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WO1997034637A2 - Therapie photodynamique dans laquelle des recepteurs d'hormones nucleaires sont utilises pour cibler des photosensibilisateurs - Google Patents

Therapie photodynamique dans laquelle des recepteurs d'hormones nucleaires sont utilises pour cibler des photosensibilisateurs Download PDF

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Publication number
WO1997034637A2
WO1997034637A2 PCT/US1997/004542 US9704542W WO9734637A2 WO 1997034637 A2 WO1997034637 A2 WO 1997034637A2 US 9704542 W US9704542 W US 9704542W WO 9734637 A2 WO9734637 A2 WO 9734637A2
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conjugate
photosensitizer
die
cells
nuclear hormone
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PCT/US1997/004542
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WO1997034637A3 (fr
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Scott C. Mohr
Rahul Ray
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Trustees Of Boston University
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Priority to AU25387/97A priority Critical patent/AU2538797A/en
Publication of WO1997034637A2 publication Critical patent/WO1997034637A2/fr
Publication of WO1997034637A3 publication Critical patent/WO1997034637A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines

Definitions

  • This invention is directed to ligand-photosensitizer conjugates and their use in photodynamic therapy.
  • this invention involves conjugation of photosensitizing agents to hormones which bind to receptors that transport them to intracellular receptors, and to photodynamic therapy of tumors characterized by the presence of these receptors.
  • cancer chemotherapy represents the major example ofthe latter procedure, with the objective being to eliminate tumor cells selectively.
  • the degree of selectivity of cancer chemotherapeutic agents varies, but typically is far from perfect, with the result being numerous very unpleasant side effects (e.g., nausea, hair-loss, weakness, anemia, etc. ). In fact, the need to restrict these side-effects limits the dosage and duration of most chemotherapeutic anti-cancer regimens.
  • Photodynamic therapy is a relatively new approach to selective cell killing which already has established itself as a useful tool for treating a variety of inoperable cancers (chiefly ofthe larynx, lung, bladder and face) as well as some forms of skin cancer.
  • the basis for this method is selective uptake of a suitable light-absorbing material into the tumor cells followed by irradiation with visible light. Absorption of light produces excited states of the colored substance(s) which in turn transfer their excitation energy to adjacent oxygen molecules, converting them into singlet-state oxygen, a highly reactive (and therefore quite toxic) chemical species.
  • the colored (chromophoric) material thus acts as a photosensitizer: in its presence a sufficient dose of visible light kills cells; absent the photosensitizer, the light has no hairnful effects.
  • a sensitizer is designed to target only certain types of cells, and secondly, the harmful effect will only be expressed when and where visible light is applied.
  • Successful application of a photosensitizer not only depends upon the degree to which it localizes selectively in the desired cell type, but also upon its photophysical properties. For example, a high yield of excited-state triplets and maximum light absorption in the range of 650-800 nm, is preferred.
  • PDT agent also called hematoporphyrin derivative or HPD
  • clinicians capitalize on the selective uptake (and retention) of the sensitizer by tumors, waiting for the attainment of an optimal ratio of the sensitizer concentration in tumor cells to the concentration in normal tissues. Then they irradiate the tumor as selectively as possible (often with skillful use of fiber-optic devices), achieving a very highly selective kill of tumor cells with minimal damage to normal cells. Such damage as does occur for normal tissues takes place only in the vicinity of the tumor, in contrast to the systemic side- effects of ordinary chemotherapeutic agents.
  • this invention provides a method for selectively killing cells having a nuclear hormone receptor by photodynamic therapy (PDT), comprising administering to a patient a conjugate comprising a photosensitizer moiety coupled to a ligand moiety which specifically binds the nuclear hormone receptor and illummating tissue containing the cell with hght having a wavelength absorbed by the photosensitizer moiety, preferably a wavelength from about 600 nm to about 850 nm.
  • the ligand moiety is a cognate nuclear hormone of the nuclear hormone receptor, or a derivative or analog thereof exhibiting at least one biological activity associated with binding to the nuclear hormone receptor.
  • the method preferably includes a waiting period after administering the conjugate for a time interval sufficient to allow physiological clearance of the conjugate from cells and tissues not expressing the nuclear hormone receptor, whereby the concentration of conjugate in the cells having the nuclear hormone receptor is greater than the concentration of conjugate in cells not having the nuclear hormone receptor (i.e., there is a therapeutic ratio having higher concentration in the target cells).
  • this invention provides a conjugate comprising a photosensitizer moiety coupled to a ligand which specifically binds to a member of the nuclear hormone receptor superfamily, the coupling preferably being by a covalent linkage, more preferably by a covalent linkage which is uncharged at physiological pH.
  • the covalent linkage includes from 1 to 10 methylene groups.
  • the conjugate according to this invention contains a ligand which is an agonist or antagonist of a receptor of the nuclear hormone receptor superfamily, more preferably, the hgand is an estrogen, androgen, progestin, glucocorticoid, mineralocorticoid, thyroid hormone, retinoid, or member of the vitamin D family.
  • the preferred photosensitizer moiety absorbs light having a wavelength of from about 600 nm to about 850 nm; more preferably, the photosensitizer is selected from the group consisting of chlorins, purpurins, benzoporphyrins, phthalocyanines, and porphines.
  • conjugate according to this invention is membrane-permeable, usually determined either by uptake of the conjugate by mammalian cells in vitro or by the conjugate having a permeability coefficient for a lecithin black lipid membrane of at least IO "10 cm/sec.
  • the present invention exploits a novel mechanism for photosensitizer localization, namely interaction with the Wgh-affinity receptors which mediate the hormonal signals transmitted by steroids (and some other hormones such as thyroxine, retinoids, and members of vitamin D family). These receptors are expressed only in specific cell types — and by their expression they confer hormone sensitivity on those cells.
  • This invention provides hormone/chromophore conjugates which have reasonable binding affinity towards the hormone receptor protein and methods of administering them to patients as specific photosensitizing agents which can direct lethal damage towards receptor-positive cell lines upon irradiation with visible hght. These hormone/chromophore conjugates bound to nuclear hormone receptors can be used as selective molecular delivery systems for photodynamic therapy.
  • Figure 1 shows the abso ⁇ tion spectrum of chlorin e 6 .
  • Figure 2 shows the molecular structure and absorption spectrum of another well-studied PDT chromophore, benzopo ⁇ hyrin derivative monoacid (BPD-MA).
  • Figure 3 shows laser dyes which can be coupled to estrogens in a fashion analogous to that for chlorin e 6 and BPD-MA.
  • Figures 4a and 4b show reaction schemes for syntiiesis of
  • ECC-1 and ECC-2 respectively.
  • FIG. 5a details a suitable synthetic scheme for testosterone- chlorin conjugate (TCC-1). This compound targets the chlorin photosensitizer moiety to androgen-receptor (AR)-positive cells.
  • Figure 5b shows a suitable synthetic scheme for preparation of tamoxifen-chlorin e 6 conjugate.
  • Figure 5c shows structures of representative photosensitizer conjugates.
  • Figures 6a and 6b are bar graphs of the dose respose of OVCAR-3 cells to CDME and ECC- 1, respectively.
  • Figures 7a and 7b are line graphs of the dose respose of OVCAR-3 cells to CDME and ECC-1, respectively.
  • Figures 8a and 8b are bar graphs ofthe dose respose of MCF-7 cells to CDME and ECC-1, respectively.
  • Figures 9a and 9b are line graphs ofthe dose respose of MCF- 7cells to CDME and ECC-1, respectively.
  • Figures 10a and 10b are bar graphs of the dose respose of EJ bladder cells to CDME and ECC-1, respectively.
  • Figures 11 a and 1 lb are line graphs of the dose respose of EJ bladder cells to CDME and ECC-1, respectively.
  • FIG 12 shows structures of ECC-2 and various related compounds and their equilibrium constants for binding to human estrogen receptor DETAILED DESCRIPTION OF THE EMBODIMENTS
  • Photodynamic therapy is a relatively new approach in treatment of cancers and has already established itself as a useful tool in the treatment of certain cancers, for example, lung, bladder and some forms of skin cancer.
  • photoactivable compounds called photosensitizers
  • the photosensitizers are activated (and cause tumor cell killing) when they are irradiated at the appropriate wavelength in the visible light region.
  • Absorption of light produces excited states ofthe photosensitizers which in turn transfer the excitation energy to adjacent oxygen molecules, converting them into the highly reactive and toxic singlet-state oxygen. Tumor destruction is generally believed to occur via the singlet-state oxygen or by the production of cytotoxic radical species from the photosensitizer itself.
  • PDT has an advantage over other anti-tumor therapies because of its selectivity: (1) sensitizers are designed which only target certain cell types 1 and (2) only those sites are affected where light with the appropriate wavelength is applied. The optimal ratio of sensitizer concentration in 5 tumor cells to the concentration in normal tissues is awaited before OTa ⁇ ating the tumor as selectively as possible. This way a highly selective killing of the tumor cells is achieved with rninimal damage to the normal tissues.
  • any type of PDT depends upon preferentially 10 localizing the photosensitizer in the target tissue or cell type, followed by selective irradiation ofthe target tissue.
  • a significant unwanted side-effect of PHOTOFRIN (PF) is prolonged cutaneous phototoxicity, necessitating prolonged protection from sunlight. Therefore, photosensitizers with more selective tumor-localizing properties are needed.
  • sensitizers In attempts to control d e 15 localization of photosensitizing chromophores, sensitizers have been bound to antibodies, microspheres, and antibody-bound lipoproteins.
  • the present invention provides another mechanism to localize photosensitizers, the interaction with the high-affinity receptors which mediate the hormonal signals transmitted by steroids and a variety of other 20 low molecular weight bioactive substances. These receptors are expressed in specific cell types and thus confer hormone sensitivity on those cells.
  • the nuclear hormone receptors can function as localizing agents to concentrate ligand-photosensitizer conjugates in particular cells. 25 Photodynamic therapy (PDT) involving illumination of these localized
  • photosensitizers are being investigated preclinically, both cationic and anionic.
  • a mixture of anionic porphyrins called Hematoporphyrin derivative (HPD) or PHOTOFRIN (PF)
  • HPD Hematoporphyrin derivative
  • PF PHOTOFRIN
  • Conjugates according to this invention are made up of a photosensitizing chromphore coupled to a ligand moiety corresponding to a low-molecular-weight lipophilic compound which is specifically bound by a member of the nuclear receptor gene superfamily.
  • the nuclear hormone receptor "superfamily” includes the proteins specific for response to the following hormones (1) estrogen (estradiol, estriol, and estrone), (2) androgen (testosterone and dihydrotestosterone), (3) progesterone, (4) mineralocorticoid (aldosterone), (5) glucocorticoid (cortisol and cortisone), (6) thyroid hormones, (7) retinoic acid, (8) vitamin D, and (9) ecdysone (insects). To date more than 150 members of the nuclear receptor superfamily have been identified, including the ones just mentioned ⁇ and their isoforms - as well as a large number of "o ⁇ han” receptors (for which there is as yet no known ligand).
  • conjugates contain ligand moieties that bind to members of the thyroid hormone receptor subfamily.
  • conjugates contemplated by this invention contain ligands which are specifically recognized by one of the steriod hormone receptors, such as estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), glucocorticoid receptor (GR), etc.
  • the mechanism of steroid hormone action consists of (a) secretion of steroid by the source tissue (ovary in the case of estrogen, for example), (b) transport through the plasma (mediated by albumin, transcortin and/or other lipophilic carrier proteins), (c) uptake through the plasma membrane of cells by simple diffusion, (d) non-covalent binding to the cognate receptor (if present) with concomitant activation of the receptor, and finally (e) interaction of the activated hormone/receptor complex with target sequences in nuclear DNA.
  • hormone receptor proteins normally reside in the cytosol of target cells until their cognate hormone appears, at which point they undergo a conformation change which exposes a nuclear localization signal that causes migration into the nucleus where they bind to appropriate hormone response elements (HRE's) in the control regions of their target genes.
  • HRE's hormone response elements
  • unliganded receptors may already be present in the nucleus when they bind to the cognate hormone.
  • a cell expressing die receptor can be made very vulnerable to irradiation in the abso ⁇ tion band ofthe chromophore.
  • the hormone derivative would also, of course, have to be successfully delivered to the target tissue and enter the cells.
  • die invention as described herein encompasses as ligands in the ligand-photosensitizer conjugate all of the vertebrate hormones which act via the same general mechanism.
  • die invention as described herein encompasses as ligands in the ligand-photosensitizer conjugate all of the vertebrate hormones which act via the same general mechanism.
  • protein structures of all this diverse family of receptors strongly suggesting a common evolutionary descent, indicating diat a methodology developed successfully to exploit any one of them as delivery vehicle for photosensitizer(s) could be transferred with minor modification to die odiers.
  • diis invention contemplates PDT med ods using conjugates which specifically bind receptors for (a) estrogens (estradiol, estriol, estrone, and synthetic analogs such as DES and tamoxifen); (b) androgens (testosterone, dihydrotestosterone, and synthetic analogs); (c) progestins (progesterone and synthetic analogs); (d) mineralocorticoids (aldosterone and analogs); (e) glucocorticoids (cortisol, cortisone, and analogs); (f) thyroid hormone; (g) retinoids (retinoic acid and analogs); and (h) vitamin D (cholecalciferol and related compounds).
  • estrogens estradiol, estriol, estrone, and synthetic analogs
  • androgens testosterone, dihydrotestosterone, and synthetic analogs
  • progestins progesterone and synthetic analogs
  • mineralocorticoids aldosterone and
  • d e insect hormone ecdysone acts via the same general mechanism and would be potentially useful as a ligand for model studies.
  • the ligands contemplated by diis invention also include analogs of d e hormones which bind to receptors of die nuclear hormone receptor superfamily, both syndietic and naturally-occurring analogs.
  • Analogs widiin d e contemplation of diis invention are agonists or antagonists of the cognate hormone of die respective nuclear hormone receptor. Agonists and antagonists will exhibit at least one biological activity associated with die nuclear hormone receptor.
  • Biological activity as contemplated herein relies on binding of a ligand for die particular receptor witii an affinity that is competitive with the cognate hormone.
  • Binding by an antagonist may simply block the hormone binding site to preclude activation ofthe receptor by the hormone.
  • Agonists will bind to the receptor in a manner that promotes some or all of die physiological activities associated with hormone action.
  • Photosensitizer conjugates according to this invention will act as agonists or antagonists of the respective nuclear hormone receptor.
  • agonist or antagonist activity is determined using in vitro assays specific for die respective hormones.
  • tiiese in vitro assays involve culturing cells known to be responsive to the hormone and measuring characteristic responses of tiiese cells to die particular hormone in culture. When the analog is included in such an assay, the activity measured for a particular hormone concentration will differ from that measured in the absence of the analog.
  • analogs may be assayed by simple binding assays which show an affinity which is competitive with die hormone, either by quantitative comparison or direct competition in the assay. Binding assays based on optical or fluorescent detection ofthe conjugates of this invention are facilitated by die presence ofthe photosensitizer. Preferred conjugates will bind to dieir receptor with an affinity tiiat is within an order of magnitude of die affinity ofthe cognate hormone.
  • die estrogen receptor ER which binds estradiol, estriol, estrone and synthetic estrogen agonists/antagonists such as diethylstilbestrol and tamoxifen.
  • PDT agent Successful application of a PDT agent depends upon (a) its photophysical (chromophoric) characteristics and (b) die degree to which it localizes selectively in die desired cell type.
  • the desirable photophysical properties include a high yield of excited-state triplets (which insures efficient generation of singlet oxygen), and maximum light abso ⁇ tion in the range of 650-800 nm (where normal mammalian tissue exhibits maximum transparency).
  • photosensitizer compounds possessing tiiese properties have been identified and studied witii a view to using them in PDT protocols. (See Amato, (1993) Science, 262: 32-33, and Moan (1990), J. Photochem. Photobiol.
  • Photosensitizers are substances which absorb light (normally in the visible range) and cause otiier substances (substrates) to undergo chemical reactions under conditions where they would not react in the absence ofthe sensitizer. Specifically, the substrates are unaffected by light in die absence of die sensitizer.
  • photosensitizers are considered to act either by a so-called Type I mechanism which involves direct reaction between the sensitizer and die substrate, or by a Type II mechanism in which die excited state ofthe photosensitizer transfers energy to ambient O 2 molecules, converting them from their ground (triplet) state to an excited (singlet) state which in turn reacts vigorously and almost indiscriminately witii surrounding organic molecules.
  • Preferred photosensitizers act via a Type II process, and more preferred photosensitizers are chosen (in part) to have a high quantum yield for singlet oxygen formation (0.7 in the case of chlorin e 6 , for example).
  • Photosensitizers of this invention preferably have a quantum yield for singlet oxygen formation of from 0.2-0.9, preferably at least 0.2, more preferably at least 0.4.
  • Photosensitizing chromophores according to this invention will absorb light of wavelengths which enhance both tissue penetration and singlet oxygen formation, usually from about 600-850nm, more preferably from 650-800nm (see Moan, 1990). It will be apparent to the skilled artisan that preferred chromophores will absorb hght having a wavelength produced by a laser suitable for use in PDT.
  • Preferred chromophores include those which have already demonstrated effectiveness in PDT.
  • One such molecule, chlorin e 6 has a useful abso ⁇ tion band at 650 nm (cf.
  • FIG 1 can be obtained as an ediylene diamine derivative (CMA, Figure 1) which affords an particularly effective tether (when combined with the propionyl group ofthe chlorin e 6 itself).
  • CMA ediylene diamine derivative
  • a variety of approaches to coupling the estrogen to die chromophore may be used, including simple reductive amination with sodium eyanoborohydride.
  • FIG. 2 shows the molecular structure and abso ⁇ tion spectrum of another well-studied PDT chromophore, benzopo ⁇ hyrin derivative monoacid (BPD-MA). It has the advantage of wavelength of maximum abso ⁇ tion in die red region somewhat longer tiian chlorin e 6 .
  • BPD-MA benzopo ⁇ hyrin derivative monoacid
  • this chromophore may be coupled to estrone in essentially the same fashion as indicated for chlorin e 6 .
  • This invention further contemplates use of a variety of different PDT agents witii varying pharmacokinetic and/or photophysical properties.
  • Figure 3 shows a selection of Kodak laser dyes which may be coupled to estrogens or other ligands in a fashion analogous to that for chlorin e 6 and BPD-MA.
  • Particularly suitable chromphores are lipophilic rather than hydrophilic. Lipophilic chromphores have been reported to be more effective sensitizers than hydrophilic chromophore, perhaps because hydrophobicity of die chromphore will facilitate transport of the chromophore through the plasma membrane of the target cell. Such chromophores will ease passage ofthe conjugate of this invention through the plasma membrane which is particularly important for concentration of the conjugate by binding to intracellular receptors.
  • chromophores which have been studied for use in various PDT protocols have properties which make them suitable as the chromophore (photosensitizer moiety) of the conjugates of this invention.
  • chromophores include chlorins, pu ⁇ urins, benzopho ⁇ hyrin derivatives, phthalocyanines, po ⁇ hines, and lipophilic dyes, such as rhodamine (see, e.g., Pass, 1993, pp. 446-447). Selection of suitable chromophores (photosensitizer moieties) meeting the criteria disclosed herein is a routine matter for the skilled artisan. Conjugation of Ligands and Photosensitizers
  • conjugate is used to describe a molecule which contains a hormone (or ligand) moiety coupled to a photosensitizer moiety such that both moieties retain tiieir intrinsic properties of significance for the intended tiierapeutic function.
  • Coupling will generally be by covalent linkage, although non-covalent connections may be used, so long as die coupling is as least as stable as the hormone- receptor complex, and the conjugate satisfies the other criteria described herein.
  • the mtrinsic properties of significance for PDT include, in die case of the hormone/ligand, high affinity for the targeted nuclear hormone receptor, and in the case of the photosensitizer, high quantum yield for singlet O 2 production at the chosen wavelength of irradiation. It is to be understood tiiat such "conjugates" are well-defined, pure chemical compounds of known structure, which may be contrasted with the mixtures of photosensitizers (HPD, PHOTOFRIN I) which were used in early photodynamic therapy experiments.
  • Conjugates according to this invention may be designed to use nuclear hormone receptors for targeting agents in PDT, by (a) preparing suitable conjugates between the receptor ligand and the photosensitizing moiety; (b) demonstrating uptake of such an agent into cells; (c) demonstrating preferential uptake of the agent into the desired cell tissue type only so as to establish a therapeutic ratio of at least 2: 1; (d) showing that irradiation of such cells kills them; and (e) proving that the metiiod works in whole animals.
  • Conjugates preferred for use with targeted laser ihurnination as discussed below will be concentrated (e.g., by binding to tiie cognate receptor) to provide tiierapeutic ratios (concentration in target tissue/concentration in surrounding normal, non-malignant, non-target tissue) of from at least about 2, more preferably at least about 10.
  • Particularly preferred photosensitizer conjugates will be concentrated in the target tissue to give a tiierapeutic ratio of at least about 25: 1 (see Moan, 1990, pages 522 and 523).
  • die steroid hormones tiiere appears not to be a specific membrane transport system to allow uptake into cells - they seem simply to diffuse through the plasma membrane. That means that a steroid- sensitizer conjugate witii suitable solubility characteristics may be expected to be able to follow the same path witiiout confronting any kind of specific permeability barrier.
  • Choice of conjugate structures can be guided (a) by die known structural basis of biological function for various steroids and analogs, which suggest portions ofthe hormone or ligand molecule which can be modified for covalent coupling without substantial impairment of nuclear hormone receptor binding, and (b) by inspection and molecular modeling studies using the known structures of the ligand-binding domains (LBDs) ofthe RXR- ⁇ , RAR- ⁇ and thyroid hormone receptors (RXR and RAR refer to retinoic acid- binding receptors), as well as any homologous LBD structures.
  • LBDs ligand-binding domains
  • RXR and RAR refer to retinoic acid- binding receptors
  • This invention therefore contemplates rational modeling of die specific ligand-binding site for any nuclear hormone receptor to develop superior PDT agents which are conjugates of a suitable photosensitizing agent and a hormone/ligand which binds to a receptor of die nuclear hormone receptor superfamily, the receptor being expressed in cells whose destruction is desired, such as cells in tumor tissue.
  • PDT agents conjugates of a suitable photosensitizing agent and a hormone/ligand which binds to a receptor of die nuclear hormone receptor superfamily, the receptor being expressed in cells whose destruction is desired, such as cells in tumor tissue.
  • ligand-photosensitizer conjugates of this invention designed to bind to die ligand binding site of a particular nuclear hormone receptor, will be routine for d e skilled artisan, and any ligand-photosensitizer conjugate prepared by such routine synthesis is within d e contemplation of this invention.
  • the photosensitizer moiety and the ligand moiety are linked by means of a "tether" (flexible chain), which may be an alkyl chain of 1 to 10 methylene groups.
  • linkages most preferred for this invention are typified by those presented with the prototype compounds (ECC-1, ECC-2a, ECC-2b, and TCC-1). They will be short-to-medium lengtii chains (including 1-10 meti ylene groups or equivalent) and will be attached to die ligand and sensitizer moieties by neutral (uncharged) functionalities which are easily established such as ether (by Michael addition) and amide (by DCC or similar coupling agent). Ester linkages are also possible, but more likely to be cleaved by enzymatic activity in vivo.
  • Inco ⁇ oration of some additional, uncharged polar functional groups in the tetiier may be desirable to enhance the uptake rates determined in tissue culture and/or animal studies.
  • Tether structures which facilitate transport ofthe conjugate in the blood stream are likewise desirable, so long as uptake of die conjugate into the target cell is not unduly compromised.
  • a preferred targeting receptor is the estrogen receptor in human cells.
  • the estrogen receptor (ER) is a steroid receptor of particular importance in cancer therapy, since it is expressed in many breast and ovarian cancer cell lines.
  • the synthetic estrogen antagonist tamoxifen is used to treat metastasized breast cancer.
  • the specificity of a syntiietic estrogen-chlorin conjugate (ECC) is used to treat ER-positive breast and ovarian cancer. Particular conjugates targeted via ti is receptor will be discussed below.
  • Synthetic conjugates containing the estrogen moiety may be modified covalently to attach suitable chromophoric groups via a short, flexible "tether". Once prepared, these compounds may be tested against breast-cancer cell lines growing in tissue culture. The test cells will be those expressing ER, while the controls will be ER-negative cells. Efficacy may be demonstrated using an ER-positive cell line growing in tissue culture. As controls, the ER-negative bladder cancer cell line and the unconjugated chlorin dimethylester (CDME) are tested. Chromophores may be linked to the estrogen framework at the
  • C-17 position preferably in the axial ( ⁇ ) orientation, though for some applications enantiomeric purity is not necessarily essential.
  • the choice of the C-17 position comes from consideration of die structures of natural hormones as well as analogs. Testosterone and estradiol, for example, differ in the A hgand specificity at tiiis position ⁇ consistent with the estrogenic activity of estrone which has a carbonyl group (rather tiian an alcohol) at C- 17. It also appears probable tiiat the D-ring portion of die ER binding pocket accommodates d e dimethylaminoethyl side chain of tamoxifen or die aziridine group in die ER affinity label ketononestrol aziridine (KNA).
  • KNA ketononestrol aziridine
  • MCF-7 a non-metastatic breast cancer line
  • ER -positive an experimental cell line
  • control cell lines may be selected from MDA- MB-435, a metastatic (ER-negative) breast cancer line, human keratinocytes (which will also yield information about potential skin phototoxicity), and T4 bladder cancer cells.
  • ECC-1 a conjugate between an estrogen moiety and chlorin e 6 , a po ⁇ hyrin derivative which acts as a PDT sensitizer, can be used to target cells which contain die estrogen receptor (e.g. , most metastatic breast cancer cells).
  • a synthetic procedure for preparing ECC-1 is shown in Figure 4a. Because of its greater bulk and hydrophilicity, ECC-1 is not taken up as well as die parent chromophore CDME or die parent hormone estradiol (see Example 1).
  • Example 1 shows that ECC-1 is taken up by mammahan cells in culture, has no apparent toxic effects in the absence of light, and is quite effective as a photosensitizer when irradiated at 664 nm.
  • chlorin e 6 dimethyl ester CDME
  • tiie estrogen conjugate was more effective in sensitizing receptor-positive cells under at least some conditions.
  • a different conjugate which has an ether linkage in the tether between the steroid and chromophore instead of die secondary amine present in ECC-1 has also been prepared.
  • ECC-2 This new compound (ECC-2) will not be protonated at physiological pH (ECC-1 is protonated under such conditions) and that difference significantiy improves its ability to cross the hydrophobic portion ofthe plasma membrane, thereby increasing the rate of uptake into cells. It may also be expected to improve binding to die estrogen receptor protein.
  • the synthesis of ECC-2 shown in Figure 4b results in a product which has the carboxyl groups on the po ⁇ hyrin nucleus present as metiiyl esters ratiier tiian free carboxylate anions (as in the case of ECC-1 at pH 7). This means that ECC-2 will have no ionic charges under physiological conditions, a condition which may be expected to improve its uptake into cells.
  • the overall reaction scheme for synthesis of ECC-2 is given in Figure 4b.
  • the key intermediate (V) has been repeatedly synthesized in high yield (>80% for each of tiie four steps) and its structure confirmed by *H- and 13 C-NMR and mass spectroscopy.
  • the synthesis may be completed by carrying out the coupling and deprotection steps (steps #5 and #6 in Figure 4b). It may be possible (even desirable ) to deprotect the phenolic hydroxyl group in the A ring ofthe estrogen moiety before performing the final coupling reaction.
  • Coupling efficiency may also be improved by changing conditions and by use of different carbodiimide reagents in place of DCC, as well as combinations of other reagents with DCC (e.g., C 6 F 5 OH).
  • die scheme in Figure 4b can be adapted to syntiiesis of a variety of homologs of die main compound, particularly those with a variable ether length (set at three methylene groups in Figure 4b).
  • Testosterone-chlorin conjugate may be used as a more definitive control, as well as a second family of potential PDT agents. These compounds target die chlorin photosensitizer moiety to androgen- receptor (AR)-positive cells.
  • Figure 5a details a suitable synthetic scheme — which, as can readily be seen, shares a number of features with the estrogen- conjugate chemistry already described.
  • TCC-1 is a lead compound as a PDT agent for androgen- receptor (AR)-containing cells, such as tiiose found in virtually all metastatic prostate cancer.
  • testosterone itself binds fairly weakly to die AR ⁇ it is, in fact, considered in many situations to be a prohormone which must be converted to 5cc-dihydrotestosterone in order to have any effect. This conversion is effected in vivo by 5 ⁇ -reductase, and the result is a molecule which binds more tightly to the AR.
  • FIG. 5b A synthesis scheme for tamoxifen-chlorin e 6 conjugate is shown in Figure 5b. This scheme shows use of Fmoc-aminocaproate as an activated donor oftiie tether extension. Structures of other representative photosensitizer conjugates are shown in Figure 5c.
  • the chemical reaction steps set forth above can be used to couple suitable chromophores to ligands which specifically bind other members ofthe nuclear hormone receptor superfamily to provide conjugates according to this invention. Selection of suitable ligands and sites on die hgand for attachment ofthe tether is within die skill of die art. For example, a study by Rink, et al. ( 1996, Proc. Natl. Acad.
  • Photodynamic therapy uses photosensitizers to damage or destroy unwanted cells in situ, such as PDT to destroy hype ⁇ lastic or neoplastic tissues by irradiation with long-wavelength visible light.
  • PDT Photodynamic therapy
  • a variety of procedures and protocols have been described as reviewed by Pass, 1993, JNCI. 85: 443-456; Rosenthal, et al., 1994; Anj Med.. 26: 405- 409; and Kessel, 1990, "Photodynamic Therapy of Neoplastic Disease", volumes I and II, CRC Press, Boca Raton, all of which are inco ⁇ orated herein by reference.
  • PDT mediods according to this invention differ primarily dirough use of particular photosensitizer conjugates targeted to cells containing receptors of the nuclear hormone receptor superfamily.
  • PDT according to diis invention is particularly useful for treatment of tumors
  • the method may be used in any tiierapeutic situation for which specific destruction of cells that express receptors of the nuclear hormone receptor superfamily is desirable.
  • Adaptation of PDT protocols to accommodate die particular wavelengths absorbed by die conjugates and to selectively illuminate die target tissues is a routine matter for the skilled clinician.
  • the ligand/photosensitizer conjugates of this invention are specific for a single type of intracellular receptor, and such ligand/photosensitizer conjugates will be taken up preferentially by cells which express the matching (cognate) receptor.
  • cells which express the matching (cognate) receptor For example, a significant fraction of metastatic breast cancer cells express ER and would be expected to assimilate and retain a suitable estrogen-related conjugate; prostate cancer cells would similarly take up a testosterone-derived conjugate; etc.
  • this invention contemplates PDT using as an agent an estrogen-chlorin conjugate (e.g., ECC-2) which does not carry ionic charges and may mus readily penetrate cell membranes.
  • ECC-2 estrogen-chlorin conjugate
  • Photosensitizer conjugates according to this invention are preferably formulated in pharmaceutical compositions containing the compound and a pharmaceutically acceptable carrier, such as physiological saline or physiological buffer.
  • a pharmaceutically acceptable carrier such as physiological saline or physiological buffer.
  • the pharmaceutical composition may contain other components so long as the other components do not reduce d e effectiveness ofthe compound according to this invention so much that the therapy is negated.
  • Pharmaceutically acceptable carriers are well known, and one skilled in the pharmaceutical art can easily select carriers suitable for particular routes of administration (Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, PA, 1985).
  • compositions or dosage forms for systemic, local or topical may include solutions, lotions, ointments, creams, gels, suppositories, sprays, aerosols, suspensions, dusting powder, impregnated bandages and dressings, liposomes, biodegradable polymers, and artificial skin.
  • Typical pharmaceutical carriers used in compositions for local or topical application include alginates, carboxymetiiylcellulose, methylcellulose, agarose, pectins, gelatins, collagen, vegetable oils, mineral oils, stearic acid, stearyl alcohol, petrolatum, polyethylene glycol, polysorbate, polylactate, polyglycolate, polyanhydrides, phospholipids, polyvinylpyrrolidone, and d e like.
  • a preferred strategy is to ao ⁇ minister these conjugates locally or topically in gels, ointments, solutions, or liposomes.
  • Liposomes containing photosensitizer conjugates according to this invention may be prepared by any of die mediods known in the art for preparation of liposomes containing inclusions. Liposomes that are particularly suited for aerosol application to the lungs are described in International Patent Publication WO 93/12756, pages 25-29, inco ⁇ orated herein by reference.
  • the concentrations of the photosensitizer conjugate in pharmaceutically acceptable carriers will typically range from 1 ⁇ M to 0.1 M.
  • the dose used in a particular formulation or application will be determined by die requirements of d e particular type of disease and the constraints imposed by die characteristics and capacities of the carrier materials. Dose administered will depend on a variety of factors, including disease type, patient age, patient weight, and tolerance of toxicity.
  • Dose will generally be chosen to achieve local peak concentrations in target cells of from 0.01 nM to 10 ⁇ M, preferably from 0.1 nM to 100 nM. Peak concentration is the maximum level of photosensitizer in cells expressing the receptor which binds die ligand moiety of die photosensitizer, counting both bound and unbound photosensitizer, following administration of the photosensitizer conjugate.
  • Binding of the ligand moiety by die target receptor will result in higher total amounts of photosensitizer conjugate in the target cell than in neighboring cells or surrounding medium at equilibrium; -erum concentrations and/or concentrations in surrounding non-targeted cells will generally be at least 2- fold lower tiian the concentration in the target cells, preferably at least 5-fold lower, and more preferably at least 10-fold lower.
  • the instantaneous level of photosensitizer in any particular cell will reflect both systemic pharmacokinetics and local kinetics for transport in and out of cells.
  • Initial dose levels may be selected based on tiieir ability to achieve ambient concentrations shown to be effective in in vitro models, such as that used to determine tiierapeutic index, and in vivo models and in clinical trials, up to maximum tolerated levels.
  • the dose of a particular photosensitizer conjugate and duration of therapy for a particular patient can be determined by die skilled clinician using standard pharmacological approaches in view ofthe present disclosure.
  • the PDT may be monitored by analysis from time to time of blood or body fluid levels of die photosensitizer conjugate according to this invention. The skilled clinician will adjust the dose and duration of therapy based on these measurements.
  • compositions containing any of the photosensitizer conjugates of this invention may be administered by parenteral (subcutaneously, intramuscularly, intravenously, intraperitoneally, intrapleurally, intravesicularly or intratiiecally), topical, oral, rectal, or nasal route, at the discretion of the clinician. Lesions in externally accessible surfaces may be treated by non-invasive administration of a photosensitizer according to this invention.
  • the compound according to diis invention is formulated in a pharmaceutical composition and applied to an externally accessible surface of a patient having a lesion in an externally accessible surface.
  • Externally accessible surfaces include all surfaces tiiat may be reached by non-invasive means (witiiout cutting or puncturing the skin), including the skin surface itself, mucus membranes, such as those covering nasal, oral, gastrointestinal, or urogenital surfaces, and pulmonary surfaces, such as the alveolar sacs.
  • Non-invasive administration includes (1) topical application to the skin in a formulation, such as an ointment or cream, which will retain the compound in a localized area; (2) oral administration; (3) nasal administration as an aerosol; (4) intravaginal application oftiie compound formulated in a suppository, cream or foam; (5) rectal administration via suppository, irrigation or other suitable means; (6) bladder irrigation; and (7) administration of aerosolized formulation of the compound to the lung. Aerosolization may be accomplished by well known means, such as the means described in International Patent Publication WO 93/12756, pages 30-32, inco ⁇ orated herein by reference.
  • PDT agents will usually be stored at low temperature (preferably frozen) in the dark under an inert atmosphere so as to minimize photooxidation and otiier degradative processes. Suitable aliquots will be thawed just prior to administration. Typically, treatment will begin with administration of the agent as a sterile solution i.v. Doses may be optimized by standard procedures for the particular type of neoplastic or hype ⁇ lastic tissue. Such optimization is a routine matter for die clinical oncologist. PHOTOFRIN doses have typically fallen into the 1-2 mg/kg range; it is contemplated that the conjugates described in diis invention will be used at considerably lower levels because of their property of specific binding to target-cell components.
  • the conjugate Once the conjugate is infused into the patient, specific binding to the cognate receptors in the cytoplasm of die cells expressing the particular nuclear hormone receptors will concentrate the photosensitizer moiety in d e target tissue. Illumination of the target tissue will generally be delayed for a suitable period to permit physiological clearance for any unbound conjugate from cells and tissues not expressing the receptor. Clearance can be monitored by measuring the concentration of the conjugate in peripheral blood or urine, usually by optical or fluorescent detection of the photosensitizer moiety. Specificity of PDT is imparted by the favorable concentration ratio of sensitizer in target tissue to sensitizer in non-target cells.
  • the time delay between administration of the PDT agent and irradiation with long-wave-length visible light can be established by suitable analytical methods, leading to procedures that can be routinely followed in all cases of the same type.
  • the optimum tiierapeutic ratio concentration of drug in target tissue/concentration in normal, non-malignant, non-target tissue
  • the time (and also for as much 4-6 weeks after administration) there may well be some cutaneous photosensitivity and it is preferable for patients to avoid excessive exposure to sunlight and other intense light sources so as to minimize erythremia.
  • Irradiation of the tissue containing the PDT agent should preferably take place during die interval of maximum therapeutic ratio. Selection of suitable light sources is discussed in Pass, 1993, page 445, inco ⁇ orated herein by reference. Depending upon the sensitizer and target, one of several procedures might be followed. If the target is a solid tumor accessible to fiber-optic devices, such devices (witii a suitable diffuser on die distal end) can be embedded in the tumor (up to a depth of several centimeters). Light from a tunable dye laser (or possibly a diode laser) could be directed into the fiber and thus to the sensitized tumor site.
  • a tunable dye laser or possibly a diode laser
  • the wavelength ofthe light would be chosen to match the abso ⁇ tion bands of the PDT chromophore (e.g., 665-675 nm in the case of chlorin e 6 ). Different procedures would be followed in the case of target tissues on or close to the skin surface, as in melanoma, psoriasis, etc. In these cases the configuration ofthe laser and/or fiber-optic light-delivery system would take into account die flat geometry ofthe target. Irradiation periods can be chosen to deliver to total dose comparable to diat currently employed with PHOTOFRIN, viz. 50-300 joules/cm 2 , usually administered over a time interval of 5-45 minutes, with 8-20 minutes being typical.
  • Routine clean-up procedures should be employed as a follow- up to the PDT treatment.
  • a bronchoscope, sigmoscope or other suitable device can be used to trim and remove damaged or necrotic tissue remaining after 24 hours or so post-treatment.
  • the photosensitizer will be further concentrated in the target cell if it is conjugated to ligands which bind to more than one receptor in the target cell. This may occur in instances where d e cell, in addition to expressing the cognate nuclear hormone receptor, also expresses ancillary enzymes which bind to die conjugates, thereby increasing their intracellular concentration still more.
  • ER-positive cells have the enzyme 17 ⁇ -hydroxy steroid dehydrogenase
  • AR (androgen receptor) containing cells have 5 ⁇ -reductase, the enzyme responsible for converting testosterone into dihydrotestosterone. Binding of photosensitizer conjugates containing the respective ligands to tiiese enzymes will lead to accumulation of additional photosensitizer in the respective cells.
  • target cells may express more than one member ofthe nuclear hormone receptor superfamily, and the enhancement may be provided by using a combination of more than one photosensitizer conjugate in a cocktail.
  • the vitamin D receptor is also present, hence applying two conjugates simultaneously should be quite feasible, and that would significantly increase the therapeutic ratio (photosensitizer concentration in target cell divided by concentration in adjacent normal tissue), preferably, both photosensitizer conjugates in such a cocktail would absorb light at the same wavelength, which may be accomplished by using conjugates of the same chromophore to different ligands.
  • Synthesis ofthe corresponding vitamin-D conjugates of chlorin e 6 are described above. Selection of suitable photosensitizer conjugates and application in a cocktail or in multiple substantially contemporaneous administrations is within die skill of the art in view oftiie present disclosure.
  • MCF-7 breast cancer cells, EJ bladder cancer cells and OVCAR-3, an ovarian cancer cell line were used.
  • the MCF-7's and OVCAR-3's are estrogen-receptor positive; the EJ cells are estrogen-receptor negative.
  • the breast cancer cell line MCF-7 and die OVCAR-3 cells were maintained in Dulbecco's Modified Eagle Media (DMEM) and RPMI, respectively, supplemented with heat-inactivated fetal bovine serum (10% v/v) and gentamicin (0.05 mg/mL) at 37°C, 5% C0 2 .
  • DMEM Dulbecco's Modified Eagle Media
  • RPMI Dulbecco's Modified Eagle Media
  • EJ cells were maintained in McCoy's 5A supplemented with heat inactivated fetal bovine serum (5% v/v) and gentamicin (0.05 mg/mL) at 37 °C, 5% C0 2 .
  • heat inactivated fetal bovine serum 5% v/v
  • gentamicin 0.05 mg/mL
  • ECC-1 was prepared according to die synthetic protocol shown in Figure 4a.
  • Ox 10 5 cancer cells were plated out in a 35 mm petri dish witii 2mL medium and placed in an incubator at 37°C and 5% C0 2 .
  • the tumor cells were incubated with 0.1, 0.25 and 0.5 ⁇ M CDME and ECC-1 for 1 hour.
  • the tumor cells were harvested witii Trypsin + EDTA and lysed in 3 mL 0.1N NaOH to extract the CDME and ECC-1. 1 mL of the extract was used for protein determination whereas the otiier 2 mL were used for measuring CDME and ECC-1 concentrations. 20 ⁇ l SDS (20%) was added to the samples to obtain a final concentration of 0.1% SDS.
  • CDME and ECC-1 concentrations were measured on a fluorescence spectrophotometer by exciting at 410 nm and measuring the emission at 664 nm.
  • the CDME and ECC-1 concentrations were calculated by using standard curves and by correcting them for the protein content of each sample.
  • the protein assay was performed using the Biorad protein assay kit.
  • ECC-1 By means of fluorescence microscopy it could be observed diat ECC-1 penetrated die cells and became concentrated in some regions, such as lysosomes in preference to others. In all three cell lines ECC-1 showed no toxicity in the absence of light, even at the highest concentration tested (0.5 ⁇ M). In this regard it was completely comparable to die control compound (CDME).
  • the survival data can also be used to estimate relative uptake.
  • the OVCAR-3 (ovarian cancer) cells appear more permeable than die EJ (bladder cancer) cells, since d e photosensitized killing in die later case is significantiy less, especially at fluences of 1, 5 and 10 J/cm 2 at a dye concentration of 0.1 ⁇ M (cf. Figs. 6a & 7a vs. Figs. 10a & I la).
  • 1.5-2.0xl0 5 tumor cells were plated out in 35 mm dishes with 2 mL medium. The tumor cells were incubated witii the photosensitizer and irradiated die next day. The concentration of CDME and ECC in die media was checked with a spectrophotometer (Hewlett Packard) using die extinction coefficient of 20,000 L/mole-cm at 664 nm. After the incubation with CDME and ECC die tumor cells were washed with PBS and maintained in PBS during irradiation with the argon pumped dye laser. A dye laser, containing DCM dye was pumped by the 514.5nm emission of a continuous wave argon ion laser.
  • the dye laser emission was tuned to 664nm, die characteristic wavelength of die photosensitizer, and passed through a 1mm diameter quartz fiber optic.
  • the Hght beam was focused witii lenses onto the sample in a 35mm petri dish.
  • the PBS was replaced for medium, and die cells were incubated for 24 hours, after which the cell survival was measured (described below). All experiments and controls were performed on a minimum of tiiree dishes and were done in duplicate.
  • the cell survival was determined by die dimetiiylthiazol- diphenyltetrazolium bromide (MTT) assay.
  • the tumor cells were incubated in 1.25 mg/mL MTT in DMEM (FCS 10%v/v) without phenol red for 4 hours.
  • the MTT solution was replaced by DMSO (100%) to dissolve the tumor cells and blue crystals prior to reading die optical density (OD) at 577 nm by using die ELISA reader.
  • a dead control consisting of cells incubated at -18 °C, was used as background value and the cell survival was expressed as percentage of the control.
  • the controls consisted of plates exposed to neither photosensitizer or light and photosensitizer without light (dark controls) and die protocol was the same as described for die treated samples.
  • EJ bladder cancer cells which do not express the estrogen receptor.
  • cell survival was slightly better for the ECC- 1 -treated cells than for the controls.
  • the exceptions were for 0.1 ⁇ M dye concentration at fluences of 5 and 10 J/cm 2 .
  • This is consistent with a slightly reduced uptake of the ECC-1, which would be expected on the basis of its greater size and additional zwitterionic charge. The differences, however, were small in all cases and fell widiin the overlapping error bars ofthe measurements.
  • ECC-1 proved more effective in six out ofthe twelve illuminated conditions. At a concentration of 0.25 ⁇ M it was superior at every fluence level tested. Under four conditions, die killing by the two dyes was essentially identical, and in the remaining conditions (0.5 ⁇ M at fluences of 5 and 10 J/cm 2 ) CDME killed more effectively tiian ECC-1.
  • ECC-1 is not toxic to the tested cell lines in the absence of light and that it does act efficiently as a letiial photosensitizer.
  • At least two factors may limit the selectivity observed here.
  • any endogenous estrogen or estrogen- mimetic compounds would competitively inhibit die binding of ECC- 1 to d e receptor active site.
  • Growth medium components such as fetal calf serum may contain such compounds, and it has also been recently noted diat items of laboratory equipment such as plastic petri dishes sometimes contain detectable amounts of estrogenic substances [J. Amer. Med. Assn. 271, 414 (1994)].
  • ECC-1 tiie features make it a less-than-ideal candidate for targeting steroid hormone receptors. For one tiling, it carries two ionic charges at physiological pH (a plus charge on the protonated secondary amine of tiie tether, where it is attached to C-17 of tiie steroid moiety, and a negatively charged carboxylate on one of die side groups of the chlorin ring system). On the basis ofthe well-known inhibitory effects of charges on the permeability of molecules through lipid bilayers (and cell membranes), tiiis feature is undesirable.
  • ECC-1 has comparatively short tether (which includes die positive charge on the secondary amine already discussed) and tiiis may not allow optimum drug/protein binding.
  • ECC-2 a second estrogen-chlorin conjugate
  • ECC-2 may be synthesized by d e route outlined in Figure 4b.
  • estrone (I) the phenol is protected with a benzyl group, then d e C-17 keto group is reduced witii LiAlH 4 to give 3- (O)-benzyl-estra-17 ⁇ -ol (III).
  • This molecule contains the estradiol moiety coupled to the po ⁇ hyrin nucleus by a 6-atom linker and may be expected to be recognized by the estrogen receptor ⁇ as inferred from tiie behavior of various otiier steroids and steroid analogs.
  • the secondary amine linkage (witii its positive charge) is replaced by an ether linkage (which will remain neutral under all pH conditions).
  • the steroid moiety is reacted widi die free carboxyl group of CDME, leaving die otiier two carboxyls oftiie po ⁇ hyrin both in die form of unaltered metiryl esters.
  • an organic compound was designed to bind to the nuclear testosterone receptor.
  • the main synthetic steps are the construction of the C3 hydroxy on the A ring, die chain elongation at C 17 in a Michael addition, followed by reduction of the chain nitrile group into an amine.
  • the final product is obtained by coupling die testosterone derivative to chlorin e 6 .
  • the compound may be used in photodynamic tiierapy (PTD) of cancer cells which are testosterone-responsive, targeting specific cell types and allowing them to be killed by irradiation with visible light.
  • the compound is selectively taken up in cancer cells, and by irradiating witii visible light, toxic, reactive singlet oxygen can be produced, resulting in cell destruction.
  • This synthesis produces a molecule capable of targeting testosterone responsive cells and potentially useful in the treatment of metastatic prostate cancer.

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Abstract

La présente invention concerne un mécanisme nouveau pour localiser les photosensibilisateurs, à savoir l'interaction avec les récepteurs de haute affinité qui régulent les signaux hormonaux transmis par les stéroïdes (et quelques autres hormones telles que la thyroxine, les rétinoïdes, et des éléments de la famille de la vitamine D). Ces récepteurs ne s'expriment que dans des types spécifiques de cellules, et confèrent par leur expression une sensibilité aux hormones auxdites cellules. La présente invention concerne des conjugués hormone/chromofore dotés d'une affinité de liaison raisonnable pour la protéine réceptrice de l'hormone, ainsi que des procédés permettant d'administrer aux patients lesdits conjugués comme agents photosensibilisateurs spécifiques capables, au moment de l'irradiation par de la lumière visible, de diriger les atteintes létales vers des lignées de cellules contenant le récepteur. Lesdits conjugués hormone/chromofore liés aux récepteurs d'hormones nucléaires peuvent être utilisés comme systèmes sélectifs d'administration de molécules dans la thérapie photodynamique.
PCT/US1997/004542 1996-03-22 1997-03-21 Therapie photodynamique dans laquelle des recepteurs d'hormones nucleaires sont utilises pour cibler des photosensibilisateurs WO1997034637A2 (fr)

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WO2000074673A1 (fr) * 1999-06-03 2000-12-14 The General Hospital Corporation Traitement et analyse de troubles proliferatifs
WO2005120588A2 (fr) * 2004-05-26 2005-12-22 The Curators Of The University Of Missouri Peptides delivres a des noyaux cellulaires
US9173950B2 (en) 2012-05-17 2015-11-03 Extend Biosciences, Inc. Vitamin D-ghrelin conjugates
US9585934B2 (en) 2014-10-22 2017-03-07 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US9616109B2 (en) 2014-10-22 2017-04-11 Extend Biosciences, Inc. Insulin vitamin D conjugates
US9789197B2 (en) 2014-10-22 2017-10-17 Extend Biosciences, Inc. RNAi vitamin D conjugates
US12233115B2 (en) 2022-09-30 2025-02-25 Extend Biosciences, Inc. Long-acting parathyroid hormone

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000074673A1 (fr) * 1999-06-03 2000-12-14 The General Hospital Corporation Traitement et analyse de troubles proliferatifs
WO2005120588A2 (fr) * 2004-05-26 2005-12-22 The Curators Of The University Of Missouri Peptides delivres a des noyaux cellulaires
WO2005120588A3 (fr) * 2004-05-26 2006-04-20 Univ Missouri Peptides delivres a des noyaux cellulaires
US9173950B2 (en) 2012-05-17 2015-11-03 Extend Biosciences, Inc. Vitamin D-ghrelin conjugates
US9289507B2 (en) 2012-05-17 2016-03-22 Extend Biosciences, Inc. Carriers for improved drug delivery
US9884124B2 (en) 2012-05-17 2018-02-06 Extend Biosciences, Inc. Carriers for improved drug delivery
US9616109B2 (en) 2014-10-22 2017-04-11 Extend Biosciences, Inc. Insulin vitamin D conjugates
US9789197B2 (en) 2014-10-22 2017-10-17 Extend Biosciences, Inc. RNAi vitamin D conjugates
US9585934B2 (en) 2014-10-22 2017-03-07 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US10406202B2 (en) 2014-10-22 2019-09-10 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US10420819B2 (en) 2014-10-22 2019-09-24 Extend Biosciences, Inc. Insulin vitamin D conjugates
US10702574B2 (en) 2014-10-22 2020-07-07 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US11116816B2 (en) 2014-10-22 2021-09-14 Extend Biosciences, Inc. Therapeutic vitamin d conjugates
US12076366B2 (en) 2014-10-22 2024-09-03 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US12233115B2 (en) 2022-09-30 2025-02-25 Extend Biosciences, Inc. Long-acting parathyroid hormone

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