Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
  • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/041—Heterocyclic compounds
  • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
  • A61K51/0453—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/0497—Organic compounds conjugates with a carrier being an organic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
  • A61K51/1258—Pills, tablets, lozenges
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2121/00—Preparations for use in therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2123/00—Preparations for testing in vivo

Definitions

• the present invention relates to compounds, compositions and methods for binding bio-affecting substances, such as therapeutic and, optionally, diagnostic agents, to bio-compatible particles, including both viable cells and viruses, and non- viable carrier particles.
• bio-affecting substances such as therapeutic and, optionally, diagnostic agents
• This invention is also directed to starting materials and intermediates used in the preparation of such compounds.
• the invention also relates to the use of the compounds for site- selective delivery of therapeutic and, optionally, diagnostic agents in vivo .
• the delivery of therapeutic agents for the treatment of diseases and other pathological conditions may be accomplished by various means. These include oral, intravenous, subcutaneous, transdermal, intramuscular administration or topical application.
• the existing modes of delivery either are unable to deliver sufficient dosages to the disease site without adverse systemtic side effects or are unable to allow sufficient retention of the therapeutic product at the disease site for a time sufficient to produce the intended therapeutic effect.
• Drugs that prevent or reduce the proliferation of pathological cell types are essential to the treatment and control of various diseases involving undesirable or uncontrolled cell proliferation.
• antiproliferatives by definition, must be toxic to certain cell types. It is often not feasible to administer these drugs systemically, because the amounts needed to control the diseased cell types may be toxic or deadly to the patient's normal cells. This difficulty could be circumvented by administering antiproliferative agents directly to the site of the undesired cell proliferation.
• a mechanism is also needed for retaining antiproliferative agents at the disease site, so that they may effectively control the proliferation of undesired cells, while being restrained from migrating and damaging normal cell types.
• Specific diseases and conditions for which site-specific delivery and retention of anti ⁇ proliferatives would be particularly effective are briefly described below. Each of these conditions involves the proliferation of a particular undesirable cell type-, and systemic administration of drug therapy for their treatment has not yielded optimal results.
• Atherosclerotic lesions which limit or obstruct coronary blood flow, are the major cause of coronary heart disease-related mortality.
• Direct intervention has been employed via percutaneous transluminal coronary angioplasty (PTCA) or coronary artery bypass graft.
• PTCA percutaneous transluminal coronary angioplasty
• coronary artery bypass graft percutaneous transluminal coronary angioplasty
• a major difficulty with PTCA is the problem of post-angioplasty closure of the vessel, both immediately after PTCA (acute reocclusion) and in the long term (restenosis) .
• Restenosis after angioplasty is a response to injury of the interior arterial wall caused by the angioplasty procedure. While the exact mechanism is still under active investigation, in general, it appears to involve proliferation of smooth muscle cells of the arterial medial layer, followed by migration of these cells to the inner (intimal) layer, where cells continue to proliferate. Proliferation usually ceases within the intima within 7-14 days post-injury.
• Heparin and Heparin Fragments Heparin is a highly anionic heterogeneous glycosaminoglycan consisting of repeating disaccharide units of ⁇ -D-glucuronic acid and N-acetyl-D- glucosamine which are extensively sulfated.
• the primary therapeutic use of heparin is as an anticoagulant.
• heparin is also known to inhibit the growth of several different cell types, including vascular smooth muscle cells (SMC) .
• SMC vascular smooth muscle cells
• Heparin binds to SMC cell surfaces via high affinity binding sites and is taken up intracellularly. Heparin can also be coupled to various artificial surfaces, such as silicone. Mylar®, Dacron®, polycarbonate, polyethylene and polypropylene, through ionic association with a tridodecyl methylammonium chloride coating. See Grode et al., Trans. Am. Soc. Artif. Int. Organs, 15: 1 (1969) .
• Colchicine is a naturally occurring alkaloid used in the therapeutic control of acute gouty arthritis. Colchicine arrests plant and animal cell division both in vitro and in vivo by preventing mitotic spindle fiber formation, thus arresting cell division in metaphase. This action of colchicine is similar to that of the vinca alkaloids, vincristine and vinblastine. Recently, it has been reported that colchicine administered daily to rabbits with atherosclerotic iliac arteries reduced the degree of restenosis observed on angiography at four (4) weeks post-balloon injury. Currier et al., Circulation, J3 , 11-66 (1989) .
• ACE Angiotensin Converting Enzyme
• Angiopeptin C. Lundergan et al. , Am. J. Cardiol., __YT_ (Suppl. B) : 132B-136B (1991)
• Cyclosporin A L. Jonasson et al., Proc. Natl. Acad. Sci., 8J5: 2303-06 (1988)
• goat anti- rabbit platelet-derived growth factor antibody G.
• systemic drug delivery such as oral administration
• oral administration involves periodic administration at fixed intervals, and consequent cyclic variations in concentration of the therapeutic agent at the disease site.
• greater than 99% of the drug ingested is typically processed through the liver or kidney, depending on the drug. This represents an opportunity for serious adverse reactions.
• Rheumatoid arthritis is a chronic disorder characterized by chronic synovitis of the joints.
• synovectomy involves the removal of inflamed soft tissue in the affected joints.
• N. Gschwend Textbook of Rheumatology, (eds. W.N. Kelly et al.), W.B. Saunders, pp. 1934-1961 (1989) .
• Synovectomy can be achieved surgically, chemically or radio-pharmaceutically. Surgical synovectomy has been demonstrated to have a palliative effect on pain. In most cases, however, there is a recurrence of the synovitis in the years following the surgery.
• Radioisotope synovectomy as an alternative to chemical synovectomy, appears to inhibit synovial proliferation.
• the use of radioisotope synovectomy is limited because, insofar as is known, delivery systems used to date require prolonged immobilization of the affected joint or use of very short-lived and commercially unfeasible isotopes to reduce to acceptable levels systemic release of radioisotope to the lymph nodes, spleen or liver.
• the leakage of radioisotope from the site of synovectomy is the primary concern in the development of this procedure for early treatment of rheumatoid arthritis.
• Ovarian cancer is the fourth leading cause of cancer death in women.
• Epithelial carcinomas account for approximately 80% to 90% of ovarian malignancies.
• Epithelial carcinomas spread through the body primarily by surface shedding or lymphatic spread. The most common type of extra-ovarian spread is transperitoneal dissemination of cells shed from the surface of the primary tumor.
• Chemotherapy is the most common form of treatment of patients with advanced ovarian cancer. Drugs currently used against ovarian cancer may prolong life by a few years. However, they present very significant side effects at the recommended systemic dose levels.
• Psoriasis is a common, chronic skin disease that progresses into an uncontrolled growth of skin keratinocytes, creating inflammation and ulceration. No comprehensive cure for psoriasis is available to date.
• Topical treatments include anti-bacterial or anti-fungal preparations, tars, phototherapy using sun exposure or ultraviolet light, or the application of topical steroids.
• Topical corticosteroids are used for psoriasis more than any other.therapeutic modality.
• topical treatments suffer the disadvantage of being easily rubbed or washed off, thus impairing their long-term efficacy.
• Use of topical corticosteroids is also limited by their tendency to penetrate into the peripheral blood vessels and thence into the general circulation, causing undesirable systemic buildup.
• a mode of administering pharmacotherapy which would permit greater concentration and retention of the therapeutic agent at the disease site without serious side effects, may prove useful in treating the .above-described conditions.
• Recent research efforts have focused on the utility of specific biological interactions, e.g. receptor-ligand or antigen-antibody interactions, in the development of target-specific therapeutic or diagnostic agents.
• Another promising area of research involves target-specific cells or vesicles (e.g., liposomes) containing an appropriate diagnostic or therapeutic agent.
• monoclonal antibodies have been used to impart target specificity to these cells or vesicles. Because it is a relatively new technology, such methods of targeted drug therapy are not yet truly effective or reliable.
• compositions are utilized for exerting a site-specific predetermined effect in vivo by stably binding the compound to a selected bioparticle having a natural or acquired affinity for the predetermined site and introducing the bioparticle in vivo, whereby the bioparticle carries the bio- affecting substance to the predetermined site to produce its intended effect.
• compounds having the capability of binding therapeutically active substances to lipid containing bioparticles, e.g. , cells or viruses.
• the compounds of the invention include a bio-affecting moiety, comprising a therapeutically active substance which is stably linked via a linking moiety to at least one hydrocarbon substituent selected so as to render the compounds sufficiently non-polar that they are capable of stable binding to lipid components of lipid- containing bio-compatible particles either in vivo or in vitro.
• the compounds optionally include a spacer moiety to provide separation between the therapeutic substance and the linking moiety, as required to mediate therapeutic activity.
• the compounds of the invention are further characterized by having varying but predictable stabilities of association with the lipid component of biomembranes.
• the compounds are sufficiently non- polar as to have a surface membrane retention coefficient (MRC) of at least .90% and a membrane binding stability of at least 30%.
• MRC surface membrane retention coefficient
• the compounds of the invention should also be sufficiently stable in use that the therapeutic agent, once delivered to the selected site, either by direct administration or via a carrier, remains there for a time and in an amount sufficient to produce its intended effect. Procedures for determining membrane retention coefficient and membrane binding stability are described in detail hereinbelow.
• the above-described compounds are used for site- selective delivery of therapeutic agents, and retention thereof at the selected site.
• the therapeutic agent has an anti-proliferative action, useful for the treatment of diseases or other pathological conditions involving cell proliferation.
• the anti-proliferative agent may comprise a radiotherapeutic substance or a chemotherapeutic substance.
• the present invention provides compounds wherein the radiotherapeutic substance comprises a chelating agent and a radiometal.
• the compounds of the invention comprise chemotherapeutic substances such as heparin, hirudin and derivatives thereof, as well as agents capable of interfering with selected intracellular functions.
• chemotherapeutic substances may be releasably conjugated to compounds of the invention.
• Such chemotherapeutic substances may be selected, for example, from the group consisting of colchicine, vinca alkaloids, taxol and derivatives thereof, which exhibit their bio-effect only upon release from the compounds. These compounds interfere with tubulin synthesis assembly, dissassembly or degradation, and/or function, which are hereinafter collectively referred to as "tubulin processes".
• tubulin processes for example, there is provided an acid-cleavable colchicine derivative, in which the colchicine analog incorporated therein is essentially inactive as long as it remains conjugated to the linker moiety. The compound is delivered to a selected site and eventually taken into the cell.
• the compounds of the invention are preferably administered by direct in vivo delivery for retention at the disease site.
• the compounds may be bound to carrier particles adapted to direct the compound to the disease site.
• Direct in vivo delivery is particularly preferred for treatment of conditions such as post-angioplasty restenosis, rheumatoid arthritis, ovarian cancer and psoriasis, and will be described in further detail hereinbelow.
• the present invention possesses a number of distinct advantages as compared with compounds and methods currently available for delivery of therapeutic agents to disease sites. Most notably, compounds of the invention may be delivered and retained at a selected site in the body by stable association with cell structures at that site.
• Another notable advantage of the present invention is that compounds may be formulated such that they are initially stably associated with the external cell membranes and subsequently taken up into the cell through normal membrane trafficking processes.
• This feature coupled with the ability to formulate compounds of the invention comprising releasably conjugated therapeutic agents, allows for the delivery of potentially toxic substances to selected cell interiors, where they may become activated and exert their therapeutic effect on those cells alone and not on other cells of the body.
• the delivery of antisense RNA or DNA may also be accomplished in this manner.
• a further advantage of this invention is that binding of the compounds described herein to cells and other bio-compatible particles occurs primarily through lipid affinity.
• the lipid regions comprise the majority of the cell surface area, it is possible to place larger numbers of lipid binding compounds, and thus a greater concentration of therapeutic agent, into the plasma membrane.
• the compounds of the invention are stably incorporated into membrane lipids due to their lipophilic character, they are relatively insoluble in normal physiological salts. Accordingly, once these compounds are bound to the membrane, they are effectively trapped there and cannot dissociate easily. Consequently, leakage from the cells is minimized, thereby minimizing undesirable systemic effects.
• Fig. 1 shows the results of tests comparing the vascular response of locally injected Substance P versus a substance P-lipophilic cyanine conjugate of the invention.
• the upper graph pertains to Substance P; the lower graph pertains to the conjugate;.
• Fig. 2 shows the dose-response relationship of each of Substance P and a Substance P-lipophilic cyanine conjugate of the invention, both in the absence and in the presence of the Substance P antagonist, [D-Pro 2 , D-Trp 7 - 9 ]-SP;
• Fig. 3 is a series of fluorescence/frequency histograms wherein the points represent individual cells; increasing distance of the points from the origin on the x axis represents increasing red fluorescence intensity (LFL2 signal) , whereas increasing distance of the points from the origin on the y axis represents increasing green fluorescence intensity (LFL1 signal) ;
• Fig. 4 is a graph representing the in vivo binding stability of a Substance P-lipophilic cyanine dye conjugate of the invention to red blood cells, determined as a function of time.
• Fig. 5 is a bar graph representation of the degree of retention of radioiodinated ( 125 I) lipophilic cyanine on four different types of artificial surfaces; silastic rubber (SIL) , polycarbonate (PC) , polyvinylchloride (PVC) and polyethylene (PE) . Stippled bars represent the amount of radiiodinated ( 125 I) lipophilic cyanine bound initially to each surface; solid bars represent the amount remaining after 6 hours of continuous blood per usion. Values associated with paired bars are the percentages retained of the initial amount of compound bound; and Fig.
• SIL silastic rubber
• PC polycarbonate
• PVC polyvinylchloride
• PE polyethylene
• FIG. 6 shows data from an experiment wherein carrier cells labeled with anti-coagulant lipophilic cyanine conjugate were tested for ability to inhibit the in vitro generation of fibrin produced by a standard concentration of thrombin.
• concentration response for unconjugated anti-coagulant is provided for comparison.
• Percentage inhibition of thrombin response (y-axis) is recorded as a function of the molar concentration of compound tested (lower x-axis; log scale) , and of the number of carrier cells per test sample (upper x-axis; log scale) .
• Bio-affecting moiety The terms bio- affecting moiety and bio-affecting substance are used interchangeably herein to refer to a wide variety of different substances useful in the therapeutic, diagnostic, prophylactic or other treatment of humans or animals. These include any substances capable of exerting a biological effect.
• Bio-compatible particle includes both viable entities, e.g. cells, both in vivo and in vitro, as well as non-viable entities, such as liposomes and lipoproteins, so long as they do not give rise to a serious adverse reaction upon administration in vivo.
• viable bio-compatible particle capable of physiological function is used herein to refer to any viable cell or membrane- containing virus.
• the term "cell” includes prokaryotic cells, such as bacteria, as well as eukaryotic cells, such as white blood cells, various tumor cells, and mammalian cells in culture, e.g. Chinese hamster ovary cells, yeast, and non-nucleated cells, such as red blood cells, red blood cell ghosts and platelets.
• prokaryotic cells such as bacteria
• eukaryotic cells such as white blood cells, various tumor cells, and mammalian cells in culture, e.g. Chinese hamster ovary cells, yeast, and non-nucleated cells, such as red blood cells, red blood cell ghosts and platelets.
• non-viable entities may be suitably substituted for viable entities provided they have a natural or acquired affinity for the intended delivery site.
• liposomes may be used as carriers for therapeutically active substances to be delivered to the liver or spleen.
• Diagnostic Agent refers to a compound or substance capable of facilitating the detection, determination or analysis of a physiological condition or state by an in vivo or in vitro test.
• the compounds of the invention may serve a dual function as reporter molecules that may be detectable from outside the body, or may be detected in a body fluid or biopsy obtained for analysis in vitro.
• Chromophore refers to a substance capable of being detected, either visually or instrumentally, by absorption of at least one selected wavelength of light.
• Therapeutically active substance refers to a substance capable of preventing, alleviating, treating or curing abnormal or pathological conditions of the living body. These include substances capable of maintaining, increasing, decreasing, limiting or destroying a physiologic body function, as well as substances for protecting a living body by inhibiting, killing, modifying or retaining a microorganism or antigen thereof. Therapeutically active substances include pharmaceutical agents or drugs with therapeutic utility.
• Chemotherapeutic substance refers to a therapeutically active substance whose therapeutic effect arises from the chemical characteristics of the • substance.
• Chemotherapeutic substances may include, for example, non-radioactive pharmaceuticals. They may include small molecules or more complex molecules such as lipids, carbohydrates, proteins or nucleic acids such as DNA or RNA.
• Radiotherapeutic substance refers to a therapeutically active substance whose therapeutic effect arises from its radioactivity. Suitable radiotherapeutic substances may comprise radioisotopic atoms.
• the bio-affecting moiety comprises a chelating agent complexed with various therapeutic radionuclides, such as 16 Re, 90 Y, 67 Cu, 177 Lu or l53 Sm.
• Antiproliferative agent refers to a therapeutically active substance capable of arresting, reducing or preventing the proliferation of cells.
• the therapeutically active substance is an antiproliferative agent, which may be a radiotherapeutic or chemotherapeutic substance.
• the compounds of the invention may comprise a diagnostic agent, such as a chromophore or radionuclide, which enables tracking and/or detection of the compound of the invention in vitro or in vivo.
• compounds of the invention may comprise, for example, a therapeutically active substance as the bio-affecting moiety and a detectable chromophore as the linking moiety.
• the diagnostic agents constituting the compounds of the invention may be selected from diverse classes of substances that are detectable by various analytical procedures known to those skilled in the art.
• Detectable fluorescent compounds are preferably cyanine dyes and their derivatives, including, e.g. oxycarbocyanine, indocarbocyanine, thiocarbocyanine or acridine dyes and derivatives thereof.
• Other useful fluorescent compounds include, for example, styrylpyridine, xanthene, phenoxazine, phenothiazine or diphenylhexatriene dyes and derivatives thereof.
• Useful diagnostic agents may also include ligands which facilitate detection, such as biotin or specific antibodies.
• Other useful diagnostic agents are chelating substances complexed with metals, which may be directly or indirectly detectable.
• a suitable chelate-metal complex may comprise an isotope selected from the transition metal series whose atomic number is from 21-49, e.g. Indium-Ill or Technetium-99m.
• Such complexes may be bound to the cell plasma membrane of carrier cells, rendering them radioactive so as to permit imaging using a gamma camera after delivery into the body.
• Chelating substances may also be complexed with an ionic species of metal which is indirectly detectable, e.g. by reason of certain effects produced thereby at the site of interest.
• Complexes of paramagnetic elements are capable of influencing the relaxation times of nearby nuclei, which is detectable by magnetic resonance imaging (MRI) .
• MRI magnetic resonance imaging
• Chelate-metal complexes comprising a metal ion selected from the transition metal series whose atomic number is 21-29 or the lanthanide series, whose atomic number is 59-66 may be suitable for such purposes.
• Compounds comprising radioisotopic atoms may also be used, if desired, in the various applications of this invention.
• a radioisotope such as 125 I, I31 I 14 C, 3 H, 35 S or 75 Se may be substituted for the more abundant but non-radioactive forms of the naturally occurring atoms present in the bio-affecting moiety, chromophore or the hydrocarbon tail portion.of the compound.
• Isotopes having non-zero spin states e.g., I9 F
• I9 F may also be introduced into the compounds of the invention, so as to make their presence detectable using MRI techniques.
• the bio- affecting moiety may comprise a chelating agent of the type described above, complexed with various therapeutic radionuclides, such as 186 Re, 90 Y or 67 Cu.
• Proteinaceous substances including proteins, glycoproteins, lipoproteins or peptides may also be coupled through suitable linking moiety to hydrocarbon tails of appropriate lengths for therapeutic applications in accordance with the present invention.
• Representative bio-affecting proteinaceous substances are immunogens, toxins, hormones, enzymes, antigens, antibodies and antibody fragments.
• Such therapeutically active proteins are beneficially conjugated to lipophilic chromophores, of the type described above, with the resultant conjugate being marked by varying but predictable stability of association with a variety of lipid-containing bioparticles, as exemplified hereinbelow.
• the bio- affecting moiety comprises a carbohydrate capable of altering the migration and circulation patterns within the body of cells to which it is bound.
• One class of carbohydrates applicable in this way includes sialic acids; another includes the glycosaminoglycans.
• a formulation comprising a sialic acid could be applied to the plasma membrane of red cells to increase the number of sialic acids on the membrane. The increase in the number of charged groups should increase the lifetime of the red cells in circulation before removal in the liver.
• the bio-affecting moiety may also be in the form of a ligand capable of binding to tissue-specific receptors or receptors on cells within target organs.
• a ligand capable of binding to tissue-specific receptors or receptors on cells within target organs.
• Compounds of the invention can be delivered directly to selected sites in the body by a variety of means, including injection, infusion, catheterization and topical application, among others.
• Compounds of the invention also may be bound to carrier bio- compatible particles, e.g., autologous, allogenic or zenogenic cells, to facilitate targeted delivery of the bi ⁇ -affecting substance.
• carrier bio- compatible particles e.g., autologous, allogenic or zenogenic cells
• the discussion set forth below refers to binding of compounds of the invention to viable cells, either by direct delivery to the disease site, or in the preparation of carrier vehicles.
• One objective of the present invention is to provide compounds to which therapeutic drugs or radioisotopes may be attached and which are soluble in the lipid bilayer that constitutes the outer membrane of cells.
• the mode of action of a compound of the invention on a cell is variable and depends on: (1) the type , of cell to which it attaches; (2) the nature, length and number of the lipophilic tails; (3) the body site being treated; (4) the nature of the bio- affecting moiety; and (5) the mechanism by which the bio-affecting moiety being attached to the compound produces its effect upon the cell.
• a compound of the invention is deposited on.the cell and initially attaches to the outer lipid bilayer of the plasma membrane. Because membrane components naturally traffic inward, these compounds will also be taken inside the cell. The rate at which this occurs will vary depending on the particular cell type, its growth state and its level of activation or stimulation.
• antiproliferative agents such as radiotherapeutic substances conjugated to a compound of the invention
• Some types of antiproliferative agents will be active whether they are localized on the outer membrane or within the cell. ' These agents emit radiation which damages the nucleus and, if the radiation is sufficiently penetrating, will inhibit growth of surrounding cells as well. Drugs that must physically interact with components inside the cell to inhibit growth will be effective only after the compound of the invention is taken into the cell.
• Another mode of action has also been designed whereby extremely toxic drugs, such as colchicine, can be conjugated to a lipophilic moiety and bound to the outer membrane in an inactive form which is not toxic to the cell. Then, as the conjugate traffics inwardly, a specially prepared linkage (described in greater detail in the following examples) allows the drug to be released from the conjugate and to exert its antiproliferative action.
• B represents a bio-affecting moiety comprising a therapeutically effective substance
• R and R represent substituents which are independently selected from the group of hydrogen, alkyl, alkenyl, alkynyl, alkaryl or aralkyl, the hydrocarbon chains of which are linear or branched, and which are unsubstituted or substituted with one or more non- polar functional groups, at least one of R and Ri comprising a hydrocarbon substituent, the chain length of which is effective to impart membrane binding capability to the compound
• R 2 represents a spacer moiety, n being 0 or 1
• non-polar functional group refers to substituents such as O- alkyl, S-alkyl, halogen, N(alkyl) 2 , Se-alkyl, N0 2 , CN, CO-alkyl, Si(alkyl) 3 , O-Si(alkyl) 3 , and the like.
• the linking moiety (L) may be a saturated or unsaturated aliphatic linker or a ring structure, including alicyclics and aromatics, which may be monocyclic, polycyclic, homocyclic, heterocyclic, fused or unfused.
• the linking moiety is a chromophore.
• Incorporation of a chromophore in the compounds of the invention facilitates tracking of the compounds in vivo.
• Useful chromophores for this purpose include: cyanine, acridine, pyridine, quinoline, xanthene, phenoxazine, phenothiazine and diphenyl hexatriene dyes and derivatives thereof.
• a preferred class of compounds within the scope of the invention are those of the formula:
• R and R x represent substituents independently selected from the group of hydrogen, alkyl, alkenyl, alkynyl, alkaryl or aralkyl, the hydrocarbon chains of which having from 1 to about 30 carbon atoms, and being linear or branched, said substituents being unsubstituted or substituted with one or more non- polar functional groups
• R 2 represents a spacer moiety of the formula: -(R 3 ) p -Q-(R-Q') q -(R 5 -Q") r -(R 6 - Q'"),-(R 7 Q* ' ' ') t/
• R 3 represents an aliphatic hydrocarbon
• X and Xj may be the same or different and represent 0, S, C(CH 3 ) 2 or Se;
• Y represents a linking group selected from
• R g is selected from H, CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 or CH(CH 3 ) 2 ;
• Z represents a substituent selected from the group H, alkyl, OH, -O-alkyl, COOH, C0NH 2 , S0 3 H, S0 2 NH 2 , CONH-alkyl, CON-(alkyl) 2 , NH-acyl, NH-alkyl, N(alkyl) 2 , SH, S-alkyl, N0 2 , halogen, Si(alkyl) 3 or O-Si(alkyl) 3 , Sn(alkyl) 3 or Hg-halogen, the alkyl groups comprising said Z substituent having from 1 to 4 carbon atoms; and A represents a biologically compatible anion.
• R or R j in formula (II) should have at least 12 carbon atoms and the sum of linear carbon atoms in R and R, should total at least 23.
• Various spacer moieties (R 2 ) may readily be incorporated between the cyanine head group and the bio-affecting moiety via suitable functionalities present on either or both the head group or the bio- affecting moiety, following well known synthetic routes.
• a large number of different types of bifunctional (homobifunctional and heterobifunctional) spacer reagents have been reported in the technical literature for such purpose. See Meth. Enz., 9_1: 580- 609 (1983) .
• spacer moieties differ according to type(s) of reactive groups, hydrophobicity or hydrophilicity, length and whether the structure connecting the reactive groups is cleavable or not.
• B represents a chelating agent complexed with a radiometal, such as rhenium or yttrium, among others.
• a radiometal such as rhenium or yttrium
• B represents heparin, hirudin, colchicine, vinblastine or analogs thereof, all of which are antiproliferative agents.
• B represents a peptide.
• a derivative of the peptide known as Substance P, falling within formula II above, has been found to bind stably to red cells and to provide a protracted therapeutic effect in circulation, as compared with the free, i.e. unconjugated, peptide. It is anticipated that enhanced bioavailability can be similarly achieved for other therapeutically active substances having relatively short lifetimes in circulation when in unconjugated form.
• B in formula I may advantageously be biotin.
• R, R X, X j , Y, Z and A are as defined above with reference to the compounds of formula II; and R 2 represents a spacer moiety of the formula:
• the amino cyanines (8) can be directly attached to therapeutic agents containing suitable functionalities (e.g., CO, COOH) to produce conjugates useful for site-specific drug delivery.
• suitable functionalities e.g., CO, COOH
• the amino cyanines (8) are very versatile intermediates for the preparation of a wide range of other cyanine derivatives capable of being conjugated to other bio-affecting moieties.
• compound (8) is an ideal molecule for reaction with a large number of homo- and hetero-bifunctional spacer moieties, to provide separation between the lipophilic cyanine moiety and the bio-affecting substance linked thereto.
• the amine substituent on the resultant cyanine derivative may be converted to other functionalities in a straight forward manner using well known reaction schemes.
• conversion to isothiocyanate functional groups may be achieved by treatment with thiophosgene according to the procedure of de Costa et al., J. of Lab. Compds. and Radiopharm.. 27 (9): 1015 (1989).
• Reaction of the amine group with 1,4- terephthalic acid di-N-hydroxysuccinimide ester provides a succinimidyl functional group joined to the cyanine head group by an OOC-aryl-CONH-alkyl spacer moiety.
• the resultant compound can be isolated and purified as a stable crystalline solid.
• Simple acylation of the substituent amino group with p-nitrophenyl iodoacetate (commercially available) in dimethylformamide provides a reactive iodo-substituent coupled to the cyanine head group by an alkyl-CONH-alkyl spacer moiety.
• Treatment of the amine group with N- hydroxysuccinimidyl-3(2-pyridyldithio)propionate provides a compound having a pyridyldithio functional group and an alkyl-CONH-alkyl spacer moiety.
• Treatment of the amine group with 7- thiobutyrolactone provides a compound having a sulfhydryl functional group and an alkyl-OCNH-alkyl spacer moiety.
• treatment of the amine group with 7-butyrolactone provides a compound having a hydroxyl functional group and an alkyl-CONH-alkyl spacer moiety.
• a lipophilic cyanine precursor prepared as generally described above, may be linked to a protein or peptide via a number of different synthetic routes, to yield compounds of formula II.
• Coupling of proteins or peptides to lipophilic linker derivatives may be through amine groups present on the proteins or peptides (i.e., terminal ⁇ -amino groups or e amino groups of lysine) .
• the carboxy1 group or thiol group may be used to couple proteins or peptides to appropriate cyanine precursors. Suitable conditions for carrying out such coupling reactions are known to those skilled in the art.
• Conjugation by way of the e-aliphatic amino group of lysine is best performed above pH 8.5, at which only a limited fraction of amines are, by equilibrium, unprotonated and reactive.
• This amino group should have high reactivity for several of the amine-reactive lipophilic linker compounds described above.
• isothiocyanate derivatized linker compounds exhibit high stability in aqueous conditions and can react with lysine side chains to form conjugates linked via a thiourea bond.
• N-hydroxy-succinimidyl ester derivatized linker compound is a particularly good reagent for reaction with lysine, since the amide conjugates thus formed are very stable.
• This reaction is preferably performed under anhydrous conditions in organic solvents, such as dimethylformamide, since hydrolysis of this particular derivative under aqueous conditions is a competing side reaction.
• a reaction whereby an N-hydroxy-succinimidyl ester derivatized linker of formula III, above, is conjugated to the amino group of the lysine residue at position 3 of the undecapeptide, substance P, is described in detail hereinbelow.
• Carboxylic acid groups present in the side chains of glutamic and aspartic acid residues, as well as on the C-terminal amino acid residue of peptides or proteins, are possible sites for selective conjugation using the above-described amine derivatized lipophilic cyanine compounds.
• This reaction may be performed by using either a water soluble carbodiimide, such as 1- ethyl-3-dimethylamino-propylcarbodiimide (commercially available) , according to a modification of the procedure of Hoare and Koshland, J. Biol. Chem..
• the compounds of the invention may be purified by various standard purification techniques making use of the size, charge or lipophilicity of the particular compound formed.
• a purification method which is particularly useful for therapeutic agents comprising peptides is that of Bohlen et al. , Int. . Rept. Prot. Research, 16: 306-10 (1980) .
• biotinylated Lipophilic Cyanines The biotinylated lipophilic cyanine compounds of the invention preferably are of the formula:
• biotinylated derivatives can be prepared via the reaction of biotin or a biotin derivative with a functionalized lipophilic cyanine compound of formula III, above.
• the biotin derivative used in this reaction preferably is of the formula:
• amino functionalized cyanine derivatives (8) of the type prepared according to Reaction Scheme 1 can be reacted according to the procedure of Hofmann, Finn and Kiso, J. Am. Chem. Soc.
• the effect of such a spacer arm may be important depending on the intended application of the biotinylated compound.
• a longer spacer arm has been shown to have a beneficial effect on the ability of the biotin conjugate to bind to the "deep" binding site of biotin in avidin.
• a variety of other biotin derivatives with reactive functional groups, such as maleimido, ⁇ - iodoacetamido, hydrazino and amino are commercially available (Molecular Probes, Eugene, OR, Handbook of Fluorescent Probes and Research Reagents, 1989-91) and could be used to couple with various of the above- described functionalized lipophilic linker compounds. Suitable reaction schemes will be apparent to those skilled in the art.
• spacer moieties could be incorporated between the cyanine and biotin moieties via suitable functionalities on both precursors. Reaction schemes for the incorporation of such spacers are known to those skilled in the art.
• Compound (13) can then be prepared from (12) by the procedure of H. Azizian et al. , J. Qrganomet. Chem. , 215: 49-58 (1981) , which involves heating (12) with bis-(tri-n-butyltin) in the presence of a catalyst, tetrakis-(triphenylphosphine) palladium (0) .
• the tributylstannyl derivative (13) can then be readily radiohalogenated under mild conditions by, for example, a modification of the procedure of Wilbur et al., J. Nuc. Med.. 30: 216-226 (1989). Introduction of radiohalogens into (12) may also be achieved by solid phase exchange using variations on the procedure of Weiss et al., J. Labelled Cmpds. & Radiopharmaceuticals. XXVI: 109-10 (1989) .
• An alternative lipophilic cyanine derivative which may be used for the introduction of radiohalogens is an analogue of (13) in which the - Sn(Bu) 3 group is replaced by -HgX, X representing halogen.
• the ring position of the halogen substituent in compound (12) may be varied by appropriate selection of starting materials.
• 6-iodo-2-methyl benzothiazole prepared according to the procedure of Bassignana et al., Spectrochemica Acta. , 19 (11) , 1885 (1963) , may be used to provide the corresponding 6-iodo derivative.
• Colchicine-cyanine conjugates with an acid cleavable linkage may be prepared by selecting a suitably functionalized active colchicine derivative
• Coupling via a cis-aconityl linkage may be achieved using the procedure described by Shen et al., Biochem. Biophvs. Res. Commun. , 102 3: 1048-1054 (1981) .
• This procedure involves coupling a free amino derivative of the drug (e.g., desacetyl colchicine) and an amino form of the lipophilic cyanine with cis- aconityl anhydride (commercially available) .
• Coupling via an acetal, orthoester or ketal linkage may be achieved using modifications of the procedures described by Srinivasachar et al. , Biochemistry. 28. 2501-2509 (1989) with appropriately functionalized colchicine and cyanine derivatives.
• Coupling via a hydrazone linkage may be performed using modifications of the procedure described by Laguzza et al. , J. Med. Chem.. 32: 548- 555 (1989) .
• This procedure involves coupling an aldehyde form of the drug with a hydrazide form of the lipophilic cyanine.
• the amino functionalized cyanine (8) is coupled with the monomethyl ester of glutaric acid (commercially available) to furnish the methyl ester derivative (14) which, upon treatment with hydrazine in methanol, provides the hydrazino derivative (15) .
• the colchicine moiety is prepared by reacting deacetyl colchicine (commercially available) with glutaric anhydride to form an acid intermediate which is then activated in situ by the addition of carbonylidiimidazole to form an acyl imidazole which, upon reduction with tetrabutyl ammonium borohydride, provides alcohol (17) .
• Oxidation of (17) with pyridinium chlorochrornate produces 7-N-(5- oxopentanoyl)deacetyl colchicine (18) which is then coupled with the hydrazino derivative (15) to furnish conjugate (19) in which the colchicine and cyanine moieties are coupled via an acid cleavable acyl hydrazone bond.
• the 7-N-(5-oxopentanoly)deacetyl colchicine produced as an intermediate in Scheme 3 is a novel compound constituting part of the present invention. If desired, a methylthio, or other chalcogen-containing group, may be substituted for the methoxy group at the 10 position of the colchicine nucleus.
• the more potent 7-N-(5- oxopentanoyl)deacetyl thiocolchicine analogue can be made from deacetylthiocolchicine (prepared as described by Shian et al., J. Pharma. Sci., 64, 646- 648 (1975)) using the same reaction sequence shown in Scheme 3 for the preparation of aldehyde (18) .
• This derivative of thiocolchicine can also be attached to the hydrazino derivative (15) using the same coupling conditions to provide an acid cleavable conjugate.
• the kinetics of release of the colchicine analogue from the conjugate can also be varied by modifying the type of hydrazone bond between the colchicine and cyanine moieties.
• antibody-drug conjugates coupled via a sulfonylphenyl hydrazone and phenyl hydrazone bond have been described by Mueller et al., Bioconjugate Chem.. 1 : 325-330 (1990) to provide slower release kinetics than the corresponding acyl hydrazone derivatives.
• Heparin-Lipophilic Cyanine Conjugates A synthetic route for a heparin-cyanine conjugate, coupled via a stable carbamate bond and which is useful in the present invention is shown in Reaction Scheme 4, and described in greater detail in the examples below.
• the isocyanate derivative (20) is not isolated or purified further and is then reacted immediately with sodium heparin in a mixed solvent of dimethylformamide/ formamide according to a modification of the procedure of Dong, "Heparinized Segmented Polyurethane Urea Surfaces with Hydrophilic Spacer Groups", Dissertation, University of Utah, 1990 to provide heparin-lipophilic cyanine conjugates (21) in which heparin is covalently attached to the cyanine through its hydroxyl group via carbamate bond formation (or through amino groups via urea bond formation) .
• a carbon spacer arm may also be incorporated between the heparin and cyanine moieties using the commercially available reagent, N-Boc-6- aminohexanoic acid N-hydroxysuccinimide ester, via reactions known to those skilled in the art.
• the number of cyanine groups per heparin molecule can of course be varied by controlling the stoichiometry of reagents in the reaction since heparin has a number of free hydroxyl groups available.
• Heparin-lipophilic cyanine conjugates may be purified from free heparin, if necessary, by -hydrophobic interaction chromatography.
• a heparin-cyanine conjugate may be prepared using a modification of the procedure described by Kin et al. , Nonthromboqenic Bioactive Surface Annals. New York Academy of Sciences, p 116- 130. This procedure involves coupling the carboxylic acid groups on heparin with an amino functionalized cyanine using a carbodiimide reagent. It has been determined by Ebert et al. , Biomaterials: Interfacial Phenomenon and Application. Adv. Chem. Ser. 99, American Chem. Soc. , Washington, D.C. (1982), that up to 20% of the carboxylic groups on heparin can be derivatized with no loss in bioactivity.
• Bifunctionalized polyaminocarboxylate chelants of structure (25) can be prepared according to procedures described in European Patent Publication Number 0353450 Al. According to Scheme 6, chelant
• M(PA-DOTA) which is used in the illustration of Scheme 6, refers to the radiometal complex comprising the polyamino-carboxylate chelator and a portion of the spacer moiety (-(CH 2 ) 2 -C 6 H 4 -NH-) in intermediate (26) shown in Scheme 6.
• the 3- dimensional structure of the radiometal complex moiety of compound 27 is expected to be similar to that described by Spinlet et al., Inoron. Chem.. 23: 4278-
• the number of linear carbons in the hydrocarbon tail(s) substituted on the compounds of the invention is an important factor in achieving the desired degree of stable association between the compounds and the surface membranes of bio-particles.
• the linear number of carbons should be 23 or greater.
• cyanine derivatives prepared in accordance with the present invention indicates that in compounds having two or more hydrocarbon tails, one of the tails should have a linear length of at least 12 carbons, with the sum of the linear carbon atoms in the hydrocarbon tails being at least 23.
• hydrocarbon tails having more than 30 linear carbon atoms may pose a problem because the bio-affecting moiety and the reactant used to provide the hydrocarbon tail may not be soluble in the same solvent, making the chemistry of joining the hydrocarbon tail to the bio-affecting moiety quite difficult.
• Positively charged linking moieties for example, cyanine, styrylpyridine, e xanthene, phenoxazine, phenothiazine or diphenylhexatriene dyes and derivatives thereof, may contribute to incorporation and retention of the compound in negatively charged membranes.
• Neutral or negatively charged linking moieties may also be useful in achieving controlled release from bio-membranes.
• Stable association between the linking moiety, the hydrocarbon tail(s) and the spacer moiety, or bio-affecting moiety, as the case may be, is essential to achieving site-selective retention of the therapeutically active substance for the requisite time and in the requisite amount to realize the therapeutic benefit which this invention provides.
• the linking moiety (L) should be selected so as to impart to the compounds of the invention the level of stability required so that the compounds will be present at the selected site of delivery for a time, and in an amount sufficient to achieve the desired therapeutic benefit.
• Compounds having the requisite associative stability imparted by the linking group can be determined on the basis of time of retention, in the case of direct administration of a therapeutically active, lipophilic conjugate, or time of circulation, in the case of delivery of the conjugate to the disease site via a carrier. That is to say, the time of retention or time of circulation of the therapeutically active, lipophilic conjugates of the invention must be greater than the time of retention or time of circulation of the therapeutically active component in unconjugated form.
• Determination of time of retention or time of circulation may be carried out in various ways known to those skilled in the art, as exemplified hereinbelow in Examples 9, 10 and 12, among others. Determination of the amount of therapeutically active, lipophilic conjugate of the invention that is required to produce the desired effect, as is often the case with therapeutic agents, is not subject to a specific procedure. This is necessarily so, due to the diversity of therapeutically active substances that may be used in the practice of this invention, the numerous disease states that are treatable therewith and the variability of the condition of the patient receiving therapy. Accordingly, the response of a patient receiving therapy in accordance with this invention will have to be monitored periodically to determine that the amount of therapeutically active substance present or accumulated at the disease site is sufficient to produce the desired therapeutic benefit.
• the compounds of the invention can be designed to bind to the outer membrane of viable cells or other bio-compatible particles without initial detrimental effect on viability, or they may be designed to exert an immediate or delayed cytostatic or cytotoxic effect.
• cytotoxic bioaffeeting moiety for example, cells are exposed to a compound of the invention at a variety of concentrations, including zero concentration as well as to a compound that is not conjugated to a cytotoxic agent. The cells are then exposed to trypan blue or propidiu iodide (F. Celada et al., Proc. Natl. Acad. Sci.. 57: 630 (1967)). These dyes are normally excluded by a living cell and only permeate the membrane of a dead cell. After the appropriate incubation time, the cells are examined with a microscope or by flow cyto eter and the percentage of stained cells (percent dead) is determined.
• Binding of the compounds of the invention to carrier cells should also exert no appreciable detrimental effect on cell functions which are important to their ability to perform as carriers for target delivery. For example, it may be important for the carrier cell to divide in order for it to perform in a given application. On the other hand, the compound used may alter some function having no effect on the division potential or other performance requirement of the cell for the contemplated application. Hence, such compounds may be considered to be without appreciable detrimental effect on cell function for purpose of its use in this invention. Procedures for determining the effect on cell functions of potential importance to the practice of this invention, produced by compounds of the invention are described in U.S. Patent No. 4,859,584, in Slezak and Horan, Blood.
• the cell binding medium must (i) be isotonic for the bio-compatible particle to which the compound is to be bound and at an iso- osmotic concentration (approximately 260-340 mOs moles for mammalian cells) so as to not cause shrinkage or swelling and possible damage to the cells and (ii) allow for the compounds of the invention to be solubilized in such a manner that they are available at consistent concentrations to incorporate into the plasma membrane of the cells. Solubility time course experiments (U.S. Patent No. 4,783,401 and M.
• the amount of the compound of the invention in the supernatant of said iso-osmotic solution at each time point is compared to a sample of such compound using ethanol as a solvent, which, although not suitable for labeling cells, serves to allow for the maximum compound solubility (total) .
• the primary goal is to incorporate as much of the therapeutic agent into the cell membrane as possible. This can be achieved by direct delivery of the therapeutic compound to the disease site or by binding the therapeutic compound to cells ex vivo and reintroducing the modified cells in vivo.
• the amount of compound incorporated into the cells should increase only to such a level that no negative alterations are noted in the carrier cell with respect to viability or capability of the cells to migrate to the desired location.
• Compounds of this invention are applied to carrier cells or other bio-compatible particles in the absence of serum and other lipid-containing materials.
• Cells are removed from the body or taken from culture and washed to be free of serum. They are suspended to form a composition including the iso-osmotic regulating agent, generally not in ionic solutions, and an appropriate concentration of a compound of the invention (10 *5 to 10" 7 M) . Binding of the compound to the cells is generally complete within ten minutes and the binding reaction may be stopped with the addition of autologous or heterologous serum.
• the cells are then washed in serum-containing media (5-10% v/v) and placed into culture or injected into the recipient depending on the application.
• Another cell binding technique involves suspension of the compounds of the invention in saline to allow for micelle formation.
• the cells are then placed into the resulting suspension and the phagocytic cells (for example, monocytes, macrophages and neutrophils) will preferentially become labeled.
• the phagocytic cells for example, monocytes, macrophages and neutrophils
• the compounds of the invention can be used to deliver radiation therapy to the site of disease.
• Cells are labeled with a compound of the invention by first forming a stable complex of a chelating compound of the invention and an appropriate radioactive ion (e.g., ⁇ Cu, 90 Y, I86 Re, alpha emitters), isolating the complex and following the general cell binding procedure described above.
• an appropriate radioactive ion e.g., ⁇ Cu, 90 Y, I86 Re, alpha emitters
• Tumor infiltrating lymphocytes may be isolated from a primary lesion, expanded in IL-2, bound to a radiotherapeutic substance as described above and injected intraveneously.
• the labeled cells track to the site of metastatic disease, and emit radiation which kills the metastatic tumor cells, thereby increasing the therapeutic effectiveness of the TILs, and perhaps decreasing the number of cells required to obtain disease regression.
• other cell types which migrate to metastatic sites may be utilized for delivery of localized radiation therapy.
• the compounds of the invention have incorporated therein proteinaceous substances, including proteins, glycoproteins, lipoproteins or. peptides as the bio-affecting moiety. These compounds are bound to cells as described supra, whereupon the hydrocarbon chains of said compounds become embedded into the plasma membrane, thereby placing the protein onto the surface of a specific cell type.
• the procedure described above may be used to bind monoclonal antibody to human fibrin to the surface of a carrier cell, e.g., red cell for delivery to the site of a fibrin clot.
• a carrier cell e.g., monocyte or lymphocyte.
• Tissue plasmihogen activator may be similarly delivered by application to the surface of a carrier cell (e.g., red cell).
• a monoclonal antibody which binds to human fibrin may be bound to the surface of a cell (e.g. , red cell) , which is then also bound with a fibrinolytic compound (e.g., tPA, Streptokinase, urokinase) .
• a fibrinolytic compound e.g., tPA, Streptokinase, urokinase
• therapeutically active proteins may be conjugated to a lipophilic chromophore in accordance with the general preparative procedures described above.
• compatible particles such as cells, viruses, liposomes or LDLs, thereby substantially prolonging the bioavailability of the proteinaceous substance in circulation.
• the protein-bound bio-compatible particles may also be isotopically labeled, as described above, using a radio-imaging compound or a magnetic resonance imaging compounds.
• the resultant bioparticle may be injected into a patient whereby the cell migrates to the disease site, which can be imaged using standard gamma scintigraphy or nuclear imaging to assess the effect of the therapeutic agent .
• an immunogen to which protective antibody production is desired, which may be a protein, glycoprotein, lipoprotein or peptide, is used as the bio-affecting moiety, optionally including a linking group, for binding to the surface of a cell (e.g., red cell, monocyte) .
• a cell e.g., red cell, monocyte
• the cell thus modified is then injected in the presence or absence of adjuvant.
• the timing interval between injections will depend upon the nature of the immunogen but generally 10 6 cells may be injected each time at intervals of not less than two weeks.
• Antibody levels to the antigen are monitored with standard Elisa procedure.
• Cellular immune levels can be measured by determining proliferative or cytotoxic responses to immunizing cells.
• sialic acids or glycosaminoglycans can be bound to the plasma membrane of a cell using the compounds of the invention.
• the specific compound is placed into iso- osmotic media as described hereinabove.
• Red cells for example, are placed into the solution, resulting in binding of the compound to the plasma membrane.
• the reaction is stopped with the addition of serum, after which the cells are washed in saline containing medium and are ready for injection. Red cells traverse the circulation and, as immature cells, they have a large amount of sialic acid on their surface.
• the amount of sialic acid per cell is reduced making it possible for the splenic and liver macrophages to recognize red cell membrane antigens, thereby removing them from circulation.
• By appropriately increasing the amount of sialic acid incorporated into the membrane of a red cell it may increase the life of the red cell in circulation.
• the ability to increase the lifetime of a red cell may be advantageous for a transplant patient or for a patient with anemia.
• bone marrow transplant patients receive the transplant it is several weeks before they are capable of making their own red blood cells. By using this technology to prolong the lifetime of their own red cells, patients can be given several marrow transplants, if need be, without having bouts of anemia.
• the anemia may result from a decrease in the lifetime of the red cell or a decrease in the rate of production of red cells. In either case, increasing the lifetime of the red cell will reduce the anemia.
• Photodynamic therapy for the cure of cancer is an area of intense research (Proceedings of SPIE- The International Society for Optical Engineering; Volume 847. "New Directions in Photodynamic Therapy", Douglas C. Neckers, Editor; (October 1987)). Many of these compounds are of the phthalo ⁇ yanine class or the hematoporphrin class. All absorb light in the 600-800 nm region and produce excited state oxygen in the process .
• a lipophilic derivative of the compound is made and then dissolved in the iso-osmotic solution.
• Tumor selective cells e.g., TIL
• TIL Tumor selective cells
• the tumor selective cells migrate to the site of the micrometastasis.
• the patient is exposed to high intensity light in the region where the photodynamic molecule absorbs and the excited state oxygen produced will kill the tumor cells.
• the carrier cells will be killed and this should generate an inflammation whereby more immune cells converge to remove the dead cells, increasing the toxicity to tumors.
• the carrier cells are responsible for more selective accumulation of therapeutic agent at the tumor site.
• direct application of compounds of the invention to tumor deposits within a body cavity may assist in retention at the site, enabling more effective intracavitary irradiation treatment at the time of surgery.
• an antiproliferative drug must be retained by the smooth muscle cells in a repaired artery for up to 7-10 days after angioplasty to prevent these events.
• Compounds of the invention comprising suitable antipro ⁇ liferative agents, may be delivered to the damaged vessel wall during angioplasty, and the antiproliferative agent conjugated to the compound may be retained by the damaged cells.
• higher doses of antiproliferative drugs can be given directly to the affected cells and can be retained at the affected site longer through the use of compounds of the invention than through systemic drug delivery.
• direct deposit of antiproliferatives via drug delivery catheters in angioplasty will permit the drug to bind to the membranes of cells at the site of the angioplasty procedure, while any unbound drug may be flushed from the artery during the procedure.
• the catheter will be removed and the drug bound to resting cells will remain in the outer membrane or traverse interstitial spaces to arrive at deeper cell layers. If those cells go into an active or growth state, the compound of the invention will move into the cells as membrane components traffic inward. Once inside cells they can exert their antiproliferative action.
• compounds of the invention comprising suitable antiproliferatives, may be delivered primarily to the disease site and the amount of drug processed by the liver or kidney at any one time is minimized, reducing the opportunity for serious adverse reactions.
• compounds of the invention useful for treatment of post-angioplasty restenosis comprise antiproliferative agents, such as heparin, hirudin, colchicine, vinca alkaloids, taxol and derivatives thereof.
• antiproliferative agents such as heparin, hirudin, colchicine, vinca alkaloids, taxol and derivatives thereof.
• heparin to smooth muscle cells in culture or by administration to animals after arterial injury results in decreased growth and reduced myointimal thickening and cell proliferation.
• Compounds of the invention comprising heparin preferably will be constructed such that they remain on the external membrane of cells of the inner arterial wall, by hydrophobic interaction with one or more cell wall components, rather than being taken up into the interior of those cells.
• colchicine comprises the bio-affecting moiety of a compound of the invention as an acid- cleavable conjugant.
• the colchicine is inactive in its conjugated form.
• uptake of the compound into intracellular acid vesicles causes the agent to be released from the compound, thereby activating it.
• active colchicine is delivered to its site of activity within the cell, and is capable of inhibiting tubulin processes therein, thus inhibiting cell division.
• Another useful colchicine-containing compound has the formula:
• antiproliferative agents contemplated for use in the practice of the present invention include: angiotensin converting enzyme (ACE) inhibitors, angiopeptin, cyclosporin A, calcium channel blockers, goat-antirabbit platelet derived growth factor antibody, Terbinafine and Trapidil, interferon-gamma and polyanions for binding of cationic growth factors.
• ACE angiotensin converting enzyme
• Therapeutic compounds of the invention are particularly well-suited for treatment of rheumatoid arthritis. They provide a means of performing chemo- or radiation synovectomy that enables the joint to retain a significant amount of therapeutic agent without significant systemic release to the lymph nodes, spleen or liver.
• a major advantage over all existing delivery systems is that compounds of the invention are delivered uniformly to the very tissue that requires treatment and are retained in those cells.
• the radioactivity emitted from the compound initiates therapeutic action on cells of the synovial membrane.
• essentially all compound in the body is found in the treated joint, and approximately 70% of the injected compound is retained there after six days. This localized and extended retention will decrease the dose of radioisotope needed for each procedure of radiation synovectomy, and will reduce the side effects caused by the systemic exposure to the radioisotope released from the joint using conventional therapies.
• Radiotherapeutic compounds of the invention that are particularly preferred for radiation synovectomy may be synthesized to incorporate an appropriate radioisotope, as exemplified below.
• a radiometal may be complexed with either (1) a nitrogen and sulphur-containing chelator or (2) a nitrogen and oxygen-containing chelator.
• the radioisotope may be selected from the group consisting of radioactive halogen, copper, yttrium, rhodium, palladium, indium, iodine, samarium, gadolinium, holmium, erbium, ytterbium, lutetium, rhenium, gold, or a combination thereof.
• a preferred compound for use in the present invention is a compound having the formula:
• L, R, Rj and R 2 are as defined for Formula I above;
• Z represents H or a metal coordination site;
• R' R" or R' CR"R'" [CR"R'"] n CR"R'"
• R" SZ wherein each R' is independently a hydrogen atom or an alkyl group, preferably a lower alkyl group, or substituted lower alkyl wherein the substituent can be any ester, R' * and R'" are independently a hydrogen atom or an alkyl group and m and n can each be zero or 1; and M represents a radiometal selected from the group consisting of rhenium, indium, copper and palladium.
• the compound of formula VIII above has the formula:
• R and ⁇ Ry are hydrocarbon substituents having from 1 about 30 carbon atoms;
• X and Xi may be the same or different and represent O, S, C(CH 3 ) 2 or Se;
• A represents a pharmaceutically acceptable anion
• Z represents H or a metal coordination site
• R' R" or R' CR"R'" [CR"R'"] n CR"R'"
• each R 1 is independently a hydrogen atom or an alkyl group, preferably a lower alkyl group, or substituted lower alkyl wherein the substituent can be any ester
• R" and R'" are independently a hydrogen atom or an alkyl group and m and n can each be zero or 1
• M represents a radiometal selected from the group consisting of rhenium, indium, copper and palladium.
• Another particularly preferred compound for use in the present invention is a compound of the formula:
• M represents a radiotherapeutic substance such as rhenium, indium, copper or palladium.
• Another useful compound is of the formula:
• R and R t axe hydrocarbon substituents having from 1 to about 30 carbon atoms;
• X and X ! may be the same or different and represent O, S, C(CH 3 ) 2 or Se;
• A represents a pharmaceutically acceptable anion;
• M represents a radiotherapeutic substance selected from the group consisting of copper, technetium, rhodium, palladium, indium, samarium, gadolinium, holmium, erbium, ytterbium, lutetium, rhenium, yttrium, gold, erbium, holmium, or a combination thereof; n is 2, 3 or 4; m is l or 2; and p is 1 to 6.
• Ovarian Cancer Compounds of the invention comprising chemotherapeutic or radiotherapeutic agents will enable high concentrations of those agents to be delivered directly to the site of ovarian tumor cell proliferation. Additionally, the therapeutic agents will be retained for longer periods of time in the peritoneal cavity, thus retarding the dissemination of tumor cells. Moreover, this can be accomplished without the significant side effects accompanying administration of large concentrations of such agents via systemic delivery systems.
• Compounds of the invention may be delivered intraperitoneally through a Tenckhoff catheter as a treatment after surgery, or by a second-look laparotomy, and as adjuvant therapy at the time of surgery.
• Acid-cleavable colchicine-containing compounds of the invention will also be useful in antiproliferative treatment of ovarian tumor cells. As noted above, it is expected that such a molecule will remain on the outer membrane of a cell in a non-toxic form. However, when the compound is taken into the cell where the chemotherapeutic drug is cleaved from the remainder of the compound, the chemotherapeutic substance can exert its antiproliferative activity.
• Psoriasis Compounds of the invention comprising corticosteroids may be used to advantage in the treatment of psoriasis. They provide greater retention of the drug at the site of a psoriatic lesion, thus enhancing the efficacy of the drug in reducing the proliferation of keratinocytes and immune cells. Moreover, retention of the compounds of the invention at the site of the lesion will prevent the penetration of possibly toxic compounds into the circulatory system. This results in a clinical benefit in that, after a two-week series of applications, the therapy should not need to be terminated because of high serum concentrations of antiproliferative drug.
• platelets or low density lipoproteins may be tracked to the site of atherosclerotic plaque deposition for early detection of atherosclerosis.
• Platelets may be isolated from the individual's blood using standard gradient techniques, then labeled with indium or technetium, as optional diagnostic moieties, and reinjected intravenously.
• a suitable procedure for binding compound of the type described herein to platelets is provided in the aforementioned U.S. Patent No. 4,762,701.
• the radioactive labeled platelets accumulate at the site of the plaque formation on arterial walls, where the gamma emission can be detected using a gamma camera.
• LDL may be purified by standard ultracentrifugation techniques and labeled with compounds of the invention, by virtue of their significant lipid content and the binding affinity of compounds of the invention for lipid regions of bio- compatible particles.
• These radiolabeled LDL will accumulate at sites of atherosclerotic plaque buildup after reinjection, allowing detection by nuclear imaging.
• Monocytes are also known to accumulate in atherosclerotic plaque, and therefore, may also be useful in detecting its formation; their only limitation is expected to be availability of suitable numbers of purified monocytes for radiolabeling.
• Platelets are also known to accumulate at sites of thrombosis (e.g. , coronary thromboses, deep vein thromboses, intravascular grafts) and at sites of acute rejection following organ transplantation. Therefore, autologous platelets isolated by standard methods and radiolabeled using compounds and methods of the invention will also allow non-invasive diagnosis of such disease processes when combined with pharmacotherapy.
• thrombosis e.g. , coronary thromboses, deep vein thromboses, intravascular grafts
• autologous platelets isolated by standard methods and radiolabeled using compounds and methods of the invention will also allow non-invasive diagnosis of such disease processes when combined with pharmacotherapy.
• radio- isotopic e.g., radioactive iodine, carbon, nitrogen, sulphur, phsophorus or selenium
• Compounds emitting gamma rays of sufficient energy using radioisotopically labeled compounds of the invention may be detected using gamma scintigraphy. If the isotope is a low energy non-penetrating beta emitter, then the compound can be used in research applications using standard beta counting techniques.
• the biotinylated lipophilic compounds of the invention can function as multi-purpose reagents. For example, such compounds may be used to cause typically non-adherent cells to adhere rapidly to a selected surface. This is important for analyses requiring immobilized cells, i.e., in monitoring of a single cell over time. If a cell population is labeled with a biotinylated compound of the invention and the resultant labeled cells are brought into contact with a surface to which streptavidin is bound, the cells will rapidly adhere to the surface. The cell analysis can immediately begin. The fluorescence associated with the biotinylated compound also provides a convenient means of visually monitoring the cell during the experiment.
• fluorescent cell labelling compounds can be used to monitor growing cells and measure the growth rate by dilution of fluorescence.
• U.S. Patent No. 4,859,584 This technique loses sensitivity after 5-8 doubling times (dependent on cell type) , as the fluorescence of the labelling compound decreases to the level of autofluorescence.
• Amplification of fluorescence can be achieved by binding a fluorochro e-conjugated streptavidin to, e.g. a biotinylated cyanine, as described herein.
• a radio-labeled streptavidin can be bound to the biotinylated compound of the invention and autoradiography can be performed to identify the labeled cells.
• biotinylated compounds of the invention are in protein binding.
• an alternative coupling mechanism is the avidin-biotin binding pair.
• target cells may be labeled with a biotinylated compound of the invention and the large protein would be conjugated to avidin or a suitable derivative of avidin, e.g., streptavidin.
• the biotinylated cells would then be exposed to the avidin-protein conjugate resulting in protein bound stably to cells.
• compositions comprising compounds of the invention may be conveniently formulated for administration with a compatible biological medium, such as salt-free isomotic solutions, or pharmaceutically acceptable liquid excipients.
• a compatible biological medium such as salt-free isomotic solutions, or pharmaceutically acceptable liquid excipients.
• the latter include various inert oils, e.g., vegetable oils such as olive oil or peanut oil, or highly refined mineral oil.
• concentration of active ingredient in the chosen medium will vary, depending on the nature of the compound and the disease or pathological condition being treated. It is especially advantageous to formulate the pharmaceutical preparation in dosage unit form for ease of administration and uniformity of dosage.
• Dosage unit form as used herein refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment.
• Each dosage unit should contain the quantity of active ingredient calculated to produce the desired therapeutic effect in association with the selected pharmaceutical carrier.
• Procedures for determining the appropriate dosage unit for inhibiting cell proliferation, or for treatment of other pathological conditions in a given class of patients are well known to those skilled in the art.
• doses of approximately 5 mCi of 90 Y (half life 2.7 days, beta energy 2.2 MeV) or 300 mCi l65 Dy (half life 2.3 hours, beta energy 1.3 MeV) administered in various colloidal forms have demonstrated clinical efficacy.
• compounds of the invention may be delivered directly into tumor tissue, into body cavities containing disseminated tumor, or into blood vessels which supply the tumor, etc.
• compounds of the invention may be delivered directly into tumor tissue, into body cavities containing disseminated tumor, or into blood vessels which supply the tumor, etc.
• an average human body weight of 70 kg. corresponds to a plasma concentration in rabbits of 28 ng/ml or 70 nM.
• the concentration of colchicine in rabbits shown to prevent restenosis was 14 times the concentration achieved in the clinical trial.
• the compounds of invention can be prepared in the above-described compatible binding media up to 100 ⁇ M, i.e., 1400 times the concentration shown to be effective in the animal study, and delivered directly to the arterial wall by catheter during an angioplasty procedure.
• the pharmaceutical preparations of the invention are preferably administered by injection, intraperitoneal infusion, or catheterization. Other modes of administration may also be effective, such as oral administration in some cases, or aerosolization.
• the pharmaceutical preparation may be administered at appropriate intervals. Due to the nature of the compounds of the invention, repeated administration is likely to be unnecessary. Methods for determining the frequency of administration of the pharmaceutical preparations are well known to those skilled in the relevant medical art. In any event, the appropriate interval in any particular case would normally depend on the condition of the patient, and the type of pathological condition being treated. The following examples are provided to describe the invention in further detail. These examples are intended to illustrate certain aspects of the invention and should in no way be construed as limiting the invention.
• Example 1 Determination of Membrane Retention Coef icient
• the membrane retention coefficient provides information regarding how well a given compound is retained in the plasma membrane of a cell and is determined as described below.
• red blood cell ghosts for use as a model membrane is achieved by centrifuging whole blood at 300 x g for 15 minutes, removal of the plasma and resuspension of the cell pellet in 0.83% (w/v) ammonium chloride.
• the ghosts are pelleted from the ammonium chloride by centrifuging at 10,000 x g for 10 minutes. This ammonium chloride washing procedure is repeated a minimum of five times to insure that complete release of hemoglobin from the cells has occured.
• the ghosts are labeled with the compound in question at a concentration allowing for detection of the labeled ghosts by instrumental analysis or fluorescent microscopic methods, and at a concentration similar to those that would be used to label cells for a specific application as described above.
• stoc solutions of the compounds in question were prepared in ethanol at a molar concentration of 2 x 10 *3 , and working dilutions of the compounds were prepared in iso-osmotic sucrose (52 g/500 ml distilled water) .
• the samples were centrifuged at 10,000 x g to pellet the ghosts and the staining solution was aspirated from the samples.
• the labeled ghosts were resuspended in 1 ml of phosphate buffered saline solution containing 10% fetal bovine serum (PBS-FBS) .
• Triplicate 20 ul e aliquots were removed from each sample for the determination of the amount of total compound present.
• the samples were centrifuged as described above and triplicate 20 ul aliquots were removed from the supernatant for quantitative determination of amount of unbound compound present.
• the supernatant was aspirated and the red cell ghost pellet was resuspended in 1.0 ml of the PBS-FBS, which was once again sampled as described above. This procedure was repeated at least six times, allowing for detection of rapidly released compounds and was monitored after times equal to or greater than 24 hours to allow for the detection of more slowly released compounds.
• the 20 ul aliquots were extracted into 3.0 ml of n-butanol by shaking. The samples were centrifuged at 3000 x g to remove membrane debris and the butanol fractions were assayed for compound concentration.
• Fluorescent compounds are assayed in this manner using peak excitation and emission wave lengths for the particular compounds being assayed to determine the fluorescence units for each sample. Radiolabeled compounds do not require butanol extraction and may be assayed directly using beta or gamma counting instrumentation.
• the comparison of the MRC values defines criteria for identification of the compounds of this invention, these criteria being: 1) the MRC values determined for each washing steps should have a value of at least about 90 and 2) the percent difference between MRC values over at least a 24 hour time period should be less than about 10%.
• the compounds identified as A- C are of the formula XII, above, in which X and X j in each compound represents C(CH 3 ) 2 and Z and Zj represent H, with R/Ri representing C-5/C-5 (compd. A) , C-lO/C-10 (cmpd. B) and C-14/C-14 (cmpd. C) ;
• the compounds identified as D-K are also of the formula XIII, in which Z and Z, represent H, with R/Rj representing C-14/C-3 (cmpd. D) , C-18/C-3 (cmpd.
• a hydrocarbon chain from an aqueous phase (e.g., the extracellular medium) to a liquid hydrocarbon phase (e.g., the hydrocarbon interior of a biomembrane) is dependent on the degree of branching, the degree of unsaturation, and the number of methylene groups in the hydrocarbon chain.
• the free energy of methylene-hydrocarbon interaction is minimal for the methylene group closest to the aqueous interface and increases for successive methylene groups, becoming approximately equal to that found for a hydrocarbon in a non-polar hydrocarbon-containing solvent for hydrocarbon groups 4 or more carbons into the lipid interior of the membrane.
• n equals number of linear hydrocarbons in first tail
• m equals number of linear hydrocarbons in second tail
• the assay for MBS is carried out by suspending a decreasing number of labeled membrane ghosts in a fixed volume of albumin-containing saline.
• the total amount of label present is determined by sampling the well mixed suspension; subsequently, the membrane ghosts are pelleted by centrifugation and the amount of label released into the supernatant is determined and expressed as a percent of total label. Percent retention is plotted against number of ghosts per ml. of suspension, and MBS is determined as the area under the curve between 5xl0 7 ghosts/ml. and 4X10 8 ghosts/ml. In this assay, a compound which exhibits infinite membrane binding stability would give an MBS of 3.5xl0 10 , and the results are expressed as a percentage of this maximal MBS.
• the following table sets forth data from MRC determinations as described in Example 1, above, and MBS determinations of the present example, respectively, on a representative sample of compounds of the invention.
• the compounds identified as O-T were of formula XII, in which X and XI represent C(CH 3 ) 2 and Z and Zj represent H, with R/R, representing C-12/C-10 (cmpd. 0) , C-22/C-12 (cmpd. P) , C-14/C-3 (cmpd. " Q) ; C-14/C-14 (cmpd. R) , and C-16/C-16 (cmpd. S) , and C-22/C-14 (cmpd.T) .
• the MBS assay enables one to discriminate among compounds which differ little in MRC but which exhibit differing stabilities of in vivo association with biomembranes.
• head groups carrying various functional groups useful in the practice of the invention e.g., radiometal chelators, proteins, peptides, radionuclides and the like
• similar effects may be determined, and the balance between head group effect and carbon equivalents needed to arrive at a desired membrane binding stability may be estimated.
• Compounds with MBS of at least about 30% or greater are expected to have utility in applications of the invention of the types described herein. It can also be observed that substituents on the head groups may have a significant effect on membrane binding stability, even though the number of carbon equivalents is kept constant.
• Table III Compounds AC and AD differ only in headgroup substituent, yet their respective MBS values are quite different.
• the compounds identified as U, W and Y are of formula XIII, above, in which X and X, in each compound represent 0 and Z and Z, represent H, with R/R, representing C-22/C-3 (cmpd. U) , C-14/C-14 (cmpd. W) and C-18/C-18 (cmpd. Y) ;
• the compounds identified as V, X and AA are also of formula XII, in which X and X, represent S and Z and Z, represent H, with R/R, representing C-22/C-3 (cmpd. V) , C-14/C-14 (cmpd. X) , and C-18/C-18 (cmpd. AA) .
• the compounds identified as AB, AC and AD are of the formula XII above, in which X and X, represent (CH 3 ) 2/ Z r represents H, and R/R-* represent C-14/C-22 (cmpd.AB) and C-14/C-3 (cmpds.AC, AD) .
• Z represents -CH 2 -NHOCH.
• Z represents H.
• AD Z represents a non- cleavable colchicine derivative as shown below.
• Reaction Scheme 1 described above.
• the numbers given in parentheses indicate the corresponding numbered reagents shown in Reaction Scheme 1.
• the product obtained had the formula of compound 8 in which X and X, represent C(CH 3 ) 2 and R/R, represent C 14 H 29 /C 22 H 45 and A represents I.
• 5-(N-phthalimidoaminomethyl)-2,3,3-(3H)- trimethylindolenine (1) was prepared by a modification of the procedure of Gale et al., Aust. J. Chem.. 30:693 (1977). 2,3,3,-(3H)-trimethylindolenine (23.85 g, 0.15 mol, Aldrich) was dissolved in 150 ml of concentrated sulfuric acid. The flask was then placed in an ice bath and N-hydroxymethyl phthalimide (26.55 g, 0.15 mol, Fluka) added portion-wise over 30 mins. The ice-bath was removed and the solution stirred at room temperature for 5 days.
• the solution was then transferred to a 250 ml Erlenmeyer flask, diluted with ethanol (20 ml) and a saturated solution of KI (20 ml) , and the mixture stirred for 30 minutes.
• the product was precipitated out by the addition of 100 ml of cold water and the resulting solution stirred for 15 minutes. The precipitate was collected by filtration, washed with distilled water and dried under high vacuum overnight.
• the title compound was also prepared according to Reaction Scheme 1.
• the numbers given in parenthesis indicate the corresponding numbered reagents shown in Reaction Scheme 1.
• the product obtained had the formula of compound (8) in which X represents C(CH 3 ) 2 , Xi represents oxygen, R/R, represent C 14 H 2 /C 22 H 45 and A represents iodide.
• 2-(3-acetonilidovinyl)-1-docosanyl-benzoxazolium iodide (6) (1.10 g, 1.53 mmol), 5-(N-formylamino ⁇ methyl)-l-tetradecyl-3,3-dimethy1-2-methylene indoline (4) (630 mgs, 1.53 mmol), prepared as in 3a., above, triethylamine (0.5 ml) and ethanol (25 ml) were heated at reflux for 1 hour. The solution was then cooled to room temperature, transferred to a Erlenmeyer flask and then diluted with ethanol (40 ml) and a saturated solution of KI (20 ml) .
• the resulting material was purified by passage through a column of octadecyl silica gel eluting with 80:20:1 (methanol: water: trifluoroacetic acid) first to remove unconjugated peptide and then 100:2:1 (methanol: water: trifluoroacetic acid) to elute the desired product.
• Fractions containing the peptide-lipophilic cyanine conjugate were combined, concentrated on the Buchi and the residue lyophilized from water (50 ml) to give pure conjugate as a purple powder (14 mgs, 40%) .
• Purity by hplc was greater than 90% with less than 0.02% free Substance P.
• the conjugate thus obtained has the following structural formula (in which the conventional three letter symbols are used to show the amino acid sequence of Substance P) :
• 2,3,3-Trimethyl-(3H)-indolenine (6.36 g, 0.04 mol, Aldrich) and n-tetradecyl-4-chlorobenzene-sulfonate (15.52 g, 0.04 mol) were heated together at 130-135°C (oil bath temp.) for 3 hours with continuous stirring.
• the crude material was then dissolved in ethanol (200 ml) and then stirred with a saturated potassium iodide solution (50 ml) for 3,0 minutes.
• Cold water 500 ml was added and the precipitate collected by filtration and washed well with cold water.
• N-Docosanyl-5-iodo-2,3,3-trimethylindolinium 4- chlorobenzenesulfonate (2.36 g, 3.0 mmol), N,N'- diphenylformamidine (0.59 g, 3.0 mmol, Aldrich) and acetic anhydride (20 ml) were placed in a 50 ml round bottom flask which was under an argon atmosphere and fitted with a reflux condensor and stirring bar. This flask was then placed in an oil bath which was preheated to a constant temperature of 170°C and the mixture refluxed for 60 minutes. The reaction flask was then cooled to room temp, and then transferred to a 500 ml Erlenmeyer flask.
• the crude product was purified by flash chromatography (silica gel, first 5% methanol in methylene chloride and then 8% methanol in methylene chloride) to furnish N-docosanyl-N'-tetradecyl-5-iodo- 3,3,3',3'-tetramethylindocarbocyanine chloride (12) (272 mg, 58%) .
• the product, compound 23, was converted to the HCl salt by the addition of an HCl gas/ethanol solution.
• An ethanolic solution of oxalic acid was added as an antioxidant, the solution evaporated, and the solid residue stored under nitrogen.
• the concentration of the product as obtained above was determined to be 252 ⁇ M by absorption at 555 nm.
• the specific activity was 186 ⁇ 21 mCi/ ⁇ mol compared to the theoretical value of 181 mCi/ ⁇ mol.
• Carbonyldiimidazole (0.197g, 1.22mmol, Aldrich) was then added to a stirred solution of the 7-(N-glutaryl) deacetyl colchicine (0.397 g, 0.83 mmol) in dimethylformamide (5 mL, freshly distilled from lithium aluminum hydride) at room temperature and the reaction mixture kept stirring at room temperature for two hours. During this time a precipitate formed in solution.
• the dimethylformamide was removed by vacumn distillation and the residue was dissolved in methylene chloride (5 mL) .
• tetrabutylammonium borohydride 250 g, 0.972 mmol, Aldrich
• Compound solutions to be studied were prepared by adding 50 ⁇ l of hydrazone conjugate solution (lmg/ml of methanol) to a screw capped vial (1ml) containing citrate phosphate buffer (200 ⁇ l) of the desired pH. The vials were kept closed and the solutions were analysed by HPLC (350nm detection) for conjugate (19) remaining and 7-N-(5-oxopentanoyl) deacetyl colchicine produced at 24h and 48h. Each hplc injection was 200 ⁇ l.
• the title compound was prepared according to the procedure generally set forth in Reaction Scheme 4 above.
• the reference numbers herein correspond to those shown in Reaction Scheme 4.
• Example 3d. The in vivo receptor pharmacology of the compound produced in Example 3d. , above, was studied in a rabbit blood perfused hind limb preparation similar to that described by U. Forstermann et al. , J. Pharmacol. EXP. Ther.. 234: 1055-61 (1987) .
• Distal perfusion pressure in the femoral arterial bed was measured (in mm Hg) by means of a micropressure transducer (Millar) passed down the perfusion tubing so as to lie just beyond the tip of the femoral catheter. Initially, pump speed was adjusted so that distal perfusion pressure approximated systemic mean arterial blood pressure after which blood flow was kept constant for the remainder of each experiment. Changes in resistance then varied directly as distal perfusion pressure in a manner similar to that described by Ohm's law, such that:
• FIG. 1 shows the qualitative similarity of the in vivo responses to substance P and the conjugate of Example 3d. Both substance P and the conjugate produce brief, dose-related decreases in hind-limb perfusion pressure when injected locally into the perfused rabbit hind-limb preparation. These dilator responses are entirely consistent with the known vascular pharmacology of substance P. See, for example, J. Beny et al., J. Physiol.. 398: 277-89 (1988) .
• Figure 2 also presents the dose-response relationships of both substance P and the conjugate during infusion of a known antagonist of substance P, [D-Pro 2 ,D-Trp 7 * 9 ]- substance P.
• This synthetic peptide with D-amino acids in 3 positions has been reported to antagonize the neuronal, behavioral, and vascular actions of endogenous substance P and exogenously administered substance P.
• the decrease in perfusion pressure to both substance P and the conjugate is inhibited by CD- Pro 2 ,D-Trp 7,9 ]-substance P, as assessed by parallel shifts to the right in the dose-response curves of both compounds when the antagonist is present.
• the conjugate of Example 3d. (100 uM) readily binds to washed rabbit red blood cells suspended in phosphate buffered saline with 1 mM EDTA and 0.5% rabbit serum albumin) when the two are incubated together ex vivo .
• the fluorescent moiety of the conjugate allows its ready detection on RBCs by flow cytometry.
• conjugate labeled rabbit RBCs are injected in the rabbit hind-limb, a reduction in perfusion pressure occurs, the magnitude of which is related to the number of injected cells. Further experiments have demonstrated that injection of 10 6 -10 9 RBCs results in graded decreases in perfusion pressure in the rabbit hind limb preparation.
• Example 5 Determination of Effect of Cell Binding on structure and Biological Interactions of Substance P Derivatized with Lipophilic Cyanine
• Figure 3C shows the fluorescent histograms of unlabeled RBCs or conjugate-labeled RBCs which are also indirectly fluorescently labeled using rabbit anti-substance P antibody as the primary reagent and then fluoresceinated goat anti-rabbit antibody as the secondary reagent.
• conjugate-labeled cells there is a significant amount of both green and red fluorescence detectable on the cells (LFL1 signal increase over Figure 3B) , demonstrating specific binding to cell-associated conjugate. This fluorescence increase is not noted with the RBCs that have not been labelled with conjugate.
• this technique demonstrates that Substance P is antigenically "recognized" on the surface of RBCs by an anti-substance P antibody, confirming the presence and conservation of structure of Substance P on red cells.
• Compounds, T, AA and AB can be prepared according to the general procedures set forth in foregoing Rreaction scheme 1 and described in Example 3 above. Recovery and viability (calculated as described in Slezak and Horan, Blood. 74: 2172-2177 (1989)) at 5, 10 and 20 ⁇ M labeling concentrations and for 5 or 10 ⁇ M for compounds AA and AB. However, viability and recovery decreased to 70-80% for compounds AA and AB at 20 ⁇ M labeling concentrations. Number of molecules of compound incorporated per cell was determined for cells labeled with 10 ⁇ M compounds.
• Relative membrane retention was determined using erythrocyte membrane ghosts prepared by NH 4 C1 lysis (Slezak and Horan, Blood, supra) . Ghosts were labeled at 4xl0 8 /ml with 10 ⁇ M compounds for 5 minutes at room temperature, using previously described procedures. However, post-labeling washes were carried out using PBS containing lroM EDTA and 5% BSA. After the final wash, ghosts were resuspended at 4xl0 8 /ml in PBS+EDTA+5% BSA and incubated for 24 hours at 37°C.
• iodinated compounds of the invention exhibited membrane retention characteristics equal or superior to the non-iodinated compound. Although their incorporation into red cell membranes was somewhat lower, as compared with equivalent concentrations of non-iodinated compound, as might be predicted on the basis of their larger molecular size and weight, it is not expected that this difference would be sufficient to alter their utility for cell labeling applications of the type described above.
• Example 8 Retention on Artificial Surfaces
• a solution of a radioiodinated ( I25 I) lipophilic cyanine, (compound 12, Reaction Scheme 2-, Example 3g. , above) in 100% ethanol was placed in contact with the lumenal -surface of short sections of several different types of plastic tubing, including medical silastic (SIL) , polycarbonate ' (PC) , ,polyvinylchloride (PVC) and polyethylene (PE) .
• SIL medical silastic
• PC polycarbonate '
• PVC polyvinylchloride
• PE polyethylene
• the sections were connected with a longer, closed loop tubing circuit containing approximately 30 ml of fresh, anticoagulated human blood.
• the tubing circuit was passed through a roller pump and the blood was circulated for a period of 6 hours.
• the tubing sections were disconnected, flushed gently with PBS to eliminate any adherent blood and counted for radioactivity.
• the results of this experiment are graphically represented in Fig. 5.
• Tubing section length, diameter, and the specific activity of the radioiodinated compound of invention were recorded so that the data could be expressed as picograms of radiiodinated compound/mm 2 of tubing lumen surface (x- axis of bar graph) .
• Figure 5 shows that PVC tubing initially bound the greatest amount of the radioiodinated compound while PE bound the least. Highest retention on the tubing after 6 hours in contact with blood occurred with PVC (97.6 ⁇ 2.1% of initial radioactivity retained) while retention was poorest with PC (56.0 ⁇ 10.4% of initial radioactivity retained) . However, average retention was in excess of 50% for all tubing and approached 100% for PVC, demonstrating an ability of the compound of the invention to remain on artificial surfaces for prolonged periods despite contact with biological fluids. The compounds of invention may, therefore, be useful in the retention of biopassivating substances on artificial surfaces.
• Example 9 In Vivo Retention of a I86 Re-Chelator Lipophilic Cyanine
• the 186 Re-chelator lipophilic cyanine conjugate was prepared essentially by the method described in
• Example 3h (Compound 24 of Reaction Scheme 5) , except that the SepPak was washed with approximately 10 mL distilled water prior to elution of product with ethanol, to remove excess gentisic acid and avoid the need for pH adjustment prior to in vivo administration. After vacuum evaporation, the concentrated stock solution brought to a final volume of approximately 25 ⁇ L in ethyl alcohol (compound 24 concentration approximately 500 ⁇ M; specific activity approximately 123 mCi/ ⁇ mole) . Preparations of compound 24 and Na 186 Re0 4 for in vivo injection containing approximately 5-6 ⁇ Ci of 186 Re were made as follows.
• Na 186 Re0 4 (NEZ301, in 0.1N NaOH) was diluted in sterile 300 mOsM Dulbe ⁇ co's phosphate buffered saline, the pH was verified to be 7.0 using pH paper, and the preparation was then re-sterilized by passing it through a 0.22 ⁇ m filter.
• Compound 24 was diluted in sterile 300 mOsM glucose.
• Sterile 50 ⁇ L Hamilton syringes were loaded with 5.0 ⁇ L of the Na ,6 Re0 4 or compound 24 preparations using sterile technique.
• mice were re- anesthetized, the right hind limb was sterilized using an alcohol swab and 5.0 ⁇ L aliquots of compound 24 or Na 186 Re0 4 were injected directly into tumor nodules approximately 5 mm in diameter. Injections were made over a period of approximately 10 seconds and the needle was held in place for an additional 5-10 seconds after the injection was completed to allow dissipation of any backpressure and minimize leakage at the injection site. Animals and counting standards were imaged using a GE Starcam 300 system immediately after injection (within 10-15 minutes) and daily thereafter for 4 days, using an energy window of 140+20 keV. After imaging was completed on days 2 and 4, groups of animals were sacrificed and various organs were collected, weighed, and cpm/gm determined for evaluation of biodistribution of 186 Re.
• Results are expressed as % of total counts injected recovered in the specified organ after correction for decay. Since organs and tumor differed significantly in size, the relative concentration of 186 Re found in each organ is compared in Table IV B. Results are expressed as the percentage of injected counts that would be found in a 1 gram block of tissue after correction for decay.
• Whole body scintigraphic images were analyzed by establishing regions of interest (ROI) corresponding to Tumor or Whole Body; counts in each region of interest were then determined as a function of time after injection. Localization of label in tumor was calculated as ratio of counts in Tumor ROI to counts in Whole Body ROI (columns 2 and 3, Table V). Percent retention of label within the body was calculated as the ratio of counts in Whole Body ROI to counts in Reference ROI (columns 4 and 5, Table 2) .
• ROI regions of interest
• Two properties of compounds of the invention are important in achieving site-selective administration and retention: a) the level of retention at the site of application when compared with unlinked therapeutic agents; and b) the pattern (heterogeneous vs. homogeneous) of their distribution after injection at a site.
• the 186 Re-chelator lipophilic cyanine conjugate (compound 24 of Reaction Scheme 5) was prepared as described in Example 3h. and the concentrated stock solution brought to a final volume of approximately 40 ⁇ L in ethyl alcohol.
• Preparations of compound 24 and Na 186 Re0 4 for in vivo injection were made as follows. Na 186 Re0 4 (NEZ301, in 0.1N NaOH) was diluted in sterile 300 mOsM Dulbecco's phosphate buffered saline, the pH was verified to be 7.0 using pH paper, and the preparation was then re-sterilized by passing it through a 0.22 ⁇ m filter.
• Compound 24 was diluted in sterile 300 mOsM glucose and the pH was adjusted to 7.0 by addition of sterile 0.1N NaOH (necessitated by the fact that no buffer was present in the glucose diluent and that gentisic acid present in the final preparation resulted in an unadjusted pH of 1-2) .
• Sterile 1.0 mL tuberculin syringes with 25G x 5/8 in. needles were weighed, loaded with approximately 0.1 L of the Na 186 Re0 4 or compound 24 preparations using • sterile technique, and reweighed to determine preinjection weight. Aliquots were also taken to determine cpm/ ⁇ L for each preparation.
• the exact volume and activity of compound 24 or Na 186 Re0 4 injected was determined by reweighing the syringe, calculating the difference between pre- and post-injection weights, (assuming a density of 1.0 g/mL for the injectate) and multiplying the measured volume in ⁇ L by the value of cpm/ ⁇ L obtained by counting aliquots of known volume of each preparation.
• a known volume of blood approximately 1 mL was withdrawn from the central ear artery. Animals were then placed in cages which enabled separate collection of urine and feces and monitored for 6 days.
• Circulating levels of compound 24 and Na 186 Re0 4 in blood and amounts excreted in urine over the 6 day post-injection period are shown in Table VI. Distribution of 186 Re in selected organs at day 6 is shown in Table VII. All results in Tables VI and VII are expressed as % of counts injected after correction for decay. The data of Tables VI and VII indicate that degree of leakage of 186 Re from the intra-articular space as measured by blood levels, urinary excretion and accumulation in other organs, is greatly decreased by administration of the radionuclide in the form of compound 24. Retention in the knee was further evaluated by determining counts in specific regions of interest (knee and/or whole body) from gamma scintigraphy images, as shown in Table VIII. These results also indicate greatly improved retention when radionuclide is administered in the form of compound 24.
• Example 12 Retention of Lipophilic Cyanine Conjugates at Intravascular site
• 125 I- substituted lipophilic cyanine prepared as described in Example 2 (compound 12, Reaction Scheme 2) was applied to the lumenal surface of the femoral arteries of anesthetized rabbits.
• the femoral artery was surgically isolated and a small, proximal side branch was cannulated with a short length of PE10 tubing.
• a 1 cm arterial segment surrounding the side branch was occluded and 20-50 ⁇ l of a solution containing 125 I- substituted compound 12 was administered through the cannula to the occluded section and allowed to remain for 5-10 min.
• the 125 I-substituted compound was then withdrawn along with the cannula, the side branch was permanently tied, the femoral artery occlusion removed to allow the return of blood flow, and the femoral incision was closed.
• 125 I-radioactivity was then monitored at selected times over the next 3 weeks with a Ludium Model 2200 Sealer Ratemeter and a Model 44-17 2 inch crystal with windows optimized for the detection of I25 I.
• AlO cells a clonal cell line originally derived from the thoracic aorta of embryonic rats, that proliferate as myoblasts and develop into cells which phenotypically resemble smooth muscle cells (B. Kimes and B. Brandt, Exptl. Cell Res. 98.:349 (1976).
• DMEM Dulbecco's Modified Eagles Medium
• FCS fetal calf serum
• Test substances were then prepared in the required concentrations in appropriate cell binding media and, after aspirating the plating medium, applied to the individual wells at 37°C for a period of only 10 minutes.
• the treatments were then aspirated, the wells washed 4 times with non-treatment containing medium and then placed in non-treatment containing DMEM with 10% FCS for the duration of the study.
• triplicate wells were aspirated, the aspirate saved, and the wells treated with 0.25 ml of 0.25% trypsin to release the adherent cells.
• Bound fluorescence was determined by subtraction of free from total after correction for background fluorescence from unstained ghosts; the percent free and bound fluorescence was determined by dividing the bound and free fluorescence by total fluorescence, respectively and multiplying by 100.
• Table X The results, presented in Table X below demonstrate that the anticoagulant conjugate of invention exhibited good membrane retention with 90.94% of fluorescence being associated with ghost membrane after 24 hours, compared with 94.96% retention with the compound of invention not containing anticoagulant.
• FITC-heparin showed only poor retention with only 37.63% of fluorescence remaining after 24 hours.

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