Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1858—Platelet-derived growth factor [PDGF]
  • A61K38/1866—Vascular endothelial growth factor [VEGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
  • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• Acute ischemia results from a sudden plaque that ruptures. If left untreated, coronary artery disease (CAD) progresses to worsening symptoms and morbidity and can result in myocardial infarction (MI) or death. It has been reported that as the population ages and as the incidence of obesity and diabetes approaches epidemic proportions, the number of patients with severe CAD will continue to grow.
• CAD coronary artery disease
• Treatment options for CAD and refractory angina include medical therapy, balloon angioplasty (with or without stenting), atherectomy and bypass surgery.
• Pharmacologic therapy is a mainstay of disease management for most forms of CAD and specifically for refractory angina.
• Pharmacological treatment includes first-line therapy with beta-blockers, calcium channel blockers, nitrates, second-line therapy with ranolazine and additional therapies to reduce the risk of MI and/or death including antiplatelet therapy, lipid lowering therapy, and angiotensin-converting enzyme (ACE) inhibitors.
• ACE angiotensin-converting enzyme
• CABG When the condition cannot be effectively treated with medicines or catheter-based angioplasty and stents, CABG may be recommended.
• CABG uses arteries and/or veins from other parts of the body to bypass the blocked coronary arteries on the surface of the heart. Despite the expense and the procedure- related patient morbidity and mortality, these procedures do not provide long-term relief of symptoms, and oftentimes repeat surgical intervention is required.
• Anatomic reasons which preclude current revascularization procedures include severe diffuse CAD, collateral- dependent myocardium, multiple coronary restenosis, chronic total coronary occlusions, degenerated saphenous vein grafts, poor distal targets or lack of conduits due to prior CABG in addition to a number of comorbidities.
• the invention provides a method of treating a cardiovascular disease in a subject in need thereof, the method comprising administering directly into the heart of the subject during Transthoracic Epicardial Procedure (TECAP) an effective amount of pharmaceutical composition comprising a viral vector comprising a therapeutic polynucleotide.
• TECAP Transthoracic Epicardial Procedure
• the pharmaceutical composition is administered through a series of 15 injections at separate delivery sites in the heart of the subject, and wherein the viral vector diffuses through substantially all of the heart.
• the viral vector is an adenoviral vector.
• the viral vector comprises a polynucleotide encoding one or more isoforms of VEGF.
• the heart of the subject is visualized throughout the procedure using a thorascope.
• a dose of the viral vector of about 1 x 10 9 vp, about 1 x 10 10 vp, about 4 x 10 10 vp or about 1 x 10 11 vp is administered.
• each injection has an injection volume of about 0.1 mL.
• the cardiovascular disease is coronary artery disease.
• the TECAP comprises making a 4-5 cm anterolateral incision in the 5th to 7th intercostal space of the subject.
• the injections are made in the left ventricle.
• FIG. 1 depicts a sample injection grid indicating spacing between injection sites in one embodiment of the invention.
• FIG. 2 depicts a schematic diagram of the study design for the study described in Example 1.
• FIG. 3 depicts the genetic structure of AdVEGF-A116A+. DETAILED DESCRIPTION
• an element means one element or more than one element.
• “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
• CAD coronary artery disease
• composition refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier.
• the pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, intramuscular, subcutaneous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
• an “effective amount” or “therapeutically effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.
• An “effective amount” of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.
• the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
• the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function.
• a pharmaceutically acceptable material, composition or carrier such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function.
• Such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body.
• Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient.
• materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline
• “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions.
• the “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the invention.
• Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.
• TECAP Transthoracic Epicardial Procedure
• treating a disease or disorder means reducing the frequency with which a symptom of the disease or disorder is experienced by a patient or improving patient ability to function.
• Disease and disorder are used interchangeably herein.
• treatment encompasses prophylaxis and/or therapy. Accordingly the compositions and methods of the present invention are not limited to therapeutic applications and can be used in prophylactic ones. Therefore “treating” or “treatment” of a state, disorder or condition includes: (i) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (ii) inhibiting the state, disorder or condition, i.e., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof, or (iii) relieving the disease, i.e. causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.
• VEGF refers to the gene or protein vascular epithelial growth factor.
• a person of skill in the art is familiar with VEGF and its isoforms. See, e.g., Yla-Herttuala et al Vascular Endothelial Growth Factors Biology and Current Status of Clinical Applications in Cardiovascular Medicine. J Am Coll Cardiol 2007;49:1015-26.
• vp or “viral particles” means as total number of functional (infectiouse) and non-functional (non-infectiouse) virus particles.
• ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
• the invention provides a method of treating a cardiovascular disease in a subject in need thereof, the method comprising administering directly into the heart of the subject a pharmaceutical composition comprising an effective amount of a viral vector comprising a therapeutic polynucleotide.
• Effective administration of VEGF to patients in need of treatment for cardiovascular disease has proven elusive. It has now been surprisingly discovered that direct injection of a viral vector comprising a therapeutic polynucleotide to the heart of a subject, thereby expresses the corresponding polypeptide in the heart of the subject, is a safe and effective method of treating cardiovascular disease.
• the cardiovascular disease may be any cardiovascular disease that may be treated by inducing angiogenesis in the subject’s heart.
• the cardiovascular disease is coronary artery disease.
• direct injection to the heart in various embodiments, allows lower doses of viral vector to provide greater benefit to the treatment of the subject and thereby avoids various disadvantages of systemic administration.
• Viral Vector comprising One or More Isoforms of VEGF
• the therapeutic agent delivered by the methods taught herein is a viral vector comprising a polynucleotide encoding one or more isoforms of VEGF and configured to express polypeptides corresponding to the one or more isoforms of VEGF in a tissue of the subject.
• the viral vector is an adenoviral vector.
• a person of skill in the art is familiar with various viral vectors that are suitable for the practice of the various embodimens of the invention.
• the viral vector is deficient in various genes that allow the wild type vector to replicate and therefore is suitable for gene transfer to a subject.
• the therapeutic polynucleotide encodes one or more of VEGF isoforms 121, 165 and 189. In various embodiments, the therapeutic polynucleotide encodes VEGF isoforms 121, 165 and 189. In various embodiments the viral vector is as described in U.S. Patent Nos. 6,518,255 and 7,368,553, each of which is hereby incorporated by reference in their entirety.
• the therapeutic agent is an adenoviral vector in formulation buffer in a suitable container closure system, such as vials, pre-filled syringes or other container.
• a suitable container closure system such as vials, pre-filled syringes or other container.
• the drug product can be in different presentations, such as lyophilized (freeze-dried) form or other physical state.
• the drug product can be composed of any combination of a drug and a device (a combination drug product) including robotic, semi- robotic or nonrobotic devices.
• the viral vector is AdVEGFXCl, a replication-deficient, recombinant human adenovirus serotype 5 (Ad5) viral vector with an expression cassette for human vascular endothelial growth factor (VEGF) that includes introns and splice sites to generate multiple naturally occurring isoforms of VEGF, including VEGF121,
• AdVEGFXCl a replication-deficient, recombinant human adenovirus serotype 5 (Ad5) viral vector with an expression cassette for human vascular endothelial growth factor (VEGF) that includes introns and splice sites to generate multiple naturally occurring isoforms of VEGF, including VEGF121,
• the VEGF expression cassette is inserted in the El region of the adenovirus backbone, which has deleted the adenovirus E1A and E3 genes, and partially deleted E1B.
• AdVEGFAl 16A+ is provided in SEQ ID NO: 1, and has been confirmed by Annotation for transgene region of the virus genome - provided in Table 1.
• AdVEGF-A116A+ The genetic structure of AdVEGF-A116A+ is shown in FIG. 3.
• the position of the human VEGF cDNA/genomic hybrid expression cassette is indicated by the grey arrow.
• Positions of the Ad early genes (E2A, E2B and E4), Ad late genes (LI, L2, L3, L4 and L5), inverted terminal repeats (ITR) and encapsidation signal (ES) are also indicated.
• SEQ ID NO: 1 DNA sequence of AdVEGFA116A+
• CTGCGAGAAG GCGTTGAGGC CTCCCTCGTT CCAGACGCGG CTGTAGACCA 11100
• the viral vector is administered by a series of injections during TECAP.
• the procedure comprises a series of intramyocardial injections to the left ventricle of the subject’s heart.
• the viral vector is administered through a series of 15 injections at separate delivery sites in the heart of the subject, wherein the viral vector diffuse through substantially all of the heart.
• each injection has an injection volume of about 0.1 mL.
• the heart of the subject may be visualized throughout the procedure using a thorascope. As described in more detail below, use of a thorascope to visualize the subject’s heart allows the administration of the viral vector through a minimally invasive procedure.
• a dose of viral vector between about 1 x 10 9 vp and about 1 x 10 11 vp is administered.
• a dose of the viral vector of about 1 x 10 9 vp, about 1 x 10 10 vp, about 4 x 10 10 vp or about 1 x 10 11 vp is administered.
• the TECAP comprises making a 4-5 cm anterolateral incision in the 5th to 7th intercostal space of the subject.
• the injections are made in the left ventricle.
• Example 1 The materials and methods employed in practicing the following examples are here described: Example 1:
• angiogenesis is a complex, normal physiologic process that includes the regulated proliferation and migration of endothelial cells, localized dissolution of the basement membrane/extracellular matrix at the site of the sprouting neovessel, the migration of endothelial cells and their coalescence into tube-like structures, the reformation of the surrounding basement membrane, and the formation of the new vessels into networks with linkage to an appropriate venous system.
• the physiologic process of angiogenesis involves several mediators that function to produce new vessels in an ordered fashion. VEGF is one of the key components that initiates this process.
• Myocardial administration of a gene coding for VEGF is a strategy using the delivery of genetic information to the myocardium to create networks of new blood vessels.
• the most direct method of transferring genes to the myocardium is by injection under direct vision. This can be accomplished by exposure of the myocardium through a thoracoscopy, left thoracotomy or sternotomy, or by minimally invasive surgery.
• the advantages of a direct injection strategy are the following: (1) compared with other delivery techniques, the highest levels of localized transgene expression can be achieved, (2) vectors can be delivered with a high degree of accuracy, (3) a number of targeted injections can be performed and (4) limited systemic spread of the vector occurs.
• VEGF is not only essential to the process of angiogenesis but, because it can be secreted from intact cells, it is ideal for gene transfer therapy aimed at improving perfusion to ischemic myocardium.
• VEGF DNA as plasmids or expressed by adenovirus gene transfer vectors
• Therapeutic angiogenesis mediated through a vector-delivered genetic message for an angiogenic factor has been studied in animal models and in clinical trials since the late 1990s. Studies have included genes for many protein angiogenic factors delivered by plasmid as well as by viral vectors (particularly adenovirus) and a variety of other administrative routes and delivery systems. In non-malignant tissues, the human VEGF gene is expressed in multiple isoforms, secondary to post-transcriptional splicing.
• the VEGF protein is capable of inducing angiogenesis, however, delivery of VEGF protein for therapeutic purposes has presented a significant challenge because the half-life of VEGF is very short, administration of high doses of VEGF is associated with hypotension and edema, and systemic administration of VEGF carries the theoretical risk of promiscuous induction of angiogenesis in tissues other than the target organ.
• the VEGF cDNA coding sequence can be used as the source of local VEGF at the site of administration.
• AdVEGF-All a precursor to XCOOl in which VEGF 121, VEGF 165 and VEGF 189 are expressed in an approximate 2:2:1 ratio, was shown to be more effective at inducing angiogenesis and hind limb blood flow than comparable vectors with cDNA for individual VEGF isoforms in the ischemic mouse hind-limb model.
• Administration of AdVEGF-All provided superior restoration of blood flow than did administration of the Ad vectors carrying cDNA coding for the individual isoforms across a specific dose range.
• the AdVEGF-All minimum effective dose (MED) based on muscle volume was approximately 10 4 to 10 5 vp (human equivalent dose [HED] ⁇ 10 8 to 10 9 pu).
• MED human equivalent dose
• This study demonstrated that a mixture of multiple Ad viral vectors each with a transgene expressing a single VEGF isoform or an Ad viral vector with a transgene coding for multiple VEGF isoforms provided a significant improvement in hindlimb flow ratio (angiogenic response) compared to administration of an Ad vector with cDNA for a single isoform. This supports the conclusion that these individual isoforms function synergistically, and that use of such a multiple-isoform drug may yield even better clinical efficacy than did the AdVEGF121 precursor.
• AdVEGF-All led to the investigation of the impact of administering an altered ratio of the major VEGF isoforms that could potentially provide further optimization of the safety profile of a candidate drug for clinical development.
• the drug candidate, XCOOl was constructed to increase the ratio of isoforms containing exon 6a.
• XCOOl was found to provide a potent angiogenic response in a similar fashion to VEGF-All but was found to have a better safety profile as measured by mouse mortality after IV dosing (no deaths were noted at doses of XCOOl that were approximately 10-fold greater than the highest proposed human dose), by slower tumor growth in a mass of Lewis lung carcinoma cells injected into mouse subcutaneous tissue after an IV injection of product, and by less pulmonary edema noted by lung weight after an intratracheal administration of AdVEGF vectors.
• liver VEGF levels may not fully explain it since liver VEGF levels were comparable between both groups.
• the mortality especially at the highest dose, may in part be due to the high amount of adenovirus that accumulates in tissues such as the spleen and liver after intravenous administration in mice. These two organs contain many immune cells, including liver Kupffer cells, splenic dendritic cells and macrophages. These cells have been assumed to be responsible for the production of inflammatory cytokines/chemokines that cause activation of an innate immune response which could lead to death. It should be noted that intramyocardial XCOOl delivery would be expected to result in less systemic product exposure than intravenous administration.
• a toxicology study was performed by the administration of XCOOl to the hearts of adult Fisher 344 rats.
• the study was comprised of 21 groups of 10 animals/group (5 males and 5 females).
• the in-life parameters (daily general health observations, clinical observations, and body weights) assessed throughout the study duration were used to help support the study objective from a clinical perspective. The results of these parameters revealed no significant differences between groups, and no findings were of clinical concern. Coagulation, clinical chemistry, and hematology results yielded no significant differences between groups and sexes. Human VEGF was not detected in rat plasma in the Day 8, Day 30, or Day 90 cohorts. Gross necropsy findings did not reveal any abnormalities attributed to a specific testing group. Organ weights and organ weight to terminal body weight ratios also revealed no significant differences between groups and sexes.
• XCOOl -related changes consisted of fibrosis of the myocardium in male and female rats in Groups 3 and 4. Mononuclear cells in the myocardium was also a common finding in hearts from rats on Day 90 and occurred in all treatment groups (including controls; Group 1). This is a well-recognized, age-related spontaneous finding in rats and was not caused by treatment with XCOOl. Observations on Days 30 and Day 90 cohort animals were consistent with resolution of XCOOl -induced inflammatory lesions found in the myocardium on Day 8. No other tissues analyzed in Day 8, Day 30, or Day 90 cohort animals revealed abnormalities specific to a testing group.
• Serum Cardiac Troponin I (cTnl) results did not demonstrate a correlation between dose escalation and increased cTnl values in Day 8, Day 30 and Day 90 cohorts. Since cTnl is a biomarker to indicate cardiac muscle injury, elevated serum cTnl values would be expected in those animals with an increased severity of chronic inflammation of the myocardium. However, this was not the case and in some instances the animals with the highest levels of cTnl had no to minimal inflammation of the myocardium.
• intra-myocardial administration of XCOOl was associated on Day 8 with chronic inflammation of the myocardium involving the free wall of the left ventricle (i.e. the site of experimental injection) that increased in incidence and severity with increasing dose of XCOOl.
• Observations on Days 30 and 90 were consistent with resolution of XCOOl -induced inflammatory lesions found in the myocardium on Day 8.
• all in-life parameters, clinical pathology, serum cTnl, plasma VEGF, and necropsy observations revealed no signs of clinical concern correlating to any of the testing groups.
• Adenovirus vectors have properties that make them ideal for the delivery of VEGF genes for therapeutic angiogenesis, namely, effective transduction of cardiovascular tissues, nonintegration into the human genome and short-term transduction. Most importantly, these vectors have an extensive track record for human gene therapy and a demonstrated safety profile at the doses being evaluated. Moreover, long-term safety (out to a median of 11.8 years post gene therapy) of VEGF isoforms with adenovirus delivered into the heart has been demonstrated.
• several attempts to use the gene encoding for VEGF in the clinic have met with limited success for a variety of potential reasons including ineffective delivery route, ineffective gene vectors, and poor choice of efficacy endpoint criteria:
• SPECT myocardial perfusion imaging has multiple limitations, including relatively long acquisition protocols and considerably poorer spatial resolution than other available modalities, limiting detection of sub-endocardial perfusion defects. Furthermore, the discordance of tracer uptake (tracer uptake does not correlate with myocardial blood flow) at higher myocardial blood flows limits sensitivity in detecting mild to moderate stenosis.
• time to 1 mm ST-segment depression as well as total exercise duration and time to moderate angina and in angina symptoms as measured by the CCS Angina Class and SAQ were all improved by VEGF gene transfer.
• Ad vectors are considered immunogenic, dose and route of administration are key factors to an immune response. Intravenous administration as opposed to an intramuscular injection would be expected to result in more systemic exposure and potentially a higher probability of an immune reaction.
• a large body of nonclinical and clinical data for Ad vectors yielding VEGF 121 and VEGF 165 transgene products with doses up to 4 x 10 10 vp has not elicited a clinically meaningful immune related safety trend or issue with follow-up to a median of 11.8 years.
• systemically administered Ad vectors are rapidly cleared from the blood of mice, with a half-life of less than 2 minutes, with large accumulation in the liver and spleen.
• liver Kupffer cells containing many immune cells, including liver Kupffer cells, splenic dendritic cells and macrophages and these cells have been assumed to be responsible for the production of inflammatory cytokines/chemokines responsible for activation of an innate immune response.
• shedding of vector or wild-type Ad was not detected in any sample (Days 2, 4, and 7) from any site (nose, throat, urine, and blood) in any subject.
• plasma VEGF levels were not above baseline values beyond Day 3 post administration.
• CAD is a chronic disease in which blood flow is obstructed through the coronary arteries that supply the heart with oxygen-rich blood leading to ischemia. Untreated, CAD usually continues to worsen. Many CAD patients have symptoms such as chest pain (angina) and fatigue, which occur when the heart is not receiving adequate oxygen. As many as 50% of patients, however, experience no symptoms until a heart attack occurs. CAD remains the leading killer of men and women in the world. Ischemic conditions of the heart require therapeutic intervention, including pharmacologic, coronary artery stenting and cardiac surgical bypass. However, there is a significant population with CAD who have refractory angina secondary to obstructive CAD, in which these interventions no longer will be effective or cannot be used.
• Ad vectors expressing individual isoforms of VEGF In marked contrast to Ad vectors expressing individual isoforms of VEGF, in preclinical studies with an ischemic hind-limb model XCOOl, an Ad5 vector expressing the cDNA/genomic hybrid of the VEGF gene, mediated nearly full recovery of blood flow at a dose of two logs less than required for the previous clinical vector AdVEGF121.
• XCOOl is not only closer to the natural expression of VEGF in the heart, but it is more powerful (per vector) than that used in prior clinical studies and is therefore likely to have an improved safety profile.
• the proposed Phase 1/2 clinical trial will be used to determine the safety and tolerability of direct administration of the vector XCOOl to the ischemic myocardium and to generate evidence regarding whether direct administration of XCOOl to the ischemic myocardium will induce growth of collateral blood vessels and improve cardiac function and QOL.
• XCOOl will be administered as a one-time therapy by TECAP to allow direct delivery of the vector to the target tissue compartment. This replicates the route of administration used in the nonclinical and clinical studies and data suggests this procedure is much more effective at delivering vector than intracoronary or endocardial catheter administration. Prior to the procedure, each subject will have their medical history, physical exam and other assessments reviewed by a team of cardiologists and cardiovascular surgeons for consensus on the suitability of the candidate for the trial.
• Ad vectors and VEGF isoforms 121 and 165 have an extensive track record for human gene therapy given intramyocardially with a demonstrated safety profile.
• long term safety (out to a median of 11.8 years after gene therapy) of VEGF isoforms with adenovirus delivered into the heart has been demonstrated (Table 5).
• Other isoforms containing exon 6a do not appear to have been studied in humans but as discussed above all exons except for exon 6 are represented in VEGF 165.
• VEGF 121 and VEGF 165 human experience and since exon 6a containing isoforms are naturally occurring and expected to have fewer systemic effects than VEGF 121 due to binding to the extracellular matrix, it is believed to be unlikely that XCOOl poses a safety risk beyond that of Ad vectors expressing VEGF 121 or VEGF 165.
• the proposed starting dose in humans 1 x 10 9 vp, is considered safe given the totality of nonclinical safety pharmacology and toxicology data with XCOOl.
• the safety of the second to fourth XCOOl doses (1 x 10 10 , 4 x 10 10 and 1 x 10 11 vp) is supported by the totality of the nonclinical and clinical experience of Ad vectors containing VEGF isoforms and by the XCOOl toxicology studies. Subjects will be monitored in the hospital for the first one to two days post XCOOl administration (or longer if deemed necessary). The XyloCor medical monitor will closely monitor all AEs/SAEs as they emerge.
• an internal safety group will review all available safety data, including the Day 7 visit of the latest subject dosed in the cohort, before any decision is made to dose another subject. If no adverse trends are observed, dosing of the next subject will commence.
• the AE/SAE profile may also require the external Independent Data Monitoring Committee (IDMC) to be part of the decision to dose the subsequent subject.
• IDMC Independent Data Monitoring Committee
• the IDMC will be reviewing all available safety data, including that of the third subject in the last cohort, up to and including their Day 7 visit, before any subsequent subject is dosed.
• the one-week dosing interval is considered appropriate given that potential safety findings from the procedure would have been expected to occur perioperatively and the kinetics of expression would have given peak systemic levels of the transgene product.
• Adding approximately 17-21 subjects to the highest tolerated dose will enable an examination of a set of loosely correlated outcome measures of ischemia (i.e., time to ST segment depression on exercise tolerance test; total perfusion deficit, myocardial blood flow and coronary flow reserve by PET; angina episodes; ischemic burden by ambulatory ECG, etc.) to make an assessment of preliminary evidence of efficacy.
• outcome measures of ischemia i.e., time to ST segment depression on exercise tolerance test; total perfusion deficit, myocardial blood flow and coronary flow reserve by PET; angina episodes; ischemic burden by ambulatory ECG, etc.
• This is a 6-month (with 6-month extension) Phase 1/2, first-in-human, multicenter, open-label, single arm dose escalation trial of XCOOl. No control group is included. Approximately 12 subjects (N 3 per cohort) who have refractory angina will be enrolled into 4 ascending dose groups (1 x 10 9 , 1 x 10 10 , 4 x 10 10 and 1 x 10 11 vp of XCOOl), followed by an expansion of the highest tolerated dose with approximately 17-21 additional subjects. XCOOl will be administered via TECAP directly to the free wall of the left ventricle of subjects.
• Eligibility Review Committee After qualifying for the study based on entry criteria and assessed by both the study cardiologist and surgeon, the Eligibility Review Committee (ERC) will review each candidate’s past medical history and screening assessments and formally clear each candidate for inclusion into the trial. Subjects will be monitored in the hospital for the first one to two days post XCOOl administration (or longer if deemed necessary). The medical monitor will closely monitor all AEs/SAEs as they emerge. Within cohorts, an internal safety group will review all available safety data, including the Day 7 visit of the latest subject dosed in the cohort, before any decision is made to dose another subject. If no adverse trends are observed, dosing of the next subject will commence.
• the AE/SAE profile may also require the external Independent Data Monitoring Committee (IDMC) to be part of the decision to dose the subsequent subject.
• IDMC Independent Data Monitoring Committee
• the IDMC will be reviewing all available safety data, including that of the third subject in the last cohort, up to and including their Day 7 visit, before any subsequent subject is dosed.
• the IDMC may recommend stopping the trial, dosing additional subjects at the current dose, proceeding to the next dose cohort, or proceeding by dosing additional subjects at a lower dose (further details are provided in the IDMC charter).
• the investigational product XCOOl is composed of the active ingredient AdVEGFXCl, a replication-deficient adenovirus serotype 5 vector containing a cDNA/genomic hybrid cassette coding for multiple isoforms of the vascular endothelial growth factor proteins.
• AdVEGFXCl active ingredient
• a replication-deficient adenovirus serotype 5 vector containing a cDNA/genomic hybrid cassette coding for multiple isoforms of the vascular endothelial growth factor proteins.
• Up to 4 doses will be studied: 1 x 10 9 , 1 x 10 10 , 4 x 10 10 and 1 x 10 11 vp of XCOOl.
• the route of administration will be one-time intramyocardial injections directly into the free wall of the left ventricle by TECAP.
• Total volume of investigational product administered will be 1.5 mL.
• IP will be delivered to the operating room as two sterile bags packaged in a non-sterile outer bag which will then be placed in a container for transport from Investigational Drug Service (IDS) to the operating room (OR).
• IDS Investigational Drug Service
• One sterile bag will contain the 14 syringes that are prefilled with 0.1 mL of prepared XCOOl, with the other sterile bag containing the 3 syringes prefilled with 0.2 mL of prepared XCOOl.
• Each sterile bag will be labelled according to institutional practice.
• the three prefilled syringes are slightly overfilled with 0.2 mL of IP to allow removal of any air bubbles and proper priming of the needle just prior to injection.
• Three 27-gauge spinal needles will also be provided.
• the injection volume will be 0.1 mL per each of the 15 intramyocardial injections distributed across the free wall of the left ventricle as described in further detail below.
• This protocol has an ascending dose escalation study design where a subject is assigned to 1 of 4 possible dose cohorts expressed as viral particles (vp) of AdVEGFXCl: 1 x 10 9 vp, 1 x 10 10 vp, 4 x 10 10 vp and 1 x 10 11 vp.
• vp viral particles
• the dose assignment and dilution worksheets will have been provided to the investigational drug pharmacist who will have the primary responsibility for receipt, short-term storage, thawing, dilution and prefilling the syringes according to Biosafety Level 2 (BSL-2) practices and usual institutional practice for parenteral sterile compounding that will include maintaining external sterility of each syringe and needle or syringe cap so that they may enter the sterile field in the operating room.
• BSL-2 Biosafety Level 2
• the site coordinator will alert the site Pharmacist as to when to start preparing IP (note that it may take up to 1.5 hours to prepare IP).
• the final investigational product will be provided in 3 mL borosilicate glass vials, with a fill volume of approximately 1.2 mL (extractable volume not less than 1.0 mL), sealed with latex-free stoppers and aluminum caps.
• Each cryovial will be labeled with the product name, concentration, fill volume and vial number; route of administration; statement “Caution: New Drug — Limited by Federal law to Investigational Use;” storage conditions; lot number; and manufacturer.
• the subject is placed in a 30° decubitus position supine with a roll under the back and the arm out 90 degrees on an arm rest to provide access to the pleural space from a more anterior approach and defibrillator patches are placed on the chest.
• the surgeon stands facing the subject’s heart with the camera-holding assistant on the same side when filming is utilized.
• the television monitor should be positioned so that the surgeon, the left ventricle of the subject and the monitor are aligned to allow the surgeon to look straight ahead when operating.
• This incision will typically be in the inframammary crease in women.
• a Tuffier Retractor (or any self-retaining retractor) is inserted. Adhesions from the lung and chest wall are taken down with electrocautery. A port may be inserted into the 7th or 8th intercostal space at the anterior axillary line for the thoracoscope if utilized or the thoracoscope can be inserted directly through the primary incision.
• the pericardium is then opened longitudinally 1 cm anterior to the phrenic nerve. If there has been a previous sternotomy, the pericardium may need to be dissected off of the epicardial surface to create a pericardial plane. This is typically easily performed, but injections may be performed trans-pericardially if dissection is deemed a prohibitive risk (needle depth and right angle placement should be adjusted accordingly).
• trans-pericardial injections can be undertaken with great care, adjusting the depth of the needle distal to the right-angle clamp to account for the pericardial thickness.
• the coronary arteries and veins should be avoided during injection. This can be facilitated by aspiration of the syringe prior to injection, which will also confirm that injections are not occurring in the ventricular chamber.
• the injections are then performed according to the procedure that follows. Once the procedure is completed direct intercostal nerve blocks could be considered with Exparel or Marcaine, and if thoracoscopy ports are placed complete cryoablation of intercostal nerves for pain control could be considered.
• the left ventricle should be blanketed with a total of 15 microinjections of 0.1 mL each of investigational product separated approximately 1.5 to 2 cm from each other (FIG. 1).
• the surgeon should emphasize injections in areas that are known to be ischemic based on all clinical information, where collateral vessel formation could potentially provide the greatest benefit. Clear cut areas of scar and thinning should be avoided.
• a long right-angled hemostat forcep is placed approximately 4-7 mm from the tip of the spinal needle to control depth of injection and to stabilize the needle over >5 beats to allow maximum absorption of investigational product. Obliquely aimed injections may help prevent less outflow when needle is removed.
• one of the prefilled syringes slightly overfilled with 0.2 mL will be used so that a 27-gauge spinal needle can be attached and primed to allow removal of any air bubbles and eliminate any dead space (only 0.1 ml is to be injected).
• the prefilled syringes (0.1 ml) will utilize the same 27-gauge spinal needle (with right-angled forcep attached). Blank injection maps will be provided to help with pre-surgery injection planning and for noting any issue with the injections in the OR.
• the heart should be inspected for any sites of needle hole bleeding. Digital pressure should be applied to any injection site with persistent bleeding. Once hemostasis is achieved, the thoracoscope is removed. A chest tube is then inserted through the port site or a separate incision and connected to a Pleurovac. The thoracotomy incision is closed in layers with absorbable suture and a sterile dressing placed over the incision. Anesthesia will be discontinued, and subject cared for following institutional guidelines for post-anesthesia care.
• a treadmill exercise protocol modified from the standard Bruce method to start at a lower workload than the standard test, will consist of multiple stages of progressively greater workloads created by increasing the percent grade and speed of the treadmill while monitoring cardiac function.
• testing will be performed twice during the screening period with each test separated in time by at least 72 hours or longer.
• the subject In order to be eligible for the trial, the subject must be able to exercise for 90 seconds to approximately 9 minutes while exhibiting > 1 mm horizontal or down-slopping ST segment depression on at least one of the tests, with the other test demonstrating > 0.5 mm ST segment depression.
• the ST segment requirement will apply to subjects in cohort 4, as well as the subjects in the expansion phase.
• the ST segment requirement will not apply for subjects in cohorts 1, 2 and 3. Subjects will be instructed to withhold taking anti-anginal medication the morning of their assessment if such medication is normally taken in the morning. Any short-acting NTG should be withheld within 4 hours of the assessment. If short-acting NTG is taken during this period or the patient is not in their usual state of health, the subject will be instructed to inform the site staff and reschedule the ETT.
• a detailed ETT protocol and independent review charter will be provided by a third-party, blinded ETT core laboratory with all the specifications on general requirements, staffing, equipment including maintenance and calibration, and test termination.
• the ETT core laboratory will be responsible for training and certification of the nurse or technician that will performing the test on subjects. Ideally, a primary nurse or technician and one back-up is identified for the duration of the trial.
• the ETT core laboratory interpretation (blinded assessor) and analysis of each test will be used for all efficacy analyses of the trial.
• the ETT core laboratory must review and approve the baseline paired ETT and confirm eligibility.
• PET scans will be performed using a whole-body PET scanner. Anti-hypertensives and beta-blockers, and calcium channel blockers will be withheld on the morning of the scan. Subjects will be allowed to continue using sublingual nitroglycerin as needed. Studies will be performed after 4 hours of fasting and 24 hours of abstinence from caffeine-containing products. The PET scan will take approximately 2.5 hours, including subject preparation.
• Myocardial perfusion will be assessed at rest and during maximal hyperemia using a standard adenosine or regadenoson infusion, and 13N-ammonia or 82Rubidium as the flow tracer.
• 13N-ammonia (-10-20 millicurie (mCi) or 82-Rubidium (-10-60 mCi)]
• list mode images will be acquired for approximately 19 minutes (13N-ammonia) or 7 minutes (82Rubidium). Fifteen or thirty minutes later, subjects will undergo a standard infusion of adenosine (0.14 mg/kg/min for 4 minutes) or regadenoson (0.4 mg bolus injection).
• a second dose of 13N- ammonia (-10-20 mCi) or 82Rubidium (-10-60 mCi) will be injected IV, and images recorded in the same manner.
• the heart rate, blood pressure, and 12-lead ECG will be recorded at baseline and throughout the infusion of adenosine or regadenoson, and at recovery.
• PET scans will be done for research (non-clinical) purposes only. For safety reasons, all PET scans will be reviewed locally by the site investigator, or his/her designee, for clinically important findings.
• the Screening PET will be read locally as part of the ERC packet. No reports or analyses will be provided to sites from the PET core laboratory and studies will not be assessed in real-time.
• the PET core laboratory will provide the following services: qualification of site equipment and study technologists; development of an imaging acquisition protocol and quick reference guide for study personnel; development of an independent review charter describing the processes, services and image interpretation; site technical training, certification and ongoing support during the conduct of the trial; tracking of imaging studies and quality review; quantitative analysis and independent overreading of all imaging studies as described below; and data management and data transfer services of the final data.
• a complete quantitative analysis of rest and stress myocardial perfusion PET images will include the following:
• Total Perfusion Deficit measures the total left ventricular perfusion deficit at rest (reflecting scarred myocardium) and during stress (reflecting both scarred + ischemic myocardium), as well as the difference between stress and rest (reflecting ischemic myocardium). TPD scores will be processed using standard software.
• LVEF left ventricular ejection fraction
• Ambulatory Electrocardiography Transient ST-segment deviation will be monitored by continuous ambulatory ECG for a period of 5 days as indicated on the Schedule of Assessments.
• the 5-day ambulatory ECG should be performed within the specified window of the nominal visit.
• the Day 1, or baseline, ambulatory ECG must be performed during any 5-day period in the screening period just prior to Day 1. Because most ischemic episodes during routine daily activities are related to increases in heart rate, it will be essential to encourage similar daily activities at the time of each ambulatory ECG recording.
• An ambulatory ECG monitoring core laboratory will provide an ambulatory ECG recorder in the form of an ePatch device.
• the core lab will also provide site technical training on its use and placement, a procedure manual and quick reference guide for study personnel, ongoing support during the trial, tracking of ambulatory ECG studies and quality review, blinded analysis in a written independent charter, data management and data transfer services of the final data.
• the criteria for an ischemic episode will be > 1 mm of horizontal or down-sloping ST segment depression lasting > 1 minute and separated from another episode by > 1 minute.
• the maximal depth of the ST segment depression during each episode will be noted to allow the calculation of an index of ST segment depression (mm) times duration (min) as the “total ischemic burden.”
• a motion biosensor device (activity tracker) will be provided to subjects to wear for 14 days to measure gross motor activity.
• the 14-day period should occur in advance of the study visit indicated in the Schedule of Assessments so that it concludes by the time of the visit and the device can be returned for interpretation by the actigraphy core laboratory.
• An actigraphy core laboratory will provide study personnel training and 24/7 technical service and support, study guide, device rental, motion assay services and data analysis in a written independent review charter.
• QOL Quality of Life
• the Seattle Angina Questionaire is the most sensitive, specific and responsive health- related quality of life instruction for coronary artery disease.
• the SAQ is self-administered and has been shown to be valid, reproducible and sensitive to clinical change.
• the SAQ quantifies subjects’ physical limitations caused by angina, the frequency of and recent changes in their symptoms, their satisfaction with treatment, and the degree to which they perceive their disease to affect their quality of life.
• Each scale is transformed to a score of 0 to 100, where higher scores indicate better function (eg, less physical limitation, less angina, and better quality of life).
• the instrument has 19 items that yields five subscale scores: physical limitation, angina stability, angina frequency, treatment satisfaction and disease perception. A change in 10 points in any of the subscales is considered to be clinically important.
• the EQ-5D-3L QOL instrument essentially consists of 2 pages: the EQ-5D descriptive system and the EQ visual analogue scale (EQ VAS).
• the EQ-5D-3L descriptive system comprises the following five dimensions: mobility, self-care, usual activities, pain/discomfort and anxiety/depression.
• the EQ VAS records the patient’s self-rated health on a vertical visual analogue scale.
• Clinical Global Impression is broken into two parts.
• Subjects will be given a paper diary to collect angina episodes and specifics about each episode (triggers, severity, treatments, etc.). There will also be a prophylactic nitroglycerine use diary. Subjects will record their anginal episodes as well as NTG use during the following intervals: 14-days prior to Day 1 visit to serve as the baseline with diary collected on Day 1; 14 days prior to the Month 3 visit with diary collected at the Month 3 visit; 14 days prior to the Month 6 visit with diary collected at the Month 6 visit; and the 2 week (14 days) period prior to the Month 12 visit. Diary collection should coincide with when the subject is wearing the activity tracker except for the Month 12 visit.
• Embodiment 1 provides a method of treating a cardiovascular disease in a subject in need thereof, the method comprising administering directly into the heart of the subject during Transthoracic Epicardial Procedure (TECAP) an effective amount of pharmaceutical composition comprising a viral vector comprising a therapeutic polynucleotide.
• TECAP Transthoracic Epicardial Procedure
• Embodiment 2 provides the method of embodiment 1, wherein the pharmaceutical composition is administered through a series of 15 injections at separate delivery sites in the heart of the subject, and wherein the viral vector diffuses through substantially all of the heart.
• Embodiment 3 provides the method according to any one of Embodiment 1 or Embodiment 2, wherein the viral vector is an adenoviral vector.
• Embodiment 4 provides the method according to any one of Embodiments 1-3, wherein the viral vector comprises a polynucleotide encoding one or more isoforms of VEGF.
• Embodiment 5 provides the method according to any one of Embodiments 1-4, wherein the heart of the subject is visualized throughout the procedure using a thorascope.
• Embodiment 6 provides the method according to claims 1-5, wherein a dose of the viral vector of about 1 x 10 9 vp, about 1 x 10 10 vp, about 4 x 10 10 vp or about 1 x 10 11 vp is administered.
• Embodiment 7 provides the method of Embodiment 2 wherein each injection has an injection volume of about 0.1 mL.
• Embodiment 8 provides the method according to any one of Embodiments 1-8, wherein the cardiovascular disease is coronary artery disease.
• Embodiment 9 provides the method according to any one of Embodiments 1-8, wherein the TECAP comprises making a 4-5 cm anterolateral incision in the 5th to 7th intercostal space of the subject.
• Embodiment 10 provides the method according to any one of emobidments 1-9, wherein the injections are made in the left ventricle.

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