WO2020046543A2 - Complexes tétrapyrrole solubles dans l'eau contenant des ligands de biladiène utiles en thérapie photodynamique - Google Patents

Complexes tétrapyrrole solubles dans l'eau contenant des ligands de biladiène utiles en thérapie photodynamique Download PDF

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WO2020046543A2
WO2020046543A2 PCT/US2019/045250 US2019045250W WO2020046543A2 WO 2020046543 A2 WO2020046543 A2 WO 2020046543A2 US 2019045250 W US2019045250 W US 2019045250W WO 2020046543 A2 WO2020046543 A2 WO 2020046543A2
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segments
segment
tetrapyrroie
complex
group
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WO2020046543A3 (fr
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Joel Rosenthal
Andrea POTOCNY
Maxwell I. MARTIN
Anthony RICE
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
    • C07F15/006—Palladium compounds
    • C07F15/0066—Palladium compounds without a metal-carbon linkage

Definitions

  • the present invention relates to substituted derivatives of iinear tetrapyrrotes (biladienes) which are capable of forming water soluble metal complexes useful as photochemotherapeutic agents in photodynamic therapy and other applications.
  • iinear tetrapyrrotes biladienes
  • Such complexes are referred to herein as “tetrapyrrole complexes” and are described in more detail below.
  • Photodynamic therapy represents a minimally invasive and highly localized treatment strategy to ablate tumors in patients with fe side effects.
  • photosensitizers embedded within tumors are activated by light and undergo intersystem crossing followed by energy transfer to molecular oxygen, resulting in the production of toxic singiet oxygen. The singlet oxygen thereby produced is capable of destroying tu or DCis.
  • photosensitizing compounds are either intratumoralty or intraveneousiy injected and circulated throughout the body prior to irradiation of a tumor site.
  • PDT is a promising treatment strategy for at least certain types of cancers and skin conditions because it is less invasive than surgical options, has fewer side effects than radiation or chemotherapy, and has been shown to stimulate antitumor responses.
  • photosensitizers often have high toxicity to healthy tissues even without light application, which limits the allowable administered dosages.
  • successful PDT requires sufficient oxygen presence in the native tissue to produce toxic l Oz.
  • the microenvironment deep within solid tumors is often hypoxic, thereby rendering the photosensltizers ineffective in these regions.
  • photosensitizers for POT are activated by short wavelengths of light ( ⁇ 600 nm) that cannot deeply penetrate tissue, resulting in uneven therapeutic effects throughout the tumor space.
  • Photosensitizers have been developed that can be activated with longer wavelengths of light for enhanced tissue penetration, but unfortunately this approach is still ineffective in hypoxic tumor regions and these photosensitizers still suffer from relatively high off-target toxicides even without light application.
  • a photosensitizer intended for use in PDT has the following
  • Water soluble diorganobi!adiene derivatives have now been developed that are appended with one or more water-solubilizing segments, in particular poly(alkylene) glycol functionalities such as a po!y(ethyiene) glycol functionality.
  • Such derivatives provide tetra pyrrole complexes which are comprised of a metal, such as Pd or Pfe, comp!exed by a 10,10-diorgano" 5,15-dfarylbiladfene ligand which bears at least one substituent comprised of a water- solubilizing segment.
  • Such functionalized complexes are capable of maintaining the attractive photophysical properties of the corresponding parent complexes under biologically relevant conditions.
  • Introduction of the poly(alky!ene) glycol functionality has been found to overcome the inherent hydrophobicity of the bi!adiene architecture.
  • the addition of this functionality endows the linear tetrapyrrole complex with water solubility while having little effect on its photophysical properties, thus generating a biocompatible compound that retains the ability to generate singlet oxygen with a high quantum yield under biologically relevant conditions.
  • functionalized bi!adiene complexes are highly nontoxic in the dark, they can serve as extremely potent chemotherapeutic agents for treatment of cancer cells and drive apoptotic cell death with a high phototoxicity index.
  • the absorption profile of Pd ⁇ QMBiil]-PEG> ⁇ for example, closely matches that of Pd[DMBtl2] and obeys the Beer- Lambert Law, suggesting that the complex does not aggregate under biologically relevant conditions. Additionally, the emission spectrum of PdlOMBtllj-PEGyso retains the fluorescence and phosphorescence features characteristic of
  • PDT photodynamic therapy
  • the Pd[DtytBiil]*PEG 7 so is biologically compatible, as it is taken up by MDA-M 8-231 triple negative breast cancer (TNBC) cells and has an EDso of only 0,354 mM when exposed to sx > 500 ntn light for 30 minutes.
  • the tetrapyrrole complexes of the present invention are useful as PDT photosensitizers for treatment of cancers.
  • Such complexes have been found to be well tolerated by cancer cells such as TNBC cells and are potent i Oi sensitizers,
  • the inventive tetrapyrrole complexes may be readiiy synthesized using a comparatively small number of steps fro commercially available starting materials and can be purified and isolated using modular methods. Further, the properties and characteristics of the complexes can be easily tuned as may be desired for particular end use applications by varying the different substituents present on the bt!adiene skeleton of the ligand.
  • Fig, i shows, in summary form, a reaction sequence which can be used to prepare a water soluble tetrapyrrole complex in accordance with the invention.
  • the tetrapyrrole complexes of the present invention may be described as compounds in which a metal such as palladium or platinum Is complexed by a 10,10- diorgano ⁇ 5,i5 ⁇ diaryi:biiadiene ligand which bears at least one substituent comprised of a water-soiubiSizing segment such as poly(oxyalkyiene) segment.
  • a metal such as palladium or platinum Is complexed by a 10,10- diorgano ⁇ 5,i5 ⁇ diaryi:biiadiene ligand which bears at least one substituent comprised of a water-soiubiSizing segment such as poly(oxyalkyiene) segment.
  • Such ligands have an a,c ⁇ biiadiene framework structure containing four linked pyrrole rings, which is non- cyclic
  • the complexed metal may be, for example, Pd or Pt, but any other metal may also be employed such as platinum group metals generally (Pd, Pt, Ru, Rh, Os, Ir), transition metals (such as Mi, Cu, Zn, Fe, Cd), Group 13 metals (e.g., Ai, In, Th, Ga), and other metals (e.g.. Mg),
  • the metal may be in Ionic form, for example as a divalent, bivalent or tetr choice ion.
  • One or more other anions may be present and -s- as so da ted with the complex if needed to compensate for any charges that may exist as a result of the compiexed metal being selected and its valency.
  • the ligand is disubstituted at the 10 position with organo groups, which may be the same as or different from each other.
  • the organo groups may be hydrocarbon groups, but could also be organo groups containing one or more heteroatoms such as halogen, nitrogen, oxygen, sulfur and so forth.
  • Suitable hydrocarbon groups include, for example, aikyi groups and/or aryi groups.
  • the alkyl groups may be Ci- 06 aikyi groups, straight chain or branched, such as methyl, ethyl, propyl, butyi and the iike.
  • Suitable ary! groups include phenyl groups and other aromatic (including hetroaromatic) or conjugated groups.
  • the aikyi or aryi groups may be substituted, for example with halo groups or other heteroatom-containing groups.
  • the 10 position is substituted with two aikyi groups (e.g., two methyl groups), two aryi groups (e.g,, two phenyi groups), or both an alky! group and an aryl group (e.g., a methyl group and a phenyi group).
  • Having organo groups present as substituents at the 10 position of the ligand helps to improve the oxidative stability of the tetrapyrroie ligand and etai complexes thereof. If the organo groups are replaced by hydrogens, the ligand can rapidly decompose in the presence of air.
  • the 10,10-diorgano-5,15-diary!bi!adiene ligand is substituted with one or more substituents comprised of a water-solubilizing segment.
  • the water-solubilizing segment(s) help to improve the water solubility, and thus the biocompatibility, of the tetrapyrroie complex, owing to the hydrop ilicity of the water-solubilizing segment.
  • water-solubilizing segment refers to a segment (moiety) within the ligan that functions to increase the solubility in water of a complex comprising such ligand as compared to the solubility in water of a complex comprising an analogous ligand that is identical in structure except that it does not comprise such a segment.
  • the water-solubilizing segment is oligomeric or polymeric in character and is comprised of two or more hydrophilic repeating units.
  • Suitable water-solubilizing segments include, for example, poSy(oxyalkyiene) segments, polysaccharide segments, polypeptide segments, poiy(thioaikySene) segments, poly(aminoalkylene) segments, polyvinylpyrrolidone segments, aliphatic polyester segments, polyamide segments, polyvinyl alcohol segments, poiyacryiic acid segments, polyacrylamide segments, polyoxazo!ine segments, aliphatic polycarbonate segments, polyphosphate segments, and
  • the water-solubilizing segment may contain an average of at least three monomer units with a combined mass of at least 200 g/moi (in another embodiment, a combined mass of at least 300 g/moi).
  • the water solubility of the resulting tetrapyrro!e complex may generally be Increased.
  • the number average molecular weight of the water-soiub!izing segment may be as high as 50,000, 40,000, 30,000, 20,000, 10,000 or 5000 g/mo!, although the use of even higher number average molecular weight water-soiubiiizing segments is possible.
  • the ligand may hear one, two, three, four or more substituents comprising water-soiubiiizing segments and such substituent or substituents may be present at any position of the biladiene skeleton, provided such positioning does not interfere with the ability of the ligand to complex with a selected metal.
  • the substituent or substituents comprising a water-soiubiiizing segment such as a poiy(oxyaikyiene) segment, may for example be substituted on one or both of the aryl groups which are present at the 5 and 15 positions of the a,c-biiadiene framework of the ligand.
  • poiy(oxyaikyiene) segment or poiy(oxyaSkyiene) segments may be an aliphatic poiyether moiety containing repeating
  • oxyalkylene units such as oxymethylene, oxyethylene, oxypropyiene and oxybuty!ene units or combinations thereof (for example, oxyethylene/oxypropyiene).
  • Such poiy(oxyaikyiene) segments may be formed, for example, by the ring-opening polymerization of cyclic ethers (such as oxetanes, epoxides, and oxoianes) and/or by the condensation of aliphatic glycols such as ethylene glycol, propylene glycol and butylene glycol.
  • cyclic ethers such as oxetanes, epoxides, and oxoianes
  • condensation of aliphatic glycols such as ethylene glycol, propylene glycol and butylene glycol.
  • poly(oxyaikyiene) segment(s) may be poly(oxyethylene) segments.
  • poly(oxyethy!ene segments may correspond to the structure -(CHiCHaO)»-, wherein n is an integer from 3 to 250.
  • n may refer to the average number of oxyethyiene repeating units per segment.
  • n may be from about 3 to about 250 on average, for example.
  • the poly(oxyaiky!ene) segment may have a number average molecular weight of at ieast 200 g/moi
  • the at ieast one substituent comprised of a water-soiubiiizing segment such as a poiy(oxyaikylene) segment may be additionally comprised of a terminal group selected from the group consisting of alkyl groups and alkyl groups substituted with at least one functional group.
  • Suitable alkyl groups include, for, example, methyl, ethyl, propyl, butyl and the like, which may be straight chain, branched or cyclic.
  • Suitable functional groups include, without limitation, -SR, NF , CO:?R, ⁇ C( ⁇ 0)NR2, -SChR,
  • the at least one substituent comprised of at least one water-solubilizing segment may be additionally comprised of a linking moiety which links (directly or indirectly) the water-solubilizing segment to the bi!adiene skeleton of the ligand (for example, to an aryl group pendant to the biladiene skeleton). Any of the linking moieties known in the art may be employed.
  • the linking moiety may for example be selected from the group consisting of:
  • X is O, S, Se, Te, NH, NR, CHs, CHR, and CRi, with R being an organo group (e.g,, an alkyl group).
  • ⁇ 0 ⁇ GH? ⁇ triazoie- wherein a nitrogen atom of the triazole ring is covalently bonded to a carbon atom of the water-solubilizing segment
  • the at least one substituent comprised of at least one water-solubilizing segment may comprise at least one biologically active group (which may be a part of the water-solubilizing segment, Sinking moiety and/or terminal group, but may also be present in the water-solubilizing segment in addition to the water-solubilizing segment, linking moiety or terminal group).
  • biologically active group can be any group that selectively promotes the accumulation, elimination, binding rate, or tightness of binding in a particular biological environment.
  • one category of biologically active groups is the substituents derived from sugars, specifically, (l) aldoses such as glyceraldehyde, erythrose, threose, ribose, arablnose, xyiose, lyxose, ailose, altrose, glucose, mannose, gufose, idose, galactose, and ta!ose; (2) ketoses such as hydroxyacetone, erythrulose, rebulose, xylulose, pslcose, fructose, verbose, and tagatose; (3) pyranoses such as giucopyranose; (4) furanoses such as fructo- furanose; (5) O-acyl derivatives such as penta-O-acetyl-a-giucose; (6) O-methyl derivatives such as methyl a-giucoside, methyl p-giucoside
  • Amino add derivatives are aiso useful biologically active groups, such as those derived from valine, leucine, isoleudne, threonine, methionine, phenylalanine, tryptophan, alanine, arginine, aspartic acid, cystine, cysteine, glutamic add, glycine, histidine, proline, serine, tyrosine, asparagine and glutamine.
  • peptides particularly pHSip peptides, cell-penetrating peptides and those peptides known to have affinity fo specific receptors, for example, oxytocin, vasopressin, bradykinin, LHRH, thrombin and the like.
  • Another useful group of biologically active groups are those derived from nucleosides, for example, ribonudeosides such as adenosine, guanosine, cytidine, and uridine; and 2'-deoxyribonudeosides, such as 2‘-deoxyadenosine, 2 > -deoxyquanosine, 2'-deoxycytidine, and 2' ⁇ deoxythymidine,
  • Another category of biologically active groups that is particularly useful is any ligand that is specific for a particular biological receptor.
  • the term "ligand specific for a receptor’’ refers to a moiety that binds a receptor at ceil surfaces, and thus contains contours and charge patterns that are complementary to those of the biological receptor.
  • the ligand is not the receptor itself, but a substance complementary to it It is well understood that a wide variety of cell types have specific receptors designed to bind hormones, growth factors, or neurotransmitters. However, while these
  • iigand specific for a receptor refers to any substance, natural or synthetic, that binds specifically to a receptor.
  • iigands examples include: (1) the steroid hormones, such as
  • progesterone, estrogens, androgens, and the adrenal cortical hormones (2) growth factors, such as epidermal growth factor, nerve growth factor, fibroblast growth factor, and the iike; (3) other protein hormones, such as human growth hormone, parathyroid hormone, and the like; (4) neurotransmitters, such as acetylcholine, serotonin, dopamine, and the iike; and (5) antibodies. Any analog of these substances that also succeeds In binding to a biological receptor is also included.
  • the ary! groups substituted at the 5 and 15 positions of the 10,10-diorgano- 5,15-diarylbiiadiene ligand may be substituted phenyl groups having one or more substituents selected from the group consisting of halogen, alkyl, nitrogen-containing substituents (e,g., amine groups), sulfur-containing substituents (e.g., thiol, thio ether), oxygen-containing substituents (e.g., hydroxyl, carboxylate, hydroxyalky!, aikoxy) and combinations thereof, subject to the proviso that at least one of the substituted phenyl groups is substituted by at least one water-so!ubiiizing segment- containing substituent (such as a polyfoxyalkyiene) segment-containing substituent).
  • substituents selected from the group consisting of halogen, alkyl, nitrogen-containing substituents (e,g., amine groups), sulfur-containing substituents (e.g., thio
  • substituted phenyi group means a phenyl group which is substituted at at least one carbon of the aromatic ring with a substituent other than hydrogen.
  • ail substituents on the substituted phenyi groups other than water-solubilizing segment-containing substituents are halogen (e,g , fluorine).
  • a water-solubilizing segment-containing substituent such as a po!y(oxyalky!ene) segment-containing substituent, is attached to the position on the phenyi group which is para to the carbon atom which attaches the phenyi group to the 5 or 15 position of the T0, i0 ⁇ diorgano ⁇ 5,15-diaryibiSadiene ligand.
  • the 10,10-dlorgano-5,15 ⁇ diaryibiiadiene ligand may be substituted at one or both of the 2 and 18 positions with a n ⁇ conjugation-extending substituent
  • a x-conjugation-extending substituent is a substituent which extends the p-conjugation present within the 10,i0-diorgano-5,15 ⁇ dlaryibiladiene ligand.
  • the a-conjugation-extending substituent may be selected from the group consisting of carbonyl-containing substituents, imine- containing substituents, aromatic substituents, vinySaromatic substituents and ethynyiaromatic substituents.
  • Suitable aromatic substituents include phenyl groups, naphthyl groups, anthreny! groups and the like ⁇ including both unsubstituted and substituted versions thereof).
  • An ethynyiaromatic substituent may have structure -CoC-Ar, wherein Ar is an aryl group such as phenyl, naphthyl or anthreny!, which may be substituted or unsubstituted.
  • a rc-conjugation-extending substituent is present at the 2 and 18 positions of the 10,10-diorgano ⁇ 5,15 ⁇ diarylbi!adiene ligand. It is also possible for a single rc-conjugation-extending substituent to bridge between the 2 and 18 positions of the 10, lO-diorgano-5, ISdiaryibtiadiene ligand (thereby making the ligand macrocydic rather than linear).
  • Exemplary ethynyiaromatic substituents include:
  • Exemplary viny!aromatic substituents include:
  • one or more of the pyrrole rings may be substituted, particularly at the 2 and/or iS position of the biladiene, for example with substituents such as bromine or other halogen.
  • substituted means that a hydrogen atom otherwise present on the biladiene skeleton is replaced by a nonhydrogen substituent.
  • the tetrapyrrole complex has a structure corresponding to Formula (I):
  • each X is independently selected from the group consisting of hydrogen, halogen, alkyl, and water-soiubifizing segment-containing substituents, subject to the proviso that at least one X is a water-solubilizing segment-containing substituent, each R is independently subjected from the group consisting of hydrogen, halogen, and p-conjugation-extending substituents, and each R' is independently selected from the group consisting of afkyi groups and aryl groups,
  • M may be Pd or Ft or any of the other meta!s previously described.
  • R" and R' may independently be methyl or phenyl.
  • the poiy(oxyafkylene) segment may have a number average molecular weight of from 300 to 2000 g/mol.
  • the poly(oxyaiky!ene) segment may be a polyoxyethylene) segment.
  • the at least one substituent comprised of a poiy(oxyaikylene) segment-containing substituent may have a terminal group selected from the group consisting of alky! groups and alkyl groups substituted with at least one functional group.
  • poly(oxyaikylene) segment-containing substituent may be linked to a phenyl group through a linking moiety.
  • the linking moiety may, for example, be selected from the group consisting of
  • X is G, S, Se, Te, NH, NR, CM:. ⁇ , CHR, and CR2, with R being an organo group such as an aikyi group.
  • Unking moieties include:
  • one or two X groups may be poly(oxyaikylene) segment-containing substituents and the remaining X groups may be fluorine.
  • the water-solubiiizing segment-containing substitutent(s) e.g. ,
  • poiy(oxyaikyiene) segment-containing substituents) may be attached to the phenyl group in the para position.
  • Each R may be a i-conjugation-extending substituent selected from the group consisting of carbonyl-containing substituents, tmine- containing substituents, aromatic substituents, viny!aromatic substituents and ethynyiaromatic substituents.
  • the water-solubiiizing segment may have structure - (O- €H20) - wherein n is from about 3 to about 250 on average.
  • the poiy ⁇ oxyaikyiene) segment-containing substituent(s) may be selected from:
  • n is from about 3 to about 250 on average.
  • the tetrapyrrole complex is characterized by having a phenyi group of Formula (P) attached at one or both of the 2 position and the 15 position of the biiadiene skeleton:
  • each X is the same or different and is selected from the group consisting of hydrogen, halogen, alkyl, nitrogen-containing substituents (e.g., amine groups), sulfur- containing substituents (e.g., thio!, fchio ether), oxygen-containing substituents (e.g., hydroxy!, carboxy!ate, hydroxyaikyl, aikoxy) and combinations thereof, and Y is a poly(oxyalkylene) segment-containing substituent (in particular, a poly(oxyethylene) segment-containing substituent). According to certain embodiments, Y is:
  • n is from about 3 to about 250 on average.
  • n may be from 3 to 250 on average.
  • the leaving group can be a halogen, for example (e.g., fluorine), and can be positioned as a para substituent on a phenyl group.
  • the nucleophile may be a thiol-containing compound, such as mercaptoacetic acid, that either already contains a poiy(oxyaikyiene) segment or contains a functional group, such as a carboxylic acid group, that is capable of being derivatized to introduce a poiy(oxyaikyiene) segment.
  • the poly(oxyaikylene) segment can be provided in the form of an amino-functionaiized po!y(oxyaikyiene) glycol, wherein the amino functionality reacts with the carboxylic acid group to form an amide linkage.
  • Carbodiimide coupling chemistry may be used, for example, to convert a mercaptoacetic acid substituent into an N- (methoxyPEG)mercaptoacetamide.
  • An -SCFbCCbH functionalized complex may be initially reacted with Af-hydroxysucdnimide (NHS) and l-ethyi-3- ⁇ 3'- dimethylaminopropyOcarbodiimide hydrochloride (EDC) to form an NHS
  • Suitable amino-functionalized poiy(oxyaikyiene) glycols include, for example, amino-functionalized poly(oxyethyiene) glycois corresponding to Formula (III);
  • nucleophilic aromatic substitution reaction examples include any of the -1? nucleophiles known in the art of nucleophilic aromatic substitution, such as hydroxyl groups and primary or secondary amino groups.
  • Another way to link a water-solubilizing segment-containing substituent to an ary! group is to first react a leaving group (e.g., a halogen such as fluorine) on an aryl group attached to one or both of the 5 or 15 positions of the tetrapyrroie (biladiene) ligand backbone with propargyl alcohol to introduce a propargyl substituent on the ary! group, then react the ethynyl functionality of the propargyl substituent with an azide- functionalized reactant also containing a water-solubilizing segment such as a poly(oxyaikyiene segment). The ethynyl functionality reacts wit the azide group to form a triazoie linkage.
  • a leaving group e.g., a halogen such as fluorine
  • Suitable azide-functionalized poiy(oxyalkylene) glycols include, for example, azide-functionalized po!y(oxyethy!ene) glycols corresponding to Formula (IV):
  • the one or more water-solubilizing segment-containing substituents need not be substituted on an aryl group attached to one or both of the 5 or 15 positions of the tetrapyrroie (biladiene) ligand backbone. Reactive functionality anywhere in a starting tetrapyrroie ligand may be utilized for the purpose of introducing a water-solubilizing segment-containing substituent.
  • Such reactive functionality may include, for example, an ethynyl group, a primary or secondary amino group, a halogen group, or a carboxylic add group
  • a reactant containing both a functional group reactive with the reactive functionality present in the starting tetrapyrroie ligand and a water-solubilizing segment may be reacted with the starting tetrapyrroie ligand.
  • the tetrapyrroie complexes of the present invention may also be prepared by first preparing a suitable 10,10 ⁇ diorgano-5,X5-dtarylbiladiene ligand which bears at least one substituent comprised of a water-solubilizing segment and then reacting such ligand with a source of the metal to be complexed (for example, a metal salt).
  • a source of the metal to be complexed for example, a metal salt.
  • Such ligands may, for example, have a structure corresponding to that of Formula (1) wherein M is replaced by two hydrogens.
  • tetrapyrroie complexes disclosed herein can be prepared as or formulated into a formulation (pharmaceutical composition) suitable for use in the treatment, therapeutic, or diagnostic methods also described herein.
  • a formulation pharmaceutical composition
  • the tetrapyrroie complexes disclosed herein can be prepared as or formulated into a formulation (pharmaceutical composition) suitable for use in the treatment, therapeutic, or diagnostic methods also described herein.
  • the formulation pharmaceutical composition
  • formulations can further comprise one or more pharmaceutically acceptable
  • excipient(s) and/or carriers in addition to one or more tetrapyrroie complexes.
  • the pharmaceuticai!y-acceptab!e excipient(s) and/or carrier(s) can be administered with the tetrapyrroie complexes disclosed above as well as possibly other components such as nanoparticles which emit heat in response to laser light.
  • the formulation can be administered in vivo in a pharmaceutically acceptable carrier.
  • the use of water or other aqueous-based carrier is preferred in at least certain embodiments.
  • the tetrapyrroie complex may be fully or partially dissolved in the carrier.
  • pharmaceutically acceptable is meant a material selected to minimize any degradation of the active ingredient(s) and to minimize any adverse side effects in the subject, as would be well known to one of ski! in the art.
  • Suitable carriers and excipients are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa, 1995, An appropriate amount of a pharmaceuticalfy-acceptab!e salt may be used in the formulation to render the formulation isotonic.
  • Examples of pharmaceutically- acceptable carriers include, but are not limited to, saline, Ringer’s solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5, It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of tetrapyrroie complex and possibly other components being administered.
  • the formulations can be administered orally, parenterally (e,g,, via intravenous injection, intraperitonea! injection, intramuscular injection, intratumoral injection, intraarterial injection), transdermalfy, extracorporeal ly, topicai!y or the like, including by topical intranasai administration or administration by inhalant, or a combination thereof.
  • “topical intranasai administration” means delivery of the formulation into the nose and nasal passages through one or both of the nostrils and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosoiization of the formulation.
  • Administration of the formulation by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism.
  • Delivery can a!so be directly to any area of the respiratory system ⁇ e.g., lungs) via intubation.
  • the exact amount of the formulation required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the type of disorder or disease being treated, the location of the diseased tissue being treated, the particular tetrapyrroie complex, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every formulation. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein,
  • Formulations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emuisions.
  • non -aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oieate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils
  • intravenous vehicles include f!uid and nutrient replenishes, electrolyte replenishes (such as those based on Ringer’s dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration of the tetrapyrroie complexes can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be deslrabie.
  • the formulations can further include, in addition to one or more tetrapyrroie complexes
  • one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like
  • Nanoparticies which emit heat in response to laser light could also be Included in the formulations, so that tetrapyrroie complex(es) and such nanoparticies may be co-administered.
  • the disclosed tetrapyrroie complexes, and formulations comprising them, can be administered to an individual to kill endogenous tissue or cells.
  • the tissue can be undesirable tissue that has arisen due to transformation, such as a tumor, cancer, or endometriosis * adipose tissue; plaques present in vascular tissue and over-proliferation such as those formed in restenosis; birthmarks and other vascular lesions of the skin; scars and adhesions; and irregularities in connective tissue or bone, such as bone spurs.
  • the term "cancer” includes a wide variety of malignant solid neoplasms. These can be caused by viral infection, naturally occurring transformation, or exposure to environmental agents. Parasitic infections and infections with
  • organisms, especially fungai, that lead to disease may also be targeted.
  • the tetrapyrrole complexes of the present invention and formulations containing such complexes can be useful for causing photodynamic damage to cancer ceils.
  • Photodynamic damages to cancer cells include, but are not limited to, preventing or reducing the developmen of a cancer, reducing the symptoms of cancer, suppressing or inhibiting the growth of an established cancer, preventing metastasis and/or invasion of an existing cancer, promoting or inducing regression of the cancer, inhibiting or suppressing the proliferation of cancerous cells, reducing angiogenesis or increasing the amount of apoptotic cancer cells, thereby treating cancer
  • the methods can include contacting a cell with an effective amount of the tetrapyrroie complex or a formulation comprising the tetrapyrrole complex as described herein.
  • an amount can be considered therapeutically effective even if the condition is not totally eradicated but improved partially.
  • the formulations can be injected directly into the target tissue, or can be administered systemicaily. More specifically, the formuiations can be administered using any suitable method including intravenous (i,v,) intra peritonea I (i.p.), intramuscular (i.m.), intratumorai (i.t), intraarterial (i.a.), topically, and/or by inhalation. Intravenous administration is particularly preferred for solid tumors, while i.p. administration is preferred for pancreatic, !iver, and gastric tumors.
  • the tetrapyrrole complexes preferentially accumulate in the cancerous tissue, and preferably actively integrate in the cancerous tissue, as opposed to surrounding healthy tissue.
  • the disclosed methods can also include the application of external ionizing radiation for the purpose of exciting the tetrapyrrole complex.
  • the rate and time at which the cancerous ceils are irradiated may depend on the results required.
  • the cancerous cells can be Irradiated at an effective fluence rate and time to cause therapeutic injury resulting in the reduction of at least one of the surface area, the depth, and the amount of the tissue affecte by the cancerous condition.
  • the irradiation regime may also be dependent on the structure of the tetrapyrrole complex, the maximum safe dose of radiation that can be tolerated by the patient, or the targeted ceil or material.
  • Embodiments of the present invention are directed to methods of inhibiting the growth of cancer ceils, in vitro or in vivo , comprising the steps of contacting the cancer cris with a tetrapyrroie complex of the present invention, and exposing the cancer ceils to an effective amount of artificiai irradiation.
  • the invention provides methods of inhibiting the growth of cancer cells, such as breast, lung, pancreas, bladder, ovarian, testicular, prostate, retinoblastoma, Wilm's tumor, adrenocarcinoma or melanoma.
  • the subject may be a human, equine, porcine, bovine, murine, canine, feline, and avian subjects.
  • Other warm biooded animals are also included with the scope of this invention.
  • the present invention also provides a method for treating a subject suffering from cancer.
  • the subject may be a human, dog, cat, mouse, rat, rabbit, horse, goat, sheep, cow, chicken.
  • the cancer may be identified as a breast, lung, pancreas, bladder, ovarian, testicular, prostate, retinoblastoma, Wilm’s tumor, adrenocarcinoma or meionoma and is generally characterized as a group of cells which over-express and/or have an over-abundance of the target.
  • This method comprises the steps of
  • Also provided is a method of inhibiting the proliferation of mammalian tumor cells which comprises the steps of contacting the mammalian tumor cells with a sufficient concentration of the tetrapyrroie complex of the invention, and exposing the mammalian tumor ceils to artificiai irradiation.
  • the subject invention further provides methods for inhibiting the growth of human tumor ceils, treating a tumor in a subject, and treating a proliferative-type disease in a subject. These methods comprise the steps of administering to the subject an effective amount of the tetrapyrroie complex of the invention.
  • the present invention also provides for a method of treating a disease state comprising administering to a target tissue of a patient a tetrapyrroie complex of the present invention and irradiating the tetrapyrroie complex, which functions as a photosensitizer, thereby killing the target tissue. Irradiation of the tetrapyrroie complex can iead to generation of singlet oxygen in proximity to the target tissue.
  • the present invention encompasses formulations (pharmaceutical
  • compositions for use in the treatment of human carcinomas comprising a pharmaceutically effective amount of one or more tetrapyrroie complexes in accordance with the present invention and a pharmaceutically acceptable carrier.
  • formulations may additionally include other drugs or antibodies effective for treating carcinomas,
  • the tetrapyrroie complexes of the invention can be administered using conventional modes of administration including, but not limited to, intravenous, intraperitoneai, oral, intraiymphatfc, or administration directly into a tumor.
  • the tetrapyrroie compiexes of the invention may be provided in a variety of dosage forms which include, but are not limited to, liquid solutions or suspension, tablets, pills, powders, suppositories, polymeric microcapsuies or microvesicles, liposomes, and injectable or infusible solutions. The form depends upon, among other things, the mode of administration and the therapeutic application.
  • an effective dose of the tetrapyrrole compiexes of this invention may be in the range of from about 1 to about 2000 mg/kg.
  • the dosage can also be from about 2 to about 1000 mg/kg, about 4 to about 400 mg/kg, or about 5 to about 100 mg/kg.
  • Adjustments in the dosage regimen may be made to optimize the tumor cell growth inhibiting and killing response, e.g., doses may be divided and administered on a daily basis or the dose reduced proportionally depending upon the situation (e.g , several divided doses may be administered dally or proportionally reduced depending on the specific therapeutic situation).
  • the tetrapyrrole complex may be administered on a one time basis or on an as-needed basis, depending upon the patent's response to previously-administered doses.
  • the dose of the tetrapyrrole complex of the invention required to achieve cures or remission may be further reduced with schedule optimization.
  • the human or other subject is preferentially exposed to artificial irradiation which is selected from the group consisting of artificial ultraviolet, infrared (IR), gamma-irradiation, x-ray and visible Sight.
  • the irradiation is IR or near-infrared (NIR)
  • the artificial irradiation is light having a wavelength of from 350 to 1000 nm.
  • the artificial Irradiation can be applied about 5 minutes to about 3 hours after administering the tetrapyrroie complex of the present invention or the artificial irradiation is applied about 10 to about 60 minutes after administering the tetrapyrroie complex of the present invention.
  • the artificial irradiation in the methods of treating cancer of the present invention, can be applied for about 10 seconds to about 60 minutes, or the artificial irradiation is applied for about 15 seconds to about 30 minutes.
  • the present invention further provides
  • compositions which comprise the tetrapyrrole compiexes of the present invention and a pharmaceutically acceptable carrier.
  • the present invention further provides a method for treating cancer in a subject having cancer comprising the steps of administering to the subject a therapeutically effective amount of a tetrapyrroie complex in accordance with the present invention.
  • tetrapyrroie complexes of the present invention and formulations containing such compounds are also useful in applications other than dynamic phototherapy, such as diagnostic imaging.
  • the tetrapyrroie complexes of the present invention may be employed in combination with nanoparticles that are capable of emitting heat when irradiated (that is, materials in nanoparticulate form that are capable of converting light into heat, sometimes referred to as light-activated heating
  • nanoparticies Such combinations make possible the implementation of dual photothermai therapy/photodynamic therapy (FTT/PDT) for treatment of cancer and other disorders.
  • FTT/PDT dual photothermai therapy/photodynamic therapy
  • PTT using light-activated heating nanoparticies
  • PDT using tetrapyrroie complexes in accordance with the present invention
  • Dual PTT/PDT in accordance with the invention may primarily induce apoptotic cell death over necrosis at low light dosages
  • nanoparticies and tetrapyrroie complexes may be introduced into a subject, either consecutively or simultaneously, by a suitable means such as intravenous injection and allowed to accumulate within targeted tissue (such as a solid tumor) based on the enhanced permeability and retention (EPR) effect. Then, sources of artificial light effective to activate each of the administered components may be applied.
  • a 700 nm to 1000 n continuous wave laser and 3S0 to 600 nm wavelength light may be applied to activate the nanoparticies (e.g., nanoparticies having a silica core and a gold shell coated by polyethylene glycol) and the tetrapyrroie complex, respectively, to produce heat an singlet oxygen that is toxic to the surrounding cancer cells.
  • the irradiation source effective to activate the nanoparticies can comprise a single emission wavelength or a range of emission wavelengths.
  • the emission wavelength range can be a wavelength range that causes minimal or no cellular damage.
  • the emission wavelength range can be in the near-infrared wavelength range, e.g., from about 750 nm to about 1250 nm.
  • the irradiation source can comprise a single emission wavelength from about 750 nm to about 1250 nm, In some embodiments, the irradiation source can be a laser with a single emission wavelength of from about 750 nm to about 1250 nm, In some embodiments, the irradiation source can be a laser with an emission wavelength range of from about 750 nm to about 1250 nm, In some embodiments, the irradiation source can be an 808 nm diode laser.
  • nanopartides useful in such PTT/PDT treatment strategies is not particularly limited and any of the nanopartides known in the PTT field may be utilized, such as gold nanospheres, hollow gold nanocages, gold nanostars and gold nanorods.
  • Suitable nanopartides include nanopartides characterized by containing non-metaS!ic cores (e.g., silica cores) and metallic shells (e.g., gold shells), which are sometimes referred to as nanoshei!s. Other types of metal-containing nanopartides may also be employed.
  • nanopartides which do not contain metaf such as graphite nanopartides, graphene nanopartides, carbon nanotubes or other carbon-based nanopartides is aiso possible.
  • Suitable nanopartides may be from 10 to 300 nm in diameter or 100 to 200 n in diameter, for example.
  • the nanopartides may be coated with or conjugated to a hydrophilic or passivating material or ligand such as poSy ⁇ ethylene glycol) (PEG). Such treatments can increase the biocompatibility of the nanopartides.
  • the hydrophilic or passivating material may be attached to the outer surface of the go!d shell using gold- thiol conjugation chemistry.
  • the nanopartides can absorb wavelengths of light in the near-infrared (NIR) spectrum.
  • the nanopartides can absorb wavelengths of light between about 750 nm and about 1250 nm.
  • the nanopartides can have a maximum; absorption peak of about 800-810 nm (in other words, the nanopartides can have a UV-vis maximum absorption peak of about 800-810 nm).
  • a tetrapyrrole complex comprising a metal compiexed by a 10,10- diorgano ⁇ 5,15 ⁇ diaryibiiadiene ligand which bears at least one substituent comprised of a water-solubilizing segment.
  • Aspect 2 The tetrapyrrole complex of Aspect 1, wherein the water-solubilizing segment is selected from the group consisting of poly ⁇ oxyaikylene) segments, polysaccharide segments, polypeptide segments, poly(thioa!kylene) segments, poly(aniinoaikyiene) segments, polyvinylpyrrolidone segments, aliphatic polyester segments, polyamide segments, polyvinyl alcohol segments, poiyacrylic a d segments, polyacrylamide segments, poiyoxazoiine segments, aliphatic polycarbonate segments, polyphosphate segments, and polyphosphazene segments.
  • Aspect 3 The tetrapyrroie complex of Aspect I, wherein the water-solubilizing segment is a poly(oxyalkylene) segment.
  • Aspect 4 The tetrapyrroie complex of any of Aspects 1 to 3, wherein at least one aryl group substituted at the 5 or 15 position of the 10,10-diorgano-5,15- diarylbiiadiene ligand bears at least one substituent comprised of a water-solubilizing segment.
  • Aspect 5 The tetrapyrroie complex of any of Aspects 1 to 4, wherein at least one ary! group substituted at the 5 or 15 position of the 10,lQ ⁇ diorgano-5,15- diaryibiiadiene ligand bears at least one substituent comprised of a wafer-so!ubi!izing segment selected from the group consisting of poiy ⁇ oxyalkylene) segments,
  • polysaccharide segments polypeptide segments, poiy(thioa!kylene) segments, poiy(aminoaikylene) segments, polyvinylpyrrolidone segments, aliphatic polyester segments, polyamide segments, polyvinyl alcohol segments, po!yacrylic acid segments, polyacrylamide segments, polyoxazoiine segments, aliphatic polycarbonate segments, polyphosphate segments, and polyphosphazene segments.
  • Aspect 6 The tetrapyrroie complex of any of Aspects 1 to 5, wherein at least one aryl group substituted at the 5 or 15 position of the 10,10-diorgano-5,15- diarylbiladiene ligand bears at least one substituent comprised of a polyfoxyalkyiene) segment.
  • Aspect 7 The tetrapyrroie complex of any of Aspects 1 to 6, wherein the metal is Pd or Pt.
  • Aspect 8 The tetrapyrroie complex of any of Aspects 1 to 7, wherein the 10,10 ⁇ diorgano-5,15-diaryibi!adiene ligand Is a iG,lQ-dialky!-5,15-d!aryibi!adiene ligand, a IQ-alkybiQ-aryl-S/lS-diarylbiiadiene ligand, or a 10,10-diaryl-5,15- diarylbiiadiene ligand.
  • Aspect 9 The tetrapyrroie complex of any of Aspects 1 to 8, wherein the 10,10-diorgano-5,15-diary!biiadiene ligand is a iQ,10-dimethyl-5,15-d!arylbiladiene ligand, a 1G- methyl, lG-phenyS-5,15-diaryibiiadiene iigand, or a 10, 10-diphenyl- 5,15- diaryibiladiene iigand.
  • the 10,10-diorgano-5,15-diary!biiadiene ligand is a iQ,10-dimethyl-5,15-d!arylbiladiene ligand, a 1G- methyl, lG-phenyS-5,15-diaryibiiadiene iigand, or a 10, 10-diphenyl- 5,15- diaryibiladiene iigand.
  • Aspect 10 The tetrapyrroie complex of any of Aspects 1 to 9, wherein the water-soiubi!izing segment is a poly(oxyaikyiene) segment having a number average molecular weight of at least 200 g/mol,
  • Aspect 11 The tetrapyrroie complex of any of Aspects 1 to 10, wherein the water-solubilizing segment is a poly(oxyethy!ene) segment.
  • Aspect 12 The tetrapyrroie complex of any of Aspects 1 to 11, wherein the at least one substituent comprised of a water-solubilizing segment is additionally comprised of a terminal group selected from the group consisting of alkyl groups and alkyl groups substituted with at least one functional group selected from -SR, NR; ; , COzR, -C( ⁇ 0 ⁇ R?, -SOJR, -PO iR, -PRs + , or -N3 ⁇ 4 + , wherein each R is independently H or an organo group.
  • Aspect 13 The tetrapyrroie complex of any of Aspects 1 to 12, wherein the at least one substituent comprised of a water-solubilizing segment is additionally comprised of a linking moiety which links the water-solubilizing segment to the 10,10- dlorgano-5, 15 ⁇ diarylbiiadiene ligand.
  • Aspec 14 The tetrapyrroie complex of Aspect 13, wherein the linking moiety is selected from the group consisting of:
  • X is O, S, Se, Te, NH, NR, CH 3 ⁇ 4 CHR, and CFh, with R being an organo group such as an alkyl group.
  • Aspect 15 The tetrapyrroie complex of any of Aspects 1 to 14, wherein the aryl groups substituted at the 5 and 15 positions of the 10,lQ-diorgano ⁇ 5,15-diaryibiiadiene ligand are substituted phenyl groups having one or more substituents selected from the group consisting of halo, alkyl, oxygen-containing substituents, sulfur-containing substituents and nitrogen-containing substituents, subject to the proviso that at least one of the substituted phenyl groups is substituted by at Ieast one water-solubilizing segment-containing substituent.
  • Aspect 16 The tetrapyrroie complex of any of Aspects 1 to 15, wherein the ary! groups substituted at the 5 and 15 positions of the lG,iG-diorgano-5,15-diaryibiiadiene ligand are substituted phenyl groups, at leas one of the substituted phenyl groups Is substituted by a po!y(oxyaikylene) segment-containing substituent, and all substituents on the substituted phenyi groups other than poly(oxyalkyiene) segment-containing substituents are fiuorine.
  • Aspect 17 The tetrapyrroie complex of any of Aspects 1 to 16, wherein the lQ,lO-diorgano-5,i5-dtarylbiiadiene iigand Is substituted at one or both of the 2 and 18 positions with a p-conjugation-extending substituent
  • Aspect 18 The tetrapyrroie complex of Aspect 17, wherein the p-conjugafcion- exfcending substituent is selected from the group consisting of carbonyl-containing substituents, imine-containing substituents, aromatic substituents, vinyiaromatic substituents and ethynyiaromatic substituents,
  • Aspect 19 The tetrapyrroie complex of Aspect 1, wherein the tetrapyrroie complex has a structure corresponding to Formula (I):
  • each X is Independently selected from the group consisting of hydrogen, halogen, alkyl, and water-solubilizing segment-containing substituents, subject to the proviso that at least one X is a water- solubilizing segment-containing substituent, each R is independently subjected from the group consisting of hydrogen, halogen, and p-conjugation-extending substituents, and each R' is independently selected from the group consisting of alkyl groups and ary! groups.
  • Aspect 20 The tetrapyrroie complex of Aspect 19, wherein M is Pd or Pt
  • Aspect 21 The tetrapyrroie complex of Aspect 19 or 20, wherein R' and R' are independently methyl or phenyl.
  • Aspect 22 The tetrapyrroie complex of any of Aspects 19 to 21, wherein the water-solubilizing segment-containing substituent(s) compfise(s) at least one water- solubilizing segment selected from the group consisting of poly ⁇ oxyaikYlene ⁇ segments, polysaccharide segments, polypeptide segments, poly(thioalkylene) segments, poiy(aminoalkyiene) segments, polyvinylpyrrolidone segments, aliphatic polyester segments, polyamide segments, polyvinyl alcohol segments, polyacrylic acid segments, polyacrylamide segments, po!yoxazoiine segments, aliphatic polycarbonate segments, polyphosphate segments, and poiyphosphazene segments.
  • the water-solubilizing segment-containing substituent(s) compfise(s) at least one water- solubilizing segment selected from the group consisting of poly ⁇ oxyaikYlene ⁇ segments, polysaccharide segments, polypeptide segments, poly(thioal
  • Aspect 23 The tetrapyrroie complex of any of Aspects 19 to 22, wherein the water-solubilizing segment-containing substituent(s) comprise(s) at least one poiy(oxyalkyiene) segment.
  • Aspect 24 The tetrapyrroie complex of Aspect 23, wherein the at least one poiy(oxya!kyiene) segment has a number average molecular weight of at least 200 g/mo! ⁇ e.g., 300 to 5000 g/mo!).
  • Aspect 25 The tetrapyrroie complex of Aspect 23 or 24,. wherein the at least one poiy(oxyalkyiene) segment is a poiy(oxyethySene) segment.
  • Aspect 26 The tetrapyrroie complex of any of Aspects 23 to 25, wherein the water-solubilizing segment-containing substituent has a terminal group selected from the group consisting of alky! groups and aiky! groups substituted with at least one functional group.
  • Aspect 27 The tetrapyrroie complex of any of Aspects 19 to 26, wherein the water-solubilizing segment-containing substituent is linked to a phenyl group through a linking moiety.
  • Aspect 28 The tetrapyrroie complex of Aspect 27, wherein the linking moiety is selected from the group consisting of;
  • X is O, S, Se, Te, NH, NR, CHa, CHR, and CR2, with R being an organo group such as an alky! group.
  • Aspect 29 The tetrapyrroie complex of any of Aspects 19 to 28, wherein at least one X group Is a poSy(oxyaikySene) segment-containing substituent and the remaining X groups are fluorine.
  • Aspect 30 The tetrapyrroie complex of any of Aspects 19 to 29, wherein each R is a p-conjugation -extending substituent selected from the group consisting of carbonyl-containing substituents, imine-containing substituents, aromatic substituents, vinylaromatic substituents and ethynyiaromatic substituents.
  • Aspect 31 The tetrapyrroie complex of Aspect 25, wherein the
  • Aspect 32 The tetrapyrroie complex of any of Aspects 19 to 31, wherein the water-soiubiiizing segment-containing substituent(s) is or are selected from:
  • n is from about 3 to about 250 on average.
  • Aspect 33 A method of using a tetrapyrroie complex in accordance with any of Aspects 1 to 32, comprising administering the tetrapyrroie complex to a patient and, after a period of time, irradiating targeted tissue of the patient with an energy source that excites the tetrapyrroie complex thereby producing a desired therapeutic response in the targeted tissue.
  • Aspect 34 The method of Aspect 33, wherein the targeted tissue comprises tumor DCis.
  • Aspect 35 The method of Aspect 33 or 34, wherein the tetrapyrroie complex is administered intravenously or intratumoraiiy.
  • Aspect 36 The method of any of Aspects 33 to 35, wherein the tetrapyrroie complex is administered intravenously as an aqueous solution.
  • a process of photodynamic therapy for treatment of diseased tissues comprising a) delivering a formulation comprised of a tetrapyrroie complex in accordance with any of Aspects 1 to 32 and a pharmaceutically acceptable vehicle to diseased tissue at a specific treatment site; b) allowing the formulation to preferentially accumulate in the diseased tissue; and c) irradiating the specific treatment site with light of a sufficient power and wavelength to activate the tetrapyrroie complex.
  • Aspect 38 The process of Aspect 37, wherein the light has a wave length of from 350 to 1000 nm,
  • Aspect 39 The process of Aspect 37 or 38, wherein the process is carried out in coordination with photothermai therapy.
  • Aspect 40 The process of Aspect 39, wherein the photothermai therapy comprises embedding within the diseased tissue nanopartides which emit heat in response to laser Sight.
  • Aspect 41 The process of Aspect 40, wherein the nanopartides are comprised of a silica-containing core and a gold-containing shell.
  • Aspect 42 formulation useful for photodynamic therapy, comprising a tetrapyrroie complex in accordance with any of Aspects 1 to 32 and a pharmaceutically acceptable vehicle.
  • Aspect 43 The formulation of Aspect 42, wherein the pharmaceutically acceptable vehicle is comprised of water.
  • a tetrapyrroie ligand wherein the tetrapyrroie ligand is a 10,10- diorgano-5,15-diaryibiiadiene ligand which bears at ieast one substituent comprised of a water-solubilizing segment.
  • Air sensitive reaction were carried out under a nitrogen atmosphere using standard schienk techniques and flasks fitted with Suba- sea! ® rubber septa.
  • Solvents used in synthesis were of reagent grade or better, and anhydrous solvents were dried before use via passage through activated alumina.
  • Phosphate buffered saiine (PBS) solutions were obtained by dissolving one PBS tablet, purchased from Sigma-Aldrich, per 200 mi of Miliipor - water.
  • AH PBS solutions were 0.0027 M in potassium chloride, 0.137 M in sodium chloride, and had a pH of 7,4, Mercaptoacetic add and the methoxy-PEG-amine were purchased from; Sigma-Aldrieh.
  • Tnethyiamine, dirnethyiformamkle, methanol, chloroform, hexane and ethyl acetate were purchased from Fisher N-hydroxysuccinimtde was manufactured by Alfa Aesar l-Ethyl -3- ⁇ 3‘ -dimethy laminopropyl )carbodlimlde hydrochloride (EDC) and
  • ethyitnchiorosiiane were manufactured by Acros. Dichioromethane was purchased from Fisher or VWR. Ail deuterated solvents were purchased from Cambridge Isotopes Laboratories. Column chromatography was carried out using 40-63 pm silica gel from Siiicyde, Thin layer chromatography (TLC) was done on precoated glass plates from Siiicyde, ⁇ and visualized with UV light.
  • FeffD Bilt was prepared according to previously published procedures Ceii culture reagents, including cell culture media components and the Ala mar blue viability reagent, were purchased from VWR and Thermo Fisher, respect! ve!y.
  • Emission spectra were collected with an automated Photon Technology International (PTI) QuantaMaster 40 f!uorometer equipped with an LPS- 2208 lamp power supply, a 75-W Xenon arc lamp and a Hamamatsu R2658 photomultiplier tube.
  • Oxygen-free solutions of Pd£D BIIi] ⁇ PEG7s « in methanol or PBS were prepared in a nitrogen-filled glovebox and transferred to 1.0 cm pathiength quartz cuvettes with screw cap closures from Firefly Scientific. The solution of
  • the solution of Pd[DMBiIl]-PEG?so in PBS was prepared at a concentration of 80 pM such that its absorbance at 460 nm closely matched that of a 32 pM standard solution of [Ru(bpy) 3 j(PFe) 2 in acetonitrile at that wavelength.
  • the sample and standard were then excited at - 460 nm and emission was monitored from 515 - 1000 n .
  • Emission spectra of the methanol and PBS solutions of PdlDMBIilj-PEChs» were remeasured after exposure of the samples to air.
  • Ail reported spectra are the average of 5 individual acquisitions collected using a step size of 1 nm and an integration time of 0.25 sec.
  • Emission quantum yields were calculated using the following expression: and G are the emission quantum yields of the sample and reference, respectively, 4 and are the integrated emission intensities of the sample and reference, respectively, A and A f3 ⁇ 4f are the measured absorbances of the sample and reference at the excitation wavelength, and h and h ⁇ 3 ⁇ 4 ⁇ are the refractive indices of the solvents used to dissolve the sample and reference, respectively.
  • O production i methanol was measured by monitoring the attenuation of fluorescence from the trapping agent l,3 ⁇ dipheny!isobenzofuran (DPBF) as it reacted with ⁇ 0?to form a non -emissive product.
  • DPBF trapping agent
  • Ru(bpy).i](PFs)i was used as the standard ( ⁇ l ⁇ 0.81)
  • Cuvettes were prepared to contain 2.0 ml of methanol that was 1.0 mM in DP8F and 10.0 pM in either Pd D Bi!l]-PEG7soor Ru(bpy)33(PF6)2.
  • a third cuvette served as a control and contained only 2,0 ml of a 1,0 mM solution of DPBF in methanol.
  • SOSG Singlet Oxygen Sensor Green
  • DA-MB-23I Ceil Culture M DA- MB --231 cells were purchased from American Type Culture Collection (ATCC) and cultured in Duibeceo’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin- streptomycin. Ceils were cultured in T75 cell culture flasks and incubated at 37- ; C in a 5% Cth humidified environment.
  • ATCC American Type Culture Collection
  • DMEM Modified Eagle Medium
  • Ceils were cultured in T75 cell culture flasks and incubated at 37- ; C in a 5% Cth humidified environment.
  • Cells were passaged between flasks or into sample plates by detaching the ceils from the flasks with Trypsin-EDTA, diluting the ceils with complete medium, and counting the cells with a hemacytometer before transferring to a new flask or well plate.
  • MDA-M6-231 cells were plated at 25,000 cell s/we! I in 24-well plates and were Incubated overnight.
  • P O Siii3"FEG?si was diluted to 0, 0,5, 1.0, or 1.5 rnM in complete cell culture media, which was then added to cells. Well plates were Incubated with
  • ceils were plated at 15,000 ceils/ well in a glass bottom 8-well plate with a removable well chamber and were incubated overnight. A solution of Pd[OMBIII]-PEi3?s (1 M) diluted in ceil culture media was added to ceils, which were then incubated for 24 hr, Ceils were then fixed with 4% formaldehyde for 15 min and rinsed 3X with IX PBS. Ceils were stained with DARI and phaiioldan to visualize cell nuclei and F-actin on the
  • MDA-MS-231 cells were plated at 10,000 cel!s/weii In black- walled 96-well plates and incubated overnight. The ceils were then treated with Pd[D BIIi] ⁇ FEG7S0 at 0-5.0 mM concentrations for the dark toxicity assays or 0- 10,0 mM concentrations for the light exposure experiments.
  • Pd[D BIIi] ⁇ FEG7S0 at 0-5.0 mM concentrations for the dark toxicity assays or 0- 10,0 mM concentrations for the light exposure experiments.
  • isohematoporphyrin (I HP) or hematoporphyrin dihydrochlorlde (HPDC) were diluted to 0-3,0 mM or 0-700,0 mM for dark toxicity or for light exposure experiments, respectively.
  • Ail solutions of photosensitizers were prepared by diluting the dried tetrapyrrole complexes in complete cell culture media.
  • well plates were covered with aluminum foil to avoid light contamination and were incubated for 48 hr prior to A!amar blue viability assays.
  • plates were covered with aluminum foil and were incubated for 0 or 24 hr prior to light exposure.
  • the 96-weli plates were placed onto a LightPad 930 (Artograph) with a Song pass (l -- 525 nrn) filter for 0, 10, 20, or 30 min.
  • EDC dimethylaminopropyljcarbodiimide hydrochloride
  • the crude reaction mixture was diluted with extra CHaCh, washed 4 times with deionized water and once with brine, and dried over Ua?SQ
  • the solvent was removed via rotary evaporation, and the remaining residue was redissolved in 6 ml of DCM, Methoxy-PEG750-amine (82 mg, 109 pmol) and triefchyiamine (81 pi, 581 pmol) were added to the resulting solution, which was stirred under air at room temperature for 18 hrs, Following removal of the solvent via rotary evaporation, the crude product was redissolved In CHaCia, washed 4 times with deionized water and once with brine and dried over NazSC .
  • the mass spectrogram of Pd[DH8i!l]-PB67$o shows a distribution of peaks between m/z ratios of 700-1050 as well as a distribution between m/z ratios of 1300 - 1800,
  • the peaks at m/z ratios between 700 and 1050 are separated by 22 mass units (half the size of an ethyiene glycol monomer), and correspond to [M + 2Na] ;“ while the peaks at m/z ratios between 1300 and 1800 are separated by 44 mass units (the size of a single ethyiene glycol monomer), and correspond to [M + Na] ,
  • PDFDMBilBrs may be converted into a water soluble tetrapyrrole complex containing one or more poly(oxyethylene) segment-containing substituents in accordance with the present invention by utilizing the synthetic chemistry described herein for the preparation of PdiDM&ni ⁇ P ?m and Pd[DMBSI2J-d1P£Gs5o. Palladium 2,18-Bis ⁇ Aryl-ef:hyriyt) ⁇ :LQ,i0-d!metfiyl-S,lS ⁇
  • Pd[DMBii2] may be converted into a water soluble tetrapyrrole complex containing one or more po!y(oxyethylene) segment-containing substituents in accordance with the present invention by utilizing the synthetic chemistry described herein for the preparation of f»d[DMBiil]-PEG7so and Pd[PMB!i2]-diPEGsso.
  • Pd[DMBit2-*Bu] may be converted into a water soluble tetrapyrro!e complex containing one or more po!y(oxyethyiene) segment-containing substituents in accordance with the present invention by utilizing the synthetic chemistry described herein for the preparation of Pd[DMBill] ⁇ PE €;?so and Pd DMB!l2]-diPE6s 5 o.
  • Pd[DMBii2-:NH2] may be converted into a water soluble tetrapyrroie complex containing one or more poiY(oxyethyiene) segment-containing substituents in accordance with the present invention by utilizing the synthetic chemistry described herein for the preparation of PdCDMBHlj-PEGzso and Pd[ DMBii2J-diPE ⁇ 35so.
  • Pd[DMBH2-NMe2] may be converted into a water soluble tetrapyrro!e complex containing one or more po!y(oxyefhyiene) segment-containing substituents in accordance with the present invention by utilizing the synthetic chemistry described herein for the preparation of Pd [ M Bs ! 1 ] ⁇ EGzss and PdiDMBsl2i-diPEGsso.
  • dfOMBi ⁇ -N hz may be converted into a water soluble tetrapyrro!e complex containing one or more poly(oxyethy!ene) segment-containing substituents in accordance with the present invention by utilizing the synthetic chemistry described herein for the preparation of d
  • Pd[ DMBiH-OCHs] may be converted into a water soiubie tetrapyrroie complex containing one or more poiy(oxyethyiene) segment-containing substituents in accordance with the present invention by utilizing the synthetic chemistry described herein for the preparation of Pd[DMBiil]-PEG7so and PdEDMBillJ-diPEGssts.
  • Pd[DMBH2-Me Ester] may be converted into a water soluble tetra pyrrole complex containing one or more poiy(oxyethyiene) segment-containing substituents in accordance with the present invention by utilizing the synthetic chemistry described herein for the preparation of PdtDM&i!l]-P£G7so and Pd[DMBii2] ⁇ diPEG5 S0 .
  • Pd[DMBit2-*Bu Ester] may be converted into a water soluble tetrapyrroie complex containing one or more poiy(oxyethylene) segment-containing substituents in accordance with the present invention by utilizing the synthetic chemistry described herein for the preparation of Pd£DMBiil]-PEG?so and PdiDMBsi2]-diPEGsso.
  • Pd[DMBil2-CN] may be converted into a water soluble tetrapyrro!e complex containing one or more po!y ⁇ oxyethyiene) segment-containing substituents in accordance with the present invention by utilizing the synthetic chemistry described herein for the preparation of P fDMB lijHPBSTso and Pd[DMBi!2
  • Pd[ DMBH2-CF3] may be converted into a water soluble tetrapyrro!e complex containing one or more polytoxyethyiene) segment-containing substituents in accordance with the present invention by utilizing the synthetic chemistry described herein for the preparation of Pd£DMBiil2 ⁇ PEG7$o and Pd[DMBt!2]-diPEGsso.
  • Pd[DMBiI2-Fc] may be converted into a water soiubie tetra pyrrole complex containing one or more po!y(oxyethyiene) segment-containing substituents in accordance with the present invention by utilizing the synthetic chemistry described herein for the preparation of f»d[DMBiil]-PEG7so and Pd[PMB!i2]-diPEGsso.
  • Pd[DMB*t23 (127 mg, 0.13 mmol) was dissolved in 25 ml of tetrahydrofuran in a 100 ml round bottomed flask, Propargyl aicohol (0.083 ml, 1,3 mmol) was added to the solution followed by KOH (42 mg, 0.75 mmol).
  • the reaction headspace was evacuated, and the solution was heated to 40 °C for 16 hours.
  • the reaction was cooled to room temperature and diluted with 50 mL of ethyl acetate.
  • the organic solution was then washed once with deionized water, once with saturated NaHGCh solution, and once with brine.
  • the solution was then dried over NazSO*, followed by removal of solvent via rotary evaporation.
  • Pd[DMBil23-bis(propargyi) (103 mg, 0,1 mmol), copper(II) su!fate pentahydrate (48 mg, 0.2 mmol) and sodium ascorbate (60 mg, 0.3 mmoi) were dissolved in 10 ml of tetrahydrofuran solution containing PEGsso-azide (326 mg, 0.54 mmol) in a 20 ml Scintillation vial. Via! was capped with a septum and the reaction headspace was evacuated and the solution was stirred at 55 °C, for 16 hours. The solution was then cooled to room temperature and poured over 100 ml of deionized water in a 250 mL separatory funnel.
  • phosphorescence from the PEGyiated derivative in PBS may be attributed to shortening of the triplet excited state lifetime via energy transfer to an FGO overtone.
  • Pd[BMBH13 SG7S0 to sensitize singlet oxygen was determined to 0.23 upon irradiation with 550 nm light
  • the apparent attenuation of F & in PBS relative to that in methanol is not surprising given that detecting 3 th in aqueous environments is more challenging due to its shorter lifetime as well as the lower solubility of oxygen in water as compared to organic solvents.
  • the aqueous F 3 ⁇ 4 measured for Pd[lDMBilt3 ⁇ PE €l7 ⁇ a is high enough to enable PDT in biological samples (vide infra).
  • TNBC Triple negative breast cancer
  • TNBC patients are unsusceptible to available targeted or hormonal therapies because the cells in these tumors do not express the necessary surface receptors. Therefore, these patients are treated with aggressive chemotherapies and surgeries that have harmful side effects and are often unsuccessful, necessitating the development of new treatment strategies for this disease. Due to its high potency and specificity, PDT has been recognized as a promising therapeutic approach for TNBC.
  • TNBC MDA-MB-231 ceils were treated with up to 5 0 mM P €f[BMBilt]-PE €s75o for 48 hrs in the dark and then subjected to an Aiamar blue cel I viability assay. MDA-MB-231 ceils were completely viable a Pd DMBIll ⁇ -PEG?so concentrations up to 0,5 mM, Further, the lethal dose required for 50% cell death was found to he LDso - 1,87 mM, an
  • Pd[DMBill]-PEG75o is iess toxic than two commercially available photosensitizers suggesting that it may ha used as a photoche otherapeutic without causing off-target side effects that often limit the efficacy of PDT agents.
  • MDA-MB-231 cells were treated with up to 1.5 mM PdED BiiiJ- EGyse for 48 hr and the cellular fluorescence was analyzed by fluorescence imaging and
  • MFI photochemotherapeutic agent
  • M DAMS- 231 T BC cells were treated with the biiadiene complex at concentrations ranging from 0-10.0 pH for 4 hours. At each concentration surveyed, cells were irradiated ⁇ l «* > 500 nm) for either 0, 10, 20 or 30 minutes. Viability assays were conducted to assess cell death 16 hours after irradiation. Ceils incubated with Pd[ DMBillJ-FBG-reo for 24 hours prior to irradiation were highly susceptible to PDT-mediated celi death compared to ceils irradiated immediately after adding Pd[DM ⁇ ili] ⁇ -BEG?sfs. More specifically., cells irradiated immediately following the addition of Pd [DM BH1] ⁇ PEG7SO required concentrations of at least 1 mM and 30 in of Sight exposure before any loss of cell viability was observed. By contrast, cells Incubated with
  • the phototoxicity index is determined to be PI ⁇
  • the phototoxicity index of Pg£DM8il 13 ⁇ PE ⁇ »?so is quite impressive as it is approximately 20Gx and 3000x higher than those of HPDC and IHP, respectively.
  • MDA-MB-231 cells were treated with 4.0, 6.0, or 8.0 pH Pd[DHBsf iJ » PEG?s» for 24 hours, irradiated for 30 min (l ® ⁇ > 500 nm) and then incubated for 1 hour prior to AnnexinV (FITC channel) and Pi (PerCP channel) staining. Control experiments in which none of the biiadiene photosensitizer were added to the ceils were also carried out as controls. For these experiments, the MDA-M8-231 cells were treated with higher PdtDMBiilj-PEG so concentrations than in the viability experiments because the cells were analyzed only 1 hour post light treatment (as opposed to overnight).

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Abstract

Les complexes de tétrapyrrole de métaux solubles dans l'eau sont utiles en thérapie photodynamique, les complexes étant capables de fonctionner en tant que photosensibilisateurs qui, activés par la lumière, conduisent à la génération d'oxygène singulet. L'oxygène singulet ainsi produit est toxique pour les cellules cancéreuses. Les ligands de tétrapyrrole présents dans ces complexes sont caractérisés par la présence d'au moins un segment de solubilisation dans l'eau (qui peut être présenté, par exemple, sous la forme d'un poly (oxyalkylène) contenant un substituant), ce qui aide à favoriser la biocompatibilité du complexe.
PCT/US2019/045250 2018-08-06 2019-08-06 Complexes tétrapyrrole solubles dans l'eau contenant des ligands de biladiène utiles en thérapie photodynamique Ceased WO2020046543A2 (fr)

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