygen carrier composition may be administered for an additional time period, e.g. 1 minute at an infusion rate of 1 ml/kg/min, prior to blood flow restoration. In a second aspect, the present invention relates to a composition comprising or consisting of:
- an oxygen carrier composition;
- a metabolic substrate; and/or
- a pH modulator agent.
Said composition is herein also referred as "the modified oxygen carrier composition of the invention".
Preferably, said composition comprises or consists of:
- an oxygen carrier composition;
- a metabolic substrate; and
- a pH modulator agent.
Said oxygen carrier composition is as described under the first aspect of the invention. A metabolic substrate may be any potential source of ATP, including fatty acids, glucose, ketone bodies, pyruvate, lactate, amino acids and even constituent proteins, as well as combinations thereof. Amino acids are not particularly limited, and can be selected for instance from the group consisting of alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, taurine, threonine, tryptophan, tyrosine, valine and combinations thereof. Preferably, amino acids are selected from the group consisting of glutamate, glutamine, asparagine and alanine, and combinations thereof.
In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, said metabolic substrate is glucose. Preferably, said metabolic substrate is glucose and said composition further comprises insulin.
The pH of the composition might be from pH 4.0 to pH 7.5, preferably from pH 6.8 to pH 7.4, more preferably from pH 7.0 to pH 7.4, for instance any pH selected from 7.0, 7.05, 7.1 , 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4. Preferably the pH of said composition is about 7.35.
A pH modulator or buffering agent may be an acid/salt buffer system. As non-limiting examples of the acid component in the buffer mixtures can be used inorganic acids (e.g., phosphate, carbonate, hydrogencarbonate), polyvalent carboxylic acids (e.g., succinic acid, maleic acid, benzoic acid), hydroxycarboxylic acids (such as glycolic acid, citric acid, malic acid or lactic acid), keto acids (eg, a-ketoglutaric acid) or sulfonic acids (e.g., 2- [ 4- (2- hydroxyethyl) -1 -piperazino] -ethanesulfonic acid (HEPES)), and amino acids (e.g., glycine, histidine, aspartic acid, phenylalanine, lysine, arginine, cysteine; including the natural L- amino acids as well as derivatives thereof), and combinations thereof. The buffer substances can either be present in the free acid form and/or in the form of the alkali, alkaline-earth or ammonium salts.
In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, said pH modulator agent is n- acetylcysteine. Preferably, said pH modulator agent is n-acetylcysteine and the pH of said composition is adjusted to 7.35.
In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, said composition comprises or consists of:
- HBOC-201 ;
- glucose;
- insulin; and - N-acetylcysteine;
wherein the pH of said composition is from 6.8 to 7.4, preferably wherein the pH of said composition is 7.35. Other pharmaceutically acceptable excipients which may be included in said composition include but are not limited to monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, trehalose, maltose and sucrose; trisaccharides such as raffinose, tetrasaccharides such as stachyose; polysaccharides such as dextran; and sugar alcohols such as mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur- containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thiosulfate; hydrophilic polymers such as polyvinylpyrrolidone or a surfactant such as polysorbate surfactants. Also, optionally, said composition may include one or more diagnostic, prophylactic and/or therapeutic agent, such as described herein.
The modified oxygen carrier composition of the invention may be suitable for in vitro or in vivo use. Preferably, said composition is a pharmaceutical composition. In a third aspect, the present invention relates to the use of the modified oxygen carrier composition of the invention as a pharmaceutical composition. It also relates to the composition or pharmaceutical composition of the invention for use as a medicament.
As used herein, a "pharmaceutical composition" refers to a composition that is pharmaceutically acceptable. The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.
Said pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art. This includes, for example, injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the pharmaceutical composition of the invention is administered to a subject by intravascular administration. Preferably, the administration is intracoronary. For such purposes, the composition may be injected or infused using a syringe, as well as other devices known in the art. For instance, when administration is intracoronary the pharmaceutical composition may be administered via the central lumen of the catheter which may have been introduced percutaneously.
The pharmaceutical composition as described in the preceding aspects has shown to be particularly suitable for administration under bloodless situations, for instance for intracoronary administration in cardiac ischemia conditions. Accordingly, in a fourth aspect, the invention relates to the composition according to the preceding aspects of the invention, for use in a method of in vivo diagnosis and/or treatment of a tissue or organ which has or is at risk of having ischemia, this includes for instance those organs which may be transplanted, such as the heart, liver or kidney. Preferably, said tissue or organ which has or is at risk of having ischemia is the myocardium.
A related aspect is directed to methods for in vivo diagnosis and/or treating a tissue or organ which has or is at risk of having ischemia with a composition or pharmaceutical composition of the present invention. In one embodiment, the method to in vivo diagnosing or treating said ischemia comprises administering to a subject a composition or pharmaceutical composition of the present invention in an amount effective to treat it.
In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention refers to the modified composition of the invention for use in a method for the preventive and/or therapeutic treatment of ischemia/reperfusion injury, for instance after acute myocardial infarction. Said method comprises administration of said modified composition before and/or after interruption of the blood flow. Preferably, it is administered after interruption of the blood flow and before blood flow restoration.
It has been found that said composition may be particularly suitable for administration to an ischemic organ, such as an ischemic myocardium. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the modified composition of the invention is administered after 15 minutes from oxygen deprivation, for instance after 20, 25, 30, 35, 40 or 45 minutes. Preferably, administration of the composition is performed after 45 minutes or more from oxygen deprivation. Oxygen deprivation may occur for instance by interruption of the blood flow.
Perfusion times and rates may be as described herein. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, said oxygen carrier composition is perfused at an infusion rate from 0.4 to 1 mL/kg/min, preferably at an infusion rate of 1 mL/kg/min.
In a further aspect, the invention relates to a method for obtaining a composition according to the preceding aspects of the invention. It may comprise mixing the components of the composition as described herein. It also may comprise adding a metabolic substrate to the oxygen carrier composition and adjusting the pH of the composition as described herein. In a particular embodiment, it comprises:
- adding a metabolic substrate to the oxygen carrier composition; and
- adjusting the pH with a pH modulator agent to a pH from 6.8 to 7.4, preferably to a pH of 7.35.
This may be performed by methods well known in the art of pharmaceutical formulation. The composition of the present invention has been found to be particularly suitable for bloodless drug delivery. Accordingly, in a further aspect, the invention relates to a delivery vehicle comprising a pharmaceutical composition as described herein. An additional aspect of the invention relates to an oxygen carrier composition as described in the preceding aspects of the invention, for use as delivery vehicle in the administration of a therapeutic and/or diagnostic agent, preferably in a method as described herein under the first aspect of the invention.
It is contemplated that any features described herein can optionally be combined with any of the embodiments of any pharmaceutical composition, kit, medical use, method of treatment, or method of manufacturing of the invention; and any embodiment discussed in this specification can be implemented with respect to any of these. It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The use of the word "a" or "an" may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one". The use of the term "another" may also refer to one or more. The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term "comprises" also encompasses and expressly discloses the terms "consists of" and "consists essentially of". As used herein, the phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. As used herein, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim except for, e.g., impurities ordinarily associated with the element or limitation.
The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
As used herein, words of approximation such as, without limitation, "about", "around", "approximately" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by ±1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15%. Accordingly, the term "about" may mean the indicated value ± 5% of its value, preferably the indicated value ± 2% of its value, most preferably the term "about" means exactly the indicated value (± 0%).
The following examples serve to illustrate the present invention and should not be construed as limiting the scope thereof.
EXAMPLES
Example 1.- Pre-oxygenated HBOC -201 intracoronary infusion strategies 1.1. Material and Methods
Protocol 1 preparation and procedures: Intracoronary infusion of HBOC-201 in healthy pigs
Pigs were sedated by intramuscular injection of ketamine (20 mg/kg), xylazine (2 mg/kg), and midazolam (0.5 mg/kg). Buprenorphine (0.03mg/kg) was used as an analgesic during the intervention. All animals were intubated and mechanically ventilated with oxygen (fraction of inspired 02 = 28%), and anesthesia was maintained by i.v. administration of ketamine (2 mg/kg/h), xylazine (0.2 mg/kg/h), and midazolam (0.2 mg/kg/h). Central venous and arterial lines were placed and a single bolus of unfractionated heparin (300 lU/kg) was administered immediately before catheter introduction. During the procedure, pigs were monitored by continuous electrocardiography, pulse-oximetry, and invasive measurement of systemic and pulmonary artery pressures (using a Swan-Ganz catheter). A conventional 0.014-inch guidewire was advanced into the LAD coronary artery. A short, highly compliant over-the-wire balloon (Helios Occlusion Balloon Catheter, LightLab) was then placed in the LAD artery proximal to the origin of the first diagonal branch and connected to the infusion pump (PHD ULTRA Series Syringe Pump, Harvard Apparatus). The guidewire was then removed and the balloon catheter was inflated (coronary occlusion). This was immediately followed by distal continuous intracoronary infusion of pre-oxygenated warm (37°C) HBOC- 201 via the central lumen of the catheter. The HBOC-201 solution was warmed using an inline clinical fluid warmer (Astotherm® plus, Model AP220S, Futuremed America) positioned immediately proximal to the intracoronary Helios balloon catheter. The HBOC-201 solution was contained within the sterile, high-pressure infusion line wrapped around the warmer's heating coil. Balloon location and state of inflation were monitored regularly by angiography. After balloon deflation, a coronary angiogram was recorded to confirm patency of the coronary artery. In the event of ventricular fibrillation, a biphasic defibrillator was used to deliver non-synchronized shocks as needed. Postoperative animal recovery and care were carried out by CNIC veterinarians and technicians.
Protocol 2 preparation and procedures: Myocardial infarction induction protocol followed by bloodless or regular reperfusion
The myocardial infarction induction protocol has been reported in previous publications of the group (Fernandez-Jimenez R et al., J Am Coll Cardiol 2015, 66:816-828; Fernandez- Jimenez R et al., J Am Coll Cardiol 2015, 66:816-828; Fernandez-Jimenez R et al., J Am Coll Cardiol 2015, 65:315-323; Garcia-Prieto J et al., Basic Res Cardiol 2014, 109:422; Garcia-Ruiz JM et al., J Am Coll Cardiol 2016, 67:2093-2104). In summary, pigs were sedated by intramuscular injection of ketamine (20 mg/kg), xylazine (2 mg/kg), and midazolam (0.5 mg/kg). Buprenorphine (0.03mg/kg) was used as an analgesic during the intervention. All animals were intubated and mechanically ventilated with oxygen (fraction of inspired 02 = 28%), and anesthesia was maintained by i.v. administration of ketamine (2 mg/kg/h), xylazine (0.2 mg/kg/h), and midazolam (0.2 mg/kg/h). Central venous and arterial lines were placed and a single bolus of unfractionated heparin (300 lU/kg) was administered immediately before catheter introduction. During the procedure, pigs were monitored by continuous electrocardiography, pulse-oximetry, and invasive measurement of systemic and pulmonary artery pressures (using a Swan-Ganz catheter). Femoral artery was percutaneously accessed and a sheath was placed. Through the sheath, a guiding catheter was placed in the origin of the left coronary artery. Through the guiding catheter, a conventional 0.014-inch guidewire was advanced into the LAD coronary artery. A short, highly compliant over-the-wire balloon (Helios Occlusion Balloon Catheter, LightLab) was then placed in the LAD artery distal to the origin of the first diagonal branch and connected to the infusion pump (PHD ULTRA Series Syringe Pump, Harvard Apparatus). The guidewire was then removed and the balloon catheter was inflated (coronary occlusion).
Oxygen carrier pre-oxygenation & infusion
The oxygen carrier used in these experiments was HBOC-201 (Hb02 Therapeutics; (Burkhoff D, Lefer DJ, Am Heart J 2015, 149:573-579 ; Cabrales P, Intaglietta M, ASAIO J 2013, 59:337-354; Jahr JS, Moallempour M, Lim JC , Expert Opin Biol Ther 2008, 8:1425- 1433 ; Te Lintel Hekkert M et al., Am J Physiol Heart Circ Physiol 2010, 298:1-11 103-1 1 13).
HBOC-201 was pre-oxygenated under sterile conditions 2 hours before each experiment. More specifically, HBOC-201 250ml bags were pre-chilled to 4°C overnight. Then, 80ml_ of 100% oxygen were injected into the HBOC-201 bag using an oxygenation tube set (Advanced Scientifics Inc., product no. B105885-1 ). Oxygenated bags were equilibrated by rocking for 90 minutes in a refrigerator at 4°C.
To avoid circulatory volume overload, the total volume of the oxygen carrier injected was 17% of the animal's total circulatory volume (12 ml/kg). Two different rates of infusion of the fixed oxygen carrier solution were tested (1 ml/Kg/min (physiological rate, resulting in 12 min of infusion) and 0.7 ml/Kg/min (reduced rate, resulting in 17 min of infusion). Pre-oxygenated warm (37°C) oxygen carrier was infused via the central lumen of the catheter. The oxygen carrier solution was warmed using an in-line clinical fluid warmer (Astotherm® plus, Model AP220S, Futuremed America) positioned immediately proximal to the intracoronary Helios balloon catheter. The HBOC-201 solution was contained within the sterile, high-pressure infusion line wrapped around the warmer's heating coil. CMR imaging protocol
CMR studies were performed at several time points to assess IS and LV performance. Pigs were anesthetized as described above, and anesthesia was maintained by continuous intravenous infusion of midazolam. CMR studies were performed using a Philips 3-Tesla Achieva Tx whole body scanner (Philips Medical Systems, Best, the Netherlands) equipped with a 32-element cardiac phased-array surface coil. Images were acquired with the use of ECG gating by operators blinded to the study arm. Segmented cine steady-state free precession (SSFP) was performed to acquire 1 1 -13 contiguous short-axis slices covering the heart from the base to the apex in order to evaluate global and regional LV motion (FOV, 280 x 280 mm; slice thickness, 8 mm without gap; TR, 2.8 ms; TE, 1 .4 ms, flip angle, 45°; cardiac phases, 25; voxel size, 1 .8 x 1 .8 mm; number of excitations [NEX], 3). Edema imaging (to quantify myocardial area at risk) was performed with a T2-weighted, triple inversion-recovery fast spin-echo sequence (T2W-STIR) (FOV, 280 x 280; 1 1 - 13 short-axis slices, thickness 8 mm without gap; TR, 2 to 3 heartbeats; TE, 80 ms; voxel size, 1 .4 x 1.4 mm; STIR delay, 210 ms; trigger delay, longest; echo-train length, 16; NEX, 2). A coil sensitivity correction algorithm for all T2W images was implemented in the scan acquisition. Late gadolinium enhancement imaging was performed 15 minutes after administration of 0.2 mmol/kg gadopentate dimeglumine, using an inversion-recovery fast gradient-echo sequence to determine IS (FOV, 280 x 280 mm; 1 1 -13 short-axis slices, thickness 8 mm without gap; TR, 5.6 ms; TE, 2.8 ms; voxel size, 1 .6 x 1 .6 mm; time interval optimized to null normal myocardium; trigger delay, longest; bandwidth, 304 Hz per pixel; NEX, 2).
CMR image analysis
All CMR images were analyzed using dedicated software (QMass MR v.7.5, Medis, Leiden, The Netherlands). Images were analyzed by two experienced observers with extensive experience in CMR analysis and blinded to the study allocation. The analysis protocol has been detailed elsewhere. (Garcia-Prieto J et al., Basic Res Cardiol 2014, 109:422) ]Briefly, LV cardiac borders (epi- and endocardial) were traced in each short-axis cine image to obtain LV end-diastolic volume (LVEDV), end-systolic volume (LVESV), LVEF and segmental wall thickening, which was calculated as (end-systolic wall thickness - end- diastolic wall thickness)/end-diastolic wall thickness x100. Values of LV volume normalized to body surface area were calculated with Brody's formula. The area of myocardium at risk (AAR) was defined as the extent of the LV showing high signal intensity on T2W-STIR images. (Aletras AH et AL, Circulation 2006, 1 13:1865-1870) IS (necrosis) was quantified from the extent of abnormally delayed gadolinium enhancement. AAR and necrosis were identified as hyperintense regions defined as regions exceeding 50% of the peak myocardial signal intensities (full width half maximum) with manual adjustment when needed. If present, a central core of hypointense signal within the area of increased signal was included in the T2W-STIR or late gadolinium enhancement analysis. IS was expressed both as a percentage of LV mass and normalized to AAR.
Statistical analysis
The distribution of continuous variables was analyzed with graphical methods. Continuous variables are expressed as median [IQR]. Categorical variables are expressed as absolute frequency (%). Comparisons among groups were performed with non-parametric methods (Kruskal-Wallis, Mann-Whitney U, and Fisher's exact test) as appropriate. Differences were considered statistically significant at p-value <0.05 (two-tailed).
1.2. Protocol 1 : Optimization of intracoronary HBOC-201 infusion rate and duration in healthy animals.
This protocol was undertaken in animals without infarction to assess the ability of healthy myocardium to tolerate prolonged coronary perfusion with pre-oxygenated HBOC-201 instead of blood. The first goal was to identify conditions for HBOC-201 intracoronary infusion during coronary occlusion that does not cause permanent myocardial damage (scar formation or persistent LVEF deterioration). We hypothesized that maintaining bloodless reperfusion for as long as possible without inducing myocardial damage (the goal of protocol 2) would maximize the opportunity to control factors implicated in reperfusion-related injury. Because the HBOC-201 solution is colloidal, the maximum top-load (no blood extraction) infusion volume is limited to the volume that can be administered without risking circulatory volume overload. This constraint requires that intracoronary infusion rate declines as infusion duration increases. The maximum volume that safely avoids circulatory overload is 17% of the animal's total circulatory volume in a 50-kg pig this corresponds to 600 ml (12 ml/kg).
Twenty healthy castrated male (-50 kg) Large-White pigs were randomized to receive the 600ml_ volume of pre-oxygenated HBOC-201 at 1 of 4 infusion rates beginning immediately after proximal left anterior descending (LAD) coronary artery occlusion, followed by blood flow restoration upon completion of the HBOC-201 infusion (Figure 1A). HBOC-201 was infused distal to the coronary occlusion site. The fixed infusion rates were 1 ml/kg/min (equating to normal coronary flow) for 12 minutes, 0.7 ml/kg/min for 17 minutes, 0.4 ml/kg/min for 30 minutes and 0.2 ml/kg/min for 60 minutes. The impact of the different perfusion protocols on LV performance was examined by serial CMR imaging performed at baseline (immediately before coronary occlusion) and at 15 minutes and 7 days after completion of HBOC-201 reperfusion and restoration of normal coronary blood flow (Figure 1A).
In brief, animals were randomized to HBOC-201 infusion at the following rates of infusion: 1 , 0.7, 0.4 or 0.2 ml/Kg/min, corresponding to infusion durations of 12, 17, 30, and 60 min. A total of 20 animals, 5 per group, underwent the procedure; however, 2 animals in the 0.2 ml/Kg/min (60min) infusion group died before completion of the 7-day CMR.
Effect of HBOC-201 infusion on hemodynamic parameters and electrical stability.
Hemodynamic data were obtained at baseline and 5 min before finishing the 600 mL HBOC- 201 infusion. Hemodynamic changes in the 1 , 0.7, and 0.4 ml/Kg/min (12, 17, and 30 min) infusion groups were characterized by increases in systemic arterial pressure, pulmonary arterial pressure, and filling pressures (right atrial pressure and pulmonary capillary wedge pressure), without significant changes in heart rate or cardiac output (Table 1 ). Pigs allocated to the 0.2 ml/Kg/min (60 min) infusion group had a much worse hemodynamic response, characterized by increased filling pressures and reduced cardiac output. In this group, there was a median [IQR] change in cardiac output of -1.61 [-1.84 to -0.97] l/min; p- value <0.05). Cardiac output change in this group was significantly higher than in the 1 ml/Kg/min (12 min) group (median [IQR] of differences: -1 .61 [-1 .84 to -0.97] l/min vs -0.26 [- 0.4 to 0.38] l/min; p-value <0.05). Full hemodynamic data are presented in Table 1 .
Table 1. Hemodynamic changes induced by different rate + duration combinations for intracoronary infusion of HBOC-201 immediately after left anterior descending artery occlusion (Protocol 1)
Coronary infusion group
12 min 17 min 30 min 60 min
(n=5) (n=5) (n=5) (n=5)
Change from baseline:
HR (bpm) 1 [-8 to 3] 0 [-3.5 to 2.5] -4 [-15.5 to 5] 20 [0.5 to 48.5]
mPAP (mmHg) 6 [4 to 21.5] * 11 [8.5 to 21.5] * 6 [2 to 11.5] -2 [-16.5 to 4.5]
mSAP (mmHg) 15 [9.5 to 26.5] * 13 [-10 to 27] 22 [5.5 to 29] * 4 [-6.5 to 18.5]
mRAP (mmHg) 4 [3-5] 6 [5 to 8.5] * 4 [4 to 5.5] * 5 [4 to 6.5] * mPCWP (mmHg) 3.5 [2 to 5] 6 [4 to 7] * 5 [4.5 to 6] * 9.5 [7.3 to 11] *#
CO (l/min) -0.26 [-0.4 to 0.38] -0.53 [-1.09 to 0.23] -0.53 [-0.83 to 0.22] -1.61 [-1.84 to -0.97] *#
Hemodynamic parameters were determined at baseline and 5 minutes before completion of intracoronary infusion with 600 mL HBOC-201. Coronary infusion groups correspond to infusion rates as follows: 12 min, 1 ml/kg/min; 17 min, 0.7 ml/kg/min; 30 min, 0.4 ml/kg/min; 60 min, 0.2 ml/kg/min.
Data are shown as median [Q1 to Q3]. HR, heart rate; mPAP, mean pulmonary arterial pressure; mSAP, mean systemic arterial pressure; mRAP, mean right atrial pressure; mPCWP, mean pulmonary capillary wedge pressure; CO, cardiac output. *p-value <0.05 compared with baseline. #p <0.05 compared with 12min infusion group.
During coronary occlusion and immediate HBOC-201 infusion, animals randomized to infusion rates of 1 , 0.7, and 0.4 ml/Kg/min (12, 17, and 30 min) did not experience arrhythmias, whereas all 5 animals randomized to HBOC-201 infusion at 0.2 ml/kg/min (60 min) developed premature ventricular complexes. Moreover, during blood flow restoration, animals randomized to 1 and 0.7 ml/Kg/min HBOC-201 (12 and 17 min) did not develop significant electrical events, whereas animals infused with HBOC-201 at 0.4 and 0.2 ml/Kg/min (30 and 60 min) developed various forms of supraventricular and ventricular arrhythmia (Table 2). Table 2. Description of arrhythmias during coronary infusion of HBOC (Protocol 1) Coronary Infusion Blood Reperfusion
Animal Arrhythmia Duration Arrhythmia Duration
12 min 1 None None
[n=5) 2 None None
3 None None
4 None None
5 None None
17 min 6 None None
[n=5) 7 None None
8 None Simple PVCs f<10/min) Min 1 to 2
9 None None
10 None None
30 min 11 Simple PACs (>10/min) Min 5 to 30 Simple PVCs (>10/min) Min 1 [n=5) 12 None Simple PVCs (>10/min) Min 1 to 2
13 None None
14 None PSVT* Min 1 to 6
15 None Complex PVCs (>10/min) Min 1 to 6
&
Episodes of NSVT Min 1 to 3
60 min 16 PSVT* Min 20 to 30 Complex PVCs (>10/min) Min 1 to 4 [n=5) & &
Simple PVCs (>10/min) Min 30 to 60 PSVT* Min 4 to 10
17 Simple PACs (<10/min) Min 27 to 36 Complex PVCs (>10/min) Min 1 to 10
& &
Simple PVCs (>10/min) Min 50 to 60 Episodes of NSVT Min 1 to 7
18 Simple PVCs (<10/min) Min 25 to 60 Complex PVCs (>10/min) Min 1 to 10
&
NSVT Min 4
19 Simple PVCs (<10/min) Min 20 to 25 Complex PVCs (>10/min) Min 1 to 2
&
AIVR Min 2 to 5
20 Simple PVCs (<10/min) Min 18 to 60 AIVR Min 1 to 3
& &
Complex PVCs (<10/min) Min 22, 36 & 48-50 Complex PVCs (>10/min) Min 4 to 6
AIVR, Accelerated idioventricular rhythm; PAC, Premature atrial complex; PVC, Premature ventricular complex; PSVT, Paroxistic supraventricular tachycardia; NSVT, nonsustained ventricular tachycardia; * reversed by vagal maneuvers.
Effect of HBOC-201 infusion on LV performance.
Intracoronary infusion of pre-oxygenated HBOC-201 results in dynamic changes in LV performance depending on the infusion strategy. At 15 minutes after blood flow restoration, all groups showed a significant reduction in LVEF compared to baseline levels (12 min infusion (median of differences [IQR] absolute points reduction in EF]: -5.8 [-8.5 to -3.8]; 17 min infusion: -6.9 [-12.1 to -4.4]; 30 min infusion: -13.7 [-14.1 to -6.3], and 60 min infusion: - 34.7 [-37.6 to -31.9]; p-value <0.05). The deterioration in LVEF between baseline and 15 min post blood-flow restoration was significantly worse in the 0.2ml/kg/min (60 min) infusion group than in the 1 ml/kg/min (12 min) infusion group (median of differences [IQR]: -34.7 [- 37.6 to -31 .9] vs. -5.8 [-8.5 to -3.8]; p-value <0.05) (Table 3). Table 3. CMR-derived parameters for the different rate + duration combinations used for HBOC-201 intracoronary infusion (Protocol 1)
Coronary infusion group
12 min 17 min 30 min 60 min **
(n=5) (n=5) (n=5) (n=5)
Baseline CMR
LVEF (%) 59.8 [56.7 to 61.4] 57.7 [56.6 to 61.4] 58.4 [53.9 to 60.6] 57.5 [55 to 62.2]
Acute CMR (15 min post infusion)
LVEF (%) 52.1 [50.7 to 56.1] 49.8 [45.7 to 56.3] 47.8 [40.8 to 51.8] 26 [18.7 to 27.4]
LVEF change (15min - Baseline) -5.8 [-8.5 to -3.8] * -6.9 [-12.1 to -4.4] * -13.7 [-14.1 to -6.3] * -34.7 [-37.6 to -31.9] *#
7-day CMR
LVEF (%) 61.1 [58.6 to 65] 62 [56.2 to 65.3] 60.1 [57.1 to 60.8] 40.7 [23.4 to 53.4]
LVEF change (7-day - Baseline) 1.5 [0.2 to 5.2] 3 [-0.5 to 4.6] 2.3 [-1.8 to 4.8] -14.3 [-31.6 to -9.2] *#
Myocardial edema (% pigs) 0 0 0 100 #
Myocadial necrosis (% pigs) 0 0 0 100 #
Data are shown as median [Q1 to Q3] or relative frequency (%); CMR, cardiac magnetic resonance;
LVEF, left ventricular ejection fraction, *p-value <0.05 compared with baseline; #p-value <0.05 compared with 12 min infusion group; **ln the 60min infusion group, 2 animals died before completion of 7-day CMR.
LVEF fully recovered by day 7 in the 1 , 0.7, and 0.4 ml/kg/min (12, 17, and 30 min) groups, with no evidence of permanent damage (indicated by the absence of myocardial edema or delayed enhancement on CMR). Conversely, animals randomized to HBOC-201 infused at 0.2 ml/kg/min (60 min) showed severe, permanent LV dysfunction relative to baseline (p <0.05 vs. animals randomized to 1 .0 ml/kg/min HBOC-201 for 12 min). The LV dysfunction was associated with significant edema and massive necrosis of the LV (Figure 2, Table 3).
1.3. Protocol 2: Study of the effect on infarct size of pre-oxygenated oxygen carrier
infusion after prolonged ischemia.
A closed-chest pig model of myocardial infarction (Fernandez-Jimenez R et al., J Am Coll Cardiol 2015, 66:816-828; Fernandez-Jimenez R et al., J Am Coll Cardiol 2015, 66:816-828;
Fernandez-Jimenez R et al., J Am Coll Cardiol 2015, 65:315-323; Garcia-Prieto J et al., Basic Res Cardiol 2014, 109:422; Garcia-Ruiz JM et al., J Am Coll Cardiol 2016, 67:2093- 2104) was used to test whether the intracoronary perfusion of an oxygen carrier after a prolonged ischemic period (and before blood flow restoration) resulted in infarct size reduction compared with regular reperfusion (i.e. blood flow restoration) at the end of the index ischemia duration (i.e., 45 minutes). Castrated male (-30 kg) Large-White pigs were subjected to myocardial infarction induced by 45 minutes of mid left anterior descending (LAD) coronary artery occlusion.
After 45 minutes of mid LAD artery occlusion, pigs were randomly assigned to regular reperfusion (direct blood flow restoration) or intracoronary infusion of pre-oxygenated oxygen carrier (at 2 different infusion rates) followed by ulterior blood flow restoration, see the treatment protocol in Figure 1 B. The injection of the pre-oxygenated oxygen carrier at the end of the 45 min ischemia time results in a prolongation of the no-blood flow period.
The primary endpoint of the study to test the effect of pre-oxygenated oxygen carrier infusion at the end of ischemia was infarct size (IS), as evaluated by delayed gadolinium enhancement cardiac magnetic resonance (CMR) 7 days after infarction (Garcia-Ruiz JM et al., J Am Coll Cardiol 2016, 67:2093-2104). Intracoronary perfusion of unmodified HBOC- 201 (pH 7.6-7.9) was shown to be deleterious as it significantly increased microvascular obstruction and infarct size (data not shown).
Study of the effect on infarct size of modified pre-oxygenated oxygen carrier infusion after prolonged ischemia.
In view of the deleterious effects observed with the unmodified HBOC-201 solution, the commercial HBOC-201 was modified as follows:
1- adjusting the pre-oxygenated HBOC-201 to a physiological pH (7.35).
The pH of the oxygenated HBOC-201 was 7.6 to 7.9. The pH of oxygenated HBOC-201 was adjusted to a physiological value (pH =7.35, hereinafter referred as "HBOC-pH") by adding N-acetyl-L-cysteine (NAC). NAC dose-pH curves were generated and the stability of the pH, %02Hb and %metHb were checked over 24 hours,
and
2- further adding glucose and insulin to support myocardial energy requirements.
The HBOC-201 solution was enriched with glucose (140 mg/dl) and insulin (Actrapid, 150 μυ/ml) by adding these components into the HBOC-201 bag before oxygenation. The pH, glucose and insulin modified HBOC-201 solution is referred herein as "HBOC-pH-Glc-lns".
A total of 36 pigs were included in this protocol. Pigs were randomized to control (regular reperfusion immediately after 45 min of ischemia) or to one of the two strategies for intracoronary oxygen carrier infusion (12 or 17 min infusion after 45 min of ischemia and before restoration of coronary blood flow), see Figure 1 B. Due to deaths before completion of the 7-day CMR, final group numbers were 8 control pigs and 9 and 13 pigs in the 12 min and 17 min HBOC-pH-Glc-lns infusion groups, respectively.
Effect of pre-oxygenated HBOC-pH-Glc-lns intracoronary infusion after prolonged myocardial ischemia on hemodynamic parameters.
During the ischemic period (before HBOC-pH-Glc-lns infusion or restoration of coronary blood flow), all 3 treatment groups (12 and 17 min HBOC-pH-Glc-lns and regular reperfusion) underwent similar significant reductions in systemic arterial pressure (SAP) and cardiac output compared with baseline, see Table 4. After blood-flow restoration, cardiac output was significantly reduced in HBOC-pH-Glc-lns groups (cardiac output median of differences [IQR]: regular reperfusion, -0.84 [-1 .01 to -0.62] l/min; 12 min HBOC-pH-Glc-lns, -1 .41 [-1 .52 to -1 .22] l/min; 17 min HBOC-pH-Glc-lns, -1.62 [-1 .93 to -1.08] l/min; p-value <0.05) (Table 4).
Table 4. Hemodynamic changes after myocardial infarction and reperfusion with HBOC-pH- Glc-lns (Protocol 2)
HBOC-pH-Glc-lns reperfusion group
Control 12 min 17 min
(n=8) (n=9) (n=13)
Change from baseline to 40 min:
HR (bpm) 7.5 [0 to 11.8] 8 [1 to 14.5] 7 [1.5 to 13]
mPAP (mmHg) 4.5 [2.3 to 6.5] 2 [1.3 to 4.5] 1 [-1.5 to 5]
mSAP (mmHg) -35.5 [-37.5 to -27.3] -22 [-31.5 to -11] -24 [-30 to -14.5] mRAP (mmHg) 4 [1.5 to 5.8] 2 [1 to 6.5] 4 [2.5 to 5.5]
mPCWP (mmHg) 5 [0.5 to 6] 6 [3 to 8] 4 [2.5 to 6]
CO (l/min) -0.95 [-1.13 to -0.75] -0.88 [-1.31 to -0.48] -1.27 [-1.62 to -0.57]
Change from baseline to 90 min:
HR (bpm) 19.5 [9.5 to 28] 36 [26 to 47.5] * 37 [9 to 46.5]
mPAP (mmHg) 4.5 [0.8 to 9.5] 6 [3.5 to 13] 6 [2 to 9.5]
mSAP (mmHg) -26 [-31.8 to -22.3] -7 [-28.3 to 1] -21.5 [-35.8 to -11] mRAP (mmHg) 3.5 [0 to 5] 6 [2 to 7] 7 [5.5 to 8] * mPCWP (mmHg) 7.5 [4.8 to 9] 7 [6 to 12.5] 8 [5.5 to 16]
CO (l/min) -0.84 [-1.01 to -0.62] -1.41 [-1.52 to -1.22] * -1.62 [-1.93 to -1.08] *
Data are shown as median [Q1 to Q3]. HR, heart rate; mPAP, mean pulmonary arterial pressure; mSAP, mean systemic arterial pressure; mRAP, mean right atrial pressure; mPCWP, mean pulmonary capillary wedge pressure; CO, cardiac output. *p-value <0.05 compared with the control group (blood-only reperfusion).
Effect of pre-oxygenated HBOC-pH-Glc-lns intracoronary infusion after prolonged myocardial ischemia on infarct size (primary outcome of the study), and LV performance. At one week after infarction, there was a non-significant trend for infarct size reduction in the 17 min oxygen carrier group: median IS (IQR), %LV: regular reperfusion, 27.1 [24.2 to 34] %; 12 min HBOC-pH-Glc-lns, 30.5 [29.4 to 37.5] %; 17 min HBOC-pH-Glc-lns, 25.9 [2.6 to 35.6] %), Figure 2. A similar non-significant trend for infarct size (IS) reduction in the 17 min oxygen carrier group was found: median (IQR) IS/AAR (%): regular reperfusion, 98.4 [77.6 to 100] %; 12 min HBOC-pH-Glc-lns, 100 [87.5 to 100] %; 17 min HBOC-pH-Glc-lns, 88.4 [68.9 to 100] %) (Figure 3).
There were no baseline between-group differences in LV function. After blood flow restoration, the 17 min HBOC-pH-Glc-lns infusion group had a significantly lower acute LVEF than the regular reperfusion group (median LVEF [IQR] regular reperfusion, 37.4 [33.8 to 45.7] %; 12 min HBOC-pH-Glc-lns, 33.6 [30.1 to 41 .8] %; 17 min HBOC-pH-Glc-lns, 31.9 [24.7 to 35.6] %; p-value <0.05), Figure 3. At the segmental level (regional contractile function), animals in the 17 min HBOC-pH-Glc-lns group had worse regional contractile function immediately after reperfusion than regular reperfusion-group animals (Table 5). At 7 and 45 days after AMI, LVEF did not differ significantly between groups, see Table 6.
Table 5. Change in CMR-measured regional left ventricular wall thickening between baseline and 90 min (post MR) by study group (Protocol 2)
HBOC-pH-Glc-lns reperfusion group
Control (N=8) 12min (N=9) 17min (N=13)
Left ventricle segments
Basal anterior
Basal anteroseptal
Basal inferoseptal
Basal inferior
Basal inferolateral
Basal anterolateral
Mid anterior
Mid anteroseptal
Mid inferoseptal
Mid inferior
Mid inferolateral
Mid anterolateral
Apical anterior
Apical septal
Apical inferior
Apical lateral
Figure imgf000050_0001
*p-value <0.05 compared to control group. Table 6. CMR-derived parameters for the rate + duration combinations used for HBOC-pH-Glc- Ins reperfusion after myocardial infarction (Protocol 2)
HBOC-pH-Glc-lns reperfusion group
Control 12 min 17 min
Baseline CMR
Sample size 8 9 13
LVEDV index (ml/m2) 93.9 [89.4 to 96.1] 101.6 [100.9 to 105.9] * 106 [101.3 to 110.3] *
LVESV index (ml/m2) 38.8 [5.6 to 49.5] 49.8 [48.8 to 55] * 49.4 [46.6 to 56.8] *
LVEF (%) 57.6 [50.4 to 58.7] 51 [49.7 to 52.5] 51 [47.8 to 55.2]
Acute CMR (90 min)
Sample size 8 9 13
Change in LVEDVi from baseline -14.4 [-23.4 to -7] -11.3 [-24 to -4.9] -13.4 [-18 to -8.1]
Change in LVESVi from baseline 5.4 [-0.4 to 19.5] 9.7 [3.5 to 18.3] 14.4 [10.1 to 19.1]
LVEF (%) 37.4 [33.8 to 45.7] 33.6 [30.1 to 41.8] 31.9 [24.7 to 35.6] *
Change in LVEF from baseline -14.6 [-24.5 to -9.9] -18 [-19.7 to -9.1] -22.7 [-25.3 to -16.2]
Edema (%LV) 40.3 [36.5 to 45.2] 39.5 [36.4 to 54.4] 41.7 [37.3 to 48.1]
7-day CMR
Sample size 8 9 13
Change in LVEDVi from baseline 32 [24.7 to 39.5] 45.2 [33.9 to 52.2] 34.7 [27.2 to 52.5]
Change in LVESVi from baseline 34.1 [31.1 to 40.6] 34.7 [28.2 to 44.6] 31.1 [24.2 to 45.4]
LVEF (%) 41.6 [37.1 to 43.6] 41 [39.9 to 45.6] 39.7 [35.7 to 43.5]
Change in LVEF from baseline -15.5 [-16.3 to -8.1] -10.2 [-13.7 to -2.6] -13.3 [-14.2 to -9.6]
Edema (%LV) 31.3 [24.5 to 37.8] 34.6 [30.3 to 36.7] 34.7 [30.7 to 37.5]
Infarct size (%LV) 27.1 [24.2 to 34] 30.5 [29.4 to 37.5] 25.9 [22.6 to 36.6]
Infarct size (%Edema) 98.4 [77.6 to 100] 100 [87.5 to 100] 88.4 [68.9 to 100]
45-day CMR
Sample size 8 7 12
Change in LVEDVi from baseline 39.7 [28.9 to 46.8] 37.2 [25 to 69.3] 35 [20.7 to 72.3]
Change in LVESVi from baseline 33.4 [27.1 to 57.7] 42.3 [29.3 to 59.6] 28.6 [14.6 to 56]
Change in LVEDVi from 7-day 11.1 [-8.1 to 19.5] 4.3 [-11.7 to 20.3] -2.1 [-15.6 to 39.3]
Change in LVESVi from 7-day 5 [-14.1 to 21.8] 12.4 [-0.7 to 19.7] -5.5 [-14.2 to 28.7]
LVEF (%) 45.8 [31.7 to 47.6] 38.5 [30.6 to 40.8] 44.6 [37.1 to 46.3]
Change in LVEF from baseline -11.9 [-21.3 to -7.2] -12.6 [-20.4 to -7.4] -7.2 [-13.9 to -3.6]
Infarct size (%LV) 21.9 [15.2 to 22.9] 18.8 [14.7 to 23.4] 19 [15.4 to 23.2]
Data are shown as median [Q1 to Q3]; CMR, cardiac magnetic resonance; LVEF, left ventricular ejection fraction; LVEDVi: left ventricle end-diastolic volume index; LVESVi: left ventricle end-systolic volume index; *p-value <0.05 compared with control group (blood-only reperfusion).
The rationale for the present study was that intracoronary infusion of the clinically approved oxygen carrier, HBOC-201 , after prolonged ischemia (and before coronary blood-flow restoration) would provide an opportunity to modulate mechanisms known or believed to play a role in reperfusion-related damage, and thus potentially reduce IS. We observed that infusion of unmodified HBOC-201 (pH 7.6 - 7.9) resulted in massive microvascular obstruction, and that infusion of HBOC-201 solution with a physiological pH (7.35) without any modification produces larger infarcts than those in control animals reperfused by directly restoring coronary blood-flow (Figure 4). However, after infusion with HBOC-201 -pH solution enriched with a metabolic substrate (glucose/insulin), IS was the same as in control animals (although a non-significant trend for reduced IS (absolute IS and IS normalized to AAR) was observed in the HBOC-pH-GI-lns groups). Our study thus does not support the original hypothesis that IS would be reduced by initial reperfusion of the post-ischemic heart with an acellular oxygen-carrier. However, we believe that our results provide the basis for future studies into the administration of protective reagents before blood-flow restoration to prevent reperfusion-related damage. No further damage ensued from deferring blood-flow restoration for 17 min (35% longer "no blood flow" duration) with an intervening intracoronary infusion of a metabolically enriched HBOC solution, and this observation represents a novel opportunity to administer therapies to ischemic tissue before blood-flow restoration.
Accordingly, our results indicate that the injection of unmodified HBOC-201 after 45 min of ischemia results in an additional damage to the myocardium. Conversely, when the oxygen carrier HBOC-201 was modified (pH brought to physiological values of 7.35, and addition of metabolic substrate (glucose and insulin)), there was no sign of additional damage despite 35% prolongation of no blood flow conditions with respect to control mice (17 min). Even more, a non-significant trend for infarct size reduction was documented. These experiments illustrate that modified oxygen carrier administration is a feasible and safe strategy. Example 2.- Pre-oxygenated oxygen carrier solution as a more efficient myocardial delivery system
A pilot study was performed in 2 healthy pigs to evaluate the usefulness of pre-oxygenated oxygen carrier solutions as myocardial delivery vehicle in no blood flow conditions. The proof of concept was done by evaluating the efficiency of the modified pre-oxygenated HBOC-201 solution of Example 1 .3 (i.e., HBOC-pH-Glc-lns) in the myocardial delivery of adeno associated virus (AAV), which is often used as vector for gene therapy purposes. Neutralizing antibodies against AAVs have previously been reported to be present in the blood of primates. Accordingly, the authors further investigated whether administration of HBOC-201 prior to and together with a GFP-encoding AVV vector enabled to prevent the vector contact with the remaining blood after coronary occlusion and resulted in an improvement of the gene transduction efficiency into the cardiomyocytes.
2.1. Material and Methods
Green Fluorescent Protein (GFP)-expressing AAV vector AAV serotype 9 particles were produced in HEK293 cells. More specifically, an AAV shuttle vector (pAcTnTGFP) was used. It is derived from the pAcTnT vector (kindly provided by Dr B.A. French; Prasad et al., Gene Therapy 201 1 , 18(l):43-52) which contains AAV2-ITRs and carries the green fluorescent protein (GFP) reporter gene under the control of the chicken troponin promoter. The nucleic acid construct was packaged into AAV9 capsids by using the triple transfection method as previously described (Prasad et al., Gene Therapy 201 1 , 18(l):43-52; Gao et al., PNAS 2002, 99 (18), 1 1854-1 1859).
Intracoronary administration of the AAV solution
Healthy pigs (i.e. not in the context of infarction) were instrumented as detailed in Example 1 above. In summary, 2 castrated male (-30 kg) Large-White pigs were anesthetized, intubated and mechanically ventilated. Femoral artery was percutaneously accessed and a sheath was placed. Through the sheath, a guiding catheter was placed in the origin of the left coronary artery.
In the control pig (IC), the AAV solution was administered through the guiding catheter without stopping blood flow.
In the pre-oxygenated oxygen carrier pig (HBOC), a conventional 0.014-inch guidewire was advanced into the LAD coronary artery through the guiding catheter. A short, highly compliant over-the-wire balloon (Helios Occlusion Balloon Catheter, LightLab) was then placed in the LAD artery distal to the origin of the first diagonal branch and connected to the infusion pump. Preoxygenated HBOC-201 with the modifications detailed in Example 1.3 above (i.e., HBOC-pH-Glc-lns) was mixed with AAV solution. In the HBOC pig, immediately after the balloon catheter was inflated (coronary occlusion), the modified pre-oxygenated oxygen was administered for 5 minutes at an infusion rate of of 1 ml/kg/min (the normal coronary blood flow). Immediately after the oxygen carrier containing the AAV solution was injected distal to occlusion site through the balloon for 7 minutes at an infusion rate of of 1 ml/kg/min. After finishing the perfusion of the volume of the modified oxygen carrier with the AAV solution, the balloon was deflated and blood flow was restored. Total occlusion time was 12 minutes.
Analysis of GFP expression
RNA isolation
Myocardial tissue stored at -80°C was crushed using a pestle and mortar on dry ice. Powdered tissue was added to 1 ml of Trizol® reagent (Invitrogen). Tissue was homogenized (Qiagen tissue homogenizer) and incubated at room temperature for 5 minutes. 200 μΙ of chloroform was added to each sample and samples were inverted vigorously and incubated for 10 minutes at room temperature. Samples were then spun at 12,000g for 15 minutes at 4°C and the aqueous phase was placed in a clean tube. A total of 1 ml of 75% ethanol was added to the samples which were then shaken and centrifuged at 7500 g for 5 minutes at 4°C. The supernatant was carefully removed and the pellet was washed with 70% ethanol and again spun at 14,000g for 15 minutes at 4°C. The supernatant was removed and the pellet was air dried and resuspended in 20μΙ of RNase free water. cDNA synthesis
cDNA was synthesized using 100 ng of isolated total RNA per sample and the High Capability cDNA Reverse Transcription kit (Applied Biosystems). Samples were run at 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 minutes and held at 4°C until removed from thethermocycler.
Quantitative Real Time PCR
Quantitative Real Time PCR (qRT-PCR) was carried out on Applied Biosystems 7900. Fast real time PCR kit (Applied Biosystems) was used to prepare the samples with eGFP primers: EGFP Fw (SEQ ID N01 : CCAGGAGCGCACCATCTTCTT) and EGFP Rv (SEQ ID N02: GTAGTGGTTGTGGGCAGCAG). The results were exported from Applied Biosystems SDS software package and analyzed using LinRegPCR. Relative Units of expression were normalized with a porcine GAPDH probe (Ss03374854_g1 , Applied Biosystems).
2.2. Results
One animal received the AAV solution via regular intracoronary administration (IC, control). More specifically, a total of 1013 particles of an AAV9 vector carrying the GFP reporter gene under the control of the troponin promoter were administered in normal saline via intracoronary injection without obstructing native coronary artery flow, by infusing the AAV solution through the catheter placed at the origin of the coronary artery. The AAV solution is thus mixed with flowing blood.
For the pig receiving HBOC, the same amount of viral particles were resuspended in pre- oxygenated HBOC. Coronary artery blood flow was stopped and HBOC-AAV was injected distal to coronary occlusion immediately after occluding the coronary artery. Since pre- oxygenated modified oxygen carrier solution is injected immediately after occluding the coronary artery, this strategy does not induce any harm to the myocardium. As per the results in Example 1 , the modified pre-oxygenated solution has shown to be safe in healthy individuals and even after a prolonged ischemia.
Pigs were sacrificed 28 days later (the time where the expression of the injected AAV is anticipated to be maximal) and total RNA was extracted from left ventricular myocardial samples isolated from the apex. GFP expression in the myocardial samples was analyzed by qRT-PCR and normalized to that of Gapdh. As can be seen in Figure 5, the myocardial expression of AAV was significantly higher (p<0.01 ) in the animal treated with HBOC.

Claims

1 . An hemoglobin-based oxygen carrier (HBOC) composition for use in a method for the in vivo diagnosis and/or the therapeutic and/or preventive treatment of a myocardial disease; wherein said method comprises the following consecutive steps:
i. temporary occluding a coronary artery and/or a coronary vein;
ii. intracoronary administering the HBOC composition prior to and/or simultaneously to the administration of a preventive, therapeutic and/or diagnostic agent; wherein said HBOC composition has been pre-oxygenated; and
wherein said agent is a transgene encoding adeno-associated virus (AAV) expression vector .
The HBOC composition for use according to claim 1 ;
wherein said method comprises the following consecutive steps:
i. temporary occluding a coronary artery, and optionally, temporary occluding a coronary vein;
ii. administering into the coronary artery downstream to the site of occlusion the HBOC composition; and
iii. administering into the coronary artery downstream to the site of occlusion the diagnostic, prophylactic and/or therapeutic agent and the HBOC composition; wherein the HBOC composition in ii) is substantially free of the diagnostic, prophylactic and/or therapeutic agent administered in iii);
wherein in step iii) the agent and the HBOC composition are administered simultaneously; and
wherein said HBOC composition in steps ii) and iii) has been pre-oxygenated.
The HBOC composition for use in a method according to any of claims 1 or 2, wherein said oxygenated HBOC has about 80% by weight or greater of oxyhemoglobin.
The HBOC composition for use in a method according to any of claims 1 to 3, wherein the HBOC composition comprises hemoglobin intra/intermolecular crosslinked with glutaradehyde.
The HBOC composition for use in a method according to any of claims 1 to 4, wherein said composition comprises hemoglobin intra/intermolecular crosslinked with glutaradehyde, and further comprises NaCI from 100 to 130 mmol/L (preferably, 1 14 mmol/L), KCI from 2.0 to 6.0 mmol/L (preferably, 4.0 mmol/L), CaCI2-2H20 from 0.5 to 2.5 mmol/L (preferably, 1 .4 mmol/L), NaOH from 10.0 to 15.0 mmol/L (preferably, 12.5 mmol/L), sodium lactate from 20.0 to 35.0 mmol/L (preferably, 27.1 mmol/L), N-acetyl-L- cysteine from 10.0 to 20.0 mmol/L (preferably, 12.3 mmol/L) and water (preferably, water for injection).
6. The HBOC composition for use in a method according to any of claims 1 to 5, wherein said composition comprises:
- a metabolic substrate; and
- a pH modulator agent;
wherein the pH of said composition is from 6.8 to 7.4, preferably wherein the pH of said composition is 7.35.
7. The HBOC composition for use according to claim 6, wherein said metabolic substrate is glucose, preferably wherein said pharmaceutical composition further comprises insulin.
8. The HBOC composition for use according to any of claims 6 or 7, wherein said pH modulator agent is N-acetylcysteine. 9. The HBOC composition for use in a method according to any of claims 1 to 8, wherein said expression vector comprises the 5'ITR and 3'ITR sequences of the human AAV2 serotype.
10. The HBOC composition for use in a method according to any of claims 1 to 9, wherein said expression vector comprises the 5'ITR and 3'ITR sequences of an AAV2 serotype and sequences encoding the capsid proteins of an AAV9 serotype (an AAV2/9 vector).
1 1 . The HBOC composition for use in a method according to any of claims 1 to 10, wherein in step iii) the agent and the HBOC composition are formulated in a single composition.
12. The HBOC composition for use in a method according to any of claims 1 to 1 1 , wherein the coronary arteries and/or the coronary veins are occluded from 1 to 15 minutes, preferably for 12 or 13 minutes.
13. The HBOC composition for use in a method according to any of claims 1 to 12, wherein the HBOC composition is administered in step ii) during 5 minutes at a rate of 1 ml/kg/min.
14. The HBOC composition for use in a method according to any of claims 1 to 13, wherein the HBOC and the agent are part of the same composition and said composition is administered in step iii) during 7 minutes at a rate of 1 ml/kg/min.
15. The HBOC composition for use in a method according to claim 12, wherein the coronary arteries and/or the coronary veins are occluded from 1 to 5 minutes, preferably of 2 or 3 minutes.
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Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001070A1 (en) 1990-07-09 1992-01-23 The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce High efficiency packaging of mutant adeno-associated virus using amber suppressions
US5139941A (en) 1985-10-31 1992-08-18 University Of Florida Research Foundation, Inc. AAV transduction vectors
US5173414A (en) 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
WO1993003769A1 (en) 1991-08-20 1993-03-04 THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES Adenovirus mediated transfer of genes to the gastrointestinal tract
US6133425A (en) 1986-06-20 2000-10-17 Northfield Laboratories, Inc Acellular red blood cell substitute
US6177403B1 (en) 1996-10-21 2001-01-23 The Trustees Of The University Of Pennsylvania Compositions, methods, and apparatus for delivery of a macromolecular assembly to an extravascular tissue of an animal
US6699231B1 (en) 1997-12-31 2004-03-02 Heartport, Inc. Methods and apparatus for perfusion of isolated tissue structure
US20100173786A1 (en) 2007-06-01 2010-07-08 Commissariat A L'energie Atomique Method for Absolute Quantification of Polypeptides
US20100209532A1 (en) 2007-06-13 2010-08-19 Opk Biotech Llc Targeted oxygen delivery via intravenous or intra-arterial infusion of oxygenated polymerized hemoglobin solutions
US20110039287A1 (en) 2006-12-28 2011-02-17 Snow Brand Milk Products Co., Ltd. Method for quantification of peptide and protein
WO2012155019A1 (en) 2011-05-12 2012-11-15 Genentech, Inc. Multiple reaction monitoring lc-ms/ms method to detect therapeutic antibodies in animal samples using framework signature pepides
WO2013016598A2 (en) 2011-07-27 2013-01-31 Sangart, Inc. Methods for reducing reperfusion injury using a hemoglobin-based carbon monoxide carrier

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5139941A (en) 1985-10-31 1992-08-18 University Of Florida Research Foundation, Inc. AAV transduction vectors
US6133425A (en) 1986-06-20 2000-10-17 Northfield Laboratories, Inc Acellular red blood cell substitute
WO1992001070A1 (en) 1990-07-09 1992-01-23 The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce High efficiency packaging of mutant adeno-associated virus using amber suppressions
US5173414A (en) 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
WO1993003769A1 (en) 1991-08-20 1993-03-04 THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES Adenovirus mediated transfer of genes to the gastrointestinal tract
US6177403B1 (en) 1996-10-21 2001-01-23 The Trustees Of The University Of Pennsylvania Compositions, methods, and apparatus for delivery of a macromolecular assembly to an extravascular tissue of an animal
US6699231B1 (en) 1997-12-31 2004-03-02 Heartport, Inc. Methods and apparatus for perfusion of isolated tissue structure
US20110039287A1 (en) 2006-12-28 2011-02-17 Snow Brand Milk Products Co., Ltd. Method for quantification of peptide and protein
US20100173786A1 (en) 2007-06-01 2010-07-08 Commissariat A L'energie Atomique Method for Absolute Quantification of Polypeptides
US20100209532A1 (en) 2007-06-13 2010-08-19 Opk Biotech Llc Targeted oxygen delivery via intravenous or intra-arterial infusion of oxygenated polymerized hemoglobin solutions
WO2012155019A1 (en) 2011-05-12 2012-11-15 Genentech, Inc. Multiple reaction monitoring lc-ms/ms method to detect therapeutic antibodies in animal samples using framework signature pepides
WO2013016598A2 (en) 2011-07-27 2013-01-31 Sangart, Inc. Methods for reducing reperfusion injury using a hemoglobin-based carbon monoxide carrier

Non-Patent Citations (58)

* Cited by examiner, † Cited by third party
Title
ALAYASH A. I., TRENDS BIOTECHNOL., vol. 32, no. 4, 2014, pages 177 - 185
ALETRAS AH ET AL., CIRCULATION, vol. 113, 2006, pages 1865 - 1870
ANDOH ET AL., CURRENT PHARMACEUTICAL DESIGN, vol. 15, 2009, pages 2066 - 2073
BERNS; BOHENZKY: "Advances in Virus Research", vol. 32, 1987, ACADEMIC PRESS, INC., pages: 243 - 307
BLACKLOW, N. R ET AL., J. NATL. CANCER INST., vol. 40, 1968, pages 319 - 327
BOEKSTEGERS P ET AL., GENE THER., vol. 7, 2000, pages 232 - 40
BOEKSTEGERS P; KUPATT C., BASIC RES CARDIOL, vol. 99, 2004, pages 373 - 81
BOEKSTEGERS P; KUPATT C., BASIC RES CARDIOL., vol. 99, 2004, pages 373 - 81
BUDHANI MK, REGULATORY TOXICOLOGY AND PHARMACOLOGY, 2016
BUI ET AL., NAT. REV. CARDIOL., vol. 8, no. 1, 2011, pages 30 - 41
BURKHOFF D; LEFER DJ, AM HEART J, vol. 149, 2015, pages 573 - 579
BURMEISTER, BRITISH JOURNAL OF ANAESTHESIA, vol. 95, no. 6, 2005, pages 737 - 45
BYRNE ET AL., GENE THERAPY, vol. 15, 2008, pages 1550 - 1557
CABRALES P; INTAGLIETTA M, ASAIO J, vol. 59, 2013, pages 337 - 354
CARTER, B. J., CURRENT OPINION IN BIOTECHNOLOGY, vol. 3, 1992, pages 533 - 539
CASWELL, AM J PHYSIOL HEART CIRC PHYSIOL, vol. 288, 2005, pages H1796 - H1801
CHEN J-Y ET AL., CLINICS, vol. 64, no. 8, 2009, pages 803 - 13
CHIRMULE, N ET AL., GENE THER., vol. 6, 1999, pages 1574 - 1583
DIB ET AL., JACC: CARDIOVASCULAR INTERVENTION, vol. 3, no. 3, 2010, pages 265 - 275
DUBE G.P. ET AL., EUROLNTERVENTION J., vol. 4, 2008, pages 161 - 165
FERNANDEZ-JIMENEZ R ET AL., J AM COLL CARDIOL, vol. 65, 2015, pages 315 - 323
FERNANDEZ-JIMENEZ R ET AL., J AM COLL CARDIOL, vol. 66, 2015, pages 816 - 828
GAO ET AL., PNAS, vol. 99, no. 18, 2002, pages 11854 - 11859
GARCIA-PRIETO J ET AL., BASIC RES CARDIOL, vol. 109, 2014, pages 422
GARCIA-RUIZ JM ET AL., J AM COLL CARDIOL, vol. 67, 2016, pages 2093 - 2104
GEORGE, AM J PHYSIOL HEART CIRC PHYSIOL, vol. 291, 2006, pages H1126 - H1137
GREEN; SAMBROOK: "Molecular Cloning: a Laboratory Manual, 4th ed", 2012, COLD SPRING HARBOR LABORATORY PRESS
GREENBURG AG; KIM HW, CRIT CARE, vol. 8, no. 2, 2004, pages 61 - 4
HAUSENLOY ET AL., BASIC RES CARDIOL, vol. 111, 2016, pages 70
HAYASE M ET AL., AM J PHYSIOL HEART CIRC PHYSIOL., vol. 288, 2005, pages 2995 - 3000
JAHR JS ET AL., AM J THER, vol. 9, 2002, pages 437 - 43
JAHR JS; MOALLEMPOUR M; LIM JC, EXPERT OPIN BIOL THER, vol. 8, 2008, pages 1425 - 1433
KAPLITT MG ET AL., ANN THORAC SURG, vol. 62, 1996, pages 1669 - 76
KATZ ET AL., J MOL CELL CARDIOL, vol. 50, no. 5, 2011, pages 766 - 776
KOTIN, R. M., HUMAN GENE THERAPY, vol. 5, 1994, pages 793 - 801
LEBKOWSKI ET AL., MOLEC. CELL. BIOL., vol. 8, 1988, pages 3988 - 3996
LEVY J H, AMERICAN COUNCIL ON SCIENCE AND HEALTH, May 2009 (2009-05-01)
LEVY JH, ANESTHESIOLOGY, vol. 92, 2000, pages 639 - 41
LINTEL, AM J PHYSIOL HEART CIRC PHYSIOL, vol. 298, 2010, pages H1103 - H1113
LOGEART D ET AL., HUM GENE THER, vol. 11, 2000, pages 1015 - 22
LOGEART D ET AL., HUM GENE THER, vol. 12, 2001, pages 1601 - 10
M. RAUH, J CHROMATOGR B ANALYT TECHNOL BIOMED LIFE SCI, vol. 883-884, 1 February 2012 (2012-02-01), pages 59 - 67
MAGOVERN CJ ET AL., ANN THORAC SURG, vol. 62, 1996, pages 425 - 33
MELIGA, EUROLNTERVENTION, vol. 4, 2008, pages 99 - 107
MUZYCZKA, N., CURRENT TOPICS IN MICROBIOL. AND IMMUNOL., vol. 158, 1992, pages 97 - 129
NOLTE, ADV. CLIN. CHEM., vol. 33, 1998, pages 201 - 235
PARSA ET AL., SEMIN THORAC CARDIOVASC SURG, vol. 15, 2003, pages 259 - 97
PRASAD ET AL., GENE THERAPY, vol. 18, no. l, 2011, pages 43 - 52
PRASAD, GENE THERAPY, vol. 18, no. l, 2011, pages 43 - 52
RAAKE PW ET AL., GENE THER., vol. 15, 2008, pages 12 - 7
REMPF, BRITISH JOURNAL OF ANAESTHESIA, vol. 103, no. 4, 2009, pages 496 - 504
SAMBROOK; RUSSELL: "Molecular Cloning: a Laboratory Manual, 3rd ed", 2001, COLD SPRING HARBOR LABORATORY PRESS
TE LINTEL HEKKERT M ET AL., AM J PHYSIOL HEART CIRC PHYSIOL, vol. 298, 2010, pages 1103 - 1113
TRENDS BIOTECHNOL, vol. 32, no. 4, 2014, pages 177 - 185
VINCENT ET AL.: "Vaccines", vol. 90, 1990, COLD SPRING HARBOR LABORATORY PRESS
WHITE ET AL., GENE THERAPY, vol. 18, 2011, pages 546 - 552
WOLFRAM; DONAHUE, JOURNAL OF THE AMERICAN HEART ASSOCIATION, 2013, pages e000119
YANG, OXIDATIVE MEDICINE AND CELLULAR LONGEVITY, 2015

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