Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives

Definitions

• This invention was made with support in part by funds from certain
• the invention is in the field of photodynamic therapy, specifically related to endometrial conditions. More particularly, the invention concerns the use of green porphyrins in photodynamic therapeutic treatment for ablation of the endometrium.
• Dysfunctional uterine bleeding, menorrhagia and endometriosis afflict approximately 0.1% of premenopausal women. Between 30,00 to 108,000 hysterectomies are performed in the United States each year for dysfunctional uterine bleeding. Complications are 0.1% mortality and 30% morbidity in addition to physical, social and psychological effects.
• Endometrial ablation has long been seen as a possible altemative to hysterectomy for dysfunctional uterine bleeding, menorrhagia, endometriosis and endometrial neoplasia. Furthermore, endometrial ablation could also be used as a means for sterilization. Since the endometrium regenerates from residual epithelium, partial ablation could provide an alternative to surgical abortion.
• Photodynamic therapy is a technique that destroys tissue through interaction between absorbed light and a photosensitizer. The process involves systemic or topical administration of a photosensitizing drug that is retained in the target tissue. When light of the appropriate wavelength and sufficient energy interacts with the sensitizer, highly reactive oxygen intermediates are generated.
• the human endometrium exhibits several features that satisfy the requirements for effective PDT: (1) it is easily accessible; (2) it is only 2-9 millimeters thick; and (3) it is surrounded by a thick myometrium which acts as a protective light barrier for intra-abdominal organs. Moreover, the thickness of the endometrium may be modulated by manipulating the hormonal state. PDT may therefore offer a simple, cost effective and safe alternative to more radical surgical procedures for treatment of endometrial disorders, sterilization and abortion.
• HPD hematoporphyrin derivative
• ALA 5-Aminolevulinic acid
• Green porphyrins Hydro-monobenzoporphrins, or "green porphyrins" (Gp), designed to absorb light at higher wavelengths, meet this requirement.
• the green porphyrins have been described in detail in U.S. patents 4,883,790; 4,920,143; 5,095,030 and 5,171 ,749, the entire contents of which are incorporated herein by reference. Due to their absorption peak at 670-780 nm, green porphyrins qualify as a drug for well vascularized tissues, such as the endometrium, as hemoglobin does not absorb a significant amount of light at this wavelength. The use of a photosensitizer with an excitation peak at a longer wavelength has important advantages when this method is used in humans.
• the human endometrium is significantly thicker (2-9 mm) than the endometrium of the rat or rabbit and the geometry of the human uterus may impose problems in light distribution.
• Several studies suggest that the endometrial surface regenerates from the residual epithelium of the gland stumps where stem cells are present (Ferenczy, A., Am J. Obstet Gynecol 1976; 124:64-74).
• the glandular crypts of the basal endometrial layer, from which new endometrial cells regenerate lie within the innermost myometrial layer. Accordingly, sufficient light must be delivered to the entire endometrium, including the innermost myometrial layer, to induce irreversible photochemical destruction.
• light penetration depth could play a critical role in achieving a sufficient optical dose throughout the endometrium.
• the penetration depth of light increases with longer wavelength. For example, at a depth of 4 mm inside the human uterine wall, the fluence rate increases by 59% for premenopausal and 71% for postmenopausal uteri when 690 nm light is used instead of 630 nm.
• green porphyrins having an absorption maxima in the range of 670-780 nm can facilitate more efficient treatment at greater tissue depths than other hematoporphyrin derivatives with absorption maxima at Iower wavelengths.
• BPD Benzoporphyrin derivatives
• BPD- MA benzoporphyrin derivative monoacid ring A
• the safety of BPD-MA has been demonstrated in mouse, rabbit, dog and rat models and in humans.
• Safety of BPD-MA was tested in the mouse by i.v. injection of 1.25- 10 mg/kg. The majority of the i.v. injected dose was cleared from the body during the first 24 hours (Richter A.M. et al., Photochem. Photobiol 1990; 52:495-500 and J. Photochem.
• the green porphyrins offer advantages in their selectivity for neovasculature such as the endometrium.
• administration of the green porphyrins in a viscous liquid such as dextran 70 (Hyskon) provides an advantageous delivery method for the drug to the endometrium.
• Photodynamic therapy practiced according to the method of the invention using topical application of green porphyrins is highly effective for endometrial ablation, without re-epithelialization in long-term follow-up.
• the absorption peak of benzoporphyrin derivative at 690 nm may offer a deeper penetration depth of light in the human endometrium.
• the invention is directed to treatment of certain conditions of the endometrium using photodynamic methods and employing green porphyrins as the photoactive compounds. These materials offer advantages of selectivity and effectiveness at low doses when employed in protocols directed to the destruction of the endometrium. Accordingly, in one aspect, the invention is directed to a method to treat disorders of the endometrium, which method comprises administering to a subject in need of such treatment, preferably topically, an amount of a green porphyrin that will localize in said endometrium; and irradiating the endometrium with light absorbed by the green porphyrin.
• the invention is directed to a method for sterilization, which method comprises administering to a subject an amount of a green porphyrin that will localize in the endometrium; and irradiating the endometrium with light absorbed by the green porphyrin. Topical administration is preferred.
• the invention is directed to a method to terminate early pregnancy, which method comprises administering to a subject, preferably topically, an amount of green porphyrin that will localize in the endometrium and irradiating the endometrium with light absorbed by the green porphyrin. Partial endometrial ablation is effected by modulating the amount of green porphyrin or the dose of light.
• the invention is directed to a formulation, for topical application to the endometrium, of an effective amount of green porphyrin in a pharmaceutical composition comprising an acceptable excipient and an agent providing suitable viscosity.
• Figure 1 shows preferred forms of the green porphyrins useful in the methods of the invention.
• Figure 2 shows log benzoporphyrin derivative fluorescence in rabbit endometrial glands, stroma, and myometrium versus time determined from fluorescence microscopic images of frozen tissue sections (three animals per time point).
• Figure 3 shows light microscopic images of untreated (A, C) and treated
• the green porphyrins are formulated into pharmaceutical compositions for topical administration to the endometrium using techniques known in the art. A summary of such pharmaceutical compositions may be found, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest edition.
• Green porphyrins and in particular BPD-MA, strongly interact with lipoproteins and are easily packaged in liposomes.
• Compositions of green porphyrins involving lipocomplexes, including liposomes, are described in U.S. Patent 5,214,036 and in U.S. Serial No. 07/832,542 filed 5 February 1992, the disclosures of both of these are incorporated herein by reference.
• Liposomal BPD can also be obtained from QLT PhotoTherapeutics, Inc., Vancouver, British Columbia.
• Formulations may include coupling to a specific binding ligand which may bind to a specific surface component of the endometrium, neoplasm or fetal tissue or by formulation with a carrier that delivers higher concentrations to the target tissue.
• Topical formulations may also contain penetrants, such as DMSO, Azone and/or additional ingredients which affect the depth of penetration.
• Topical formulations will be in the form of liquids or gels. Suitable excipients are, for example, water, saline, dextrose, dextran 70, glycerol and the like. Low-viscosity solutions are known to pass easily through the fallopian tubes into the abdominal cavity. Viscous liquids are preferred because they minimize the risk of retrograde spillage through the cervix and passage through the fallopian tubes into the abdominal cavity.
• these compositions may also contain minor amounts of nontoxic, auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth.
• Hyskon R is viscous, hydrophilic, branched polysaccharide routinely used for uterine distention during hysteroscopy. Other viscous solutions and gel forms could also be used.
• the dose of green porphyrin can vary widely depending on the condition to be treated; the physical delivery system in which it is carried, such as in the form of liposomes, the presence or absence of penetration enhancing agents, whether it is coupled to a target-specific ligand, such as an antibody or an immunologically active fragment, the thickness of the endometrium, the individual subject and the judgment of the practitioner. Partial endometrial ablation for termination of early pregnancy will require Iower doses than complete endometrial ablation.
• the dose of green porphyrin used is within the range of from about 0.04 to about 20 mg/kg, preferably from about 0.04-2.0 mg/kg. This range is merely suggestive, as the number of variables in regard to an individual treatment regime is large and considerable excursions from these recommended values are expected.
• the various parameters used for effective, selective photodynamic therapy in the invention are interrelated. Therefore, the dose should also be adjusted with respect to other parameters, for example, fluence, irradiance, duration ofthe light used in photodynamic therapy, and time interval between administration of the dose and the therapeutic irradiation. All of these parameters should be adjusted to produce significant damage to endometrial or neoplastic tissue without significant damage to the surrounding tissue.
• an effective dose of green porphyrin is topically applied to the subject through intra-uterine administration.
• Intra-uterine drug application and light delivery may be performed transcervically or by laparotomy. Transcervical delivery is preferred.
• a period of time is allowed to elapse before the tissue is irradiated. This time period allows for accumulation of the green porphyrin in the target tissue.
• the optimum time following green porphyrin administration until light treatment can vary widely depending on variables such as the green porphyrin used, whether penetration enhancing agents have been included, the thickness of the endometrium, etc.
• the time of light irradiation after administration of the green porphyrin may be important as one way of maximizing the selectivity of the treatment, thus minimizing damage to structures other than the target tissues.
• BPD-MA Benzoporphyrin Derivative Monoacid Ring A
• the endometrium is exposed to light at the wavelength of maximum absorbence of the green porphyrin, usually between about 670 and 780 nm.
• a wavelength in this range is especially preferred for enhanced penetration. Shorter wavelengths can also be used, if desired, for convenience. In many cases, absorption spectra show appreciable absorption down to the low 600 nm range as well as at shorter wavelengths.
• the fluence during the irradiating treatment can vary widely, depending on type of tissue (endometrium, neoplasm or fetal), depth of target tissue, but preferably varies from about 10-200 Joules/cm 2 .
• the irradiance typically varies from about 100-900 mW/cm 2 , with the range between about 100-600 mW/cm 2 being preferred. However, the use of higher irradiances may be selected as effective and having the advantage of shortening treatment times.
• the present inventor has developed an analytical model to predict optical fluence rate distributions when cylindrical optical applicators are placed in the uterine lumen.
• a similar strategy is used to predict drug levels at various times after topical application of various photosensitizers.
• the results of the model calculations are applied to determine the optimal time after drug application for irradiation and to estimate the effective photodynamic dose.
• Theoretical calculations are compared to frozen tissue fluorescence studies and absolute fluence rate measurements made in fresh, surgically removed human uteri. The results show that cylindrical optical applicators inserted into the human uterus can provide a light dose that is sufficient to cause photodynamic destruction of the entire endometrium.
• the actual depth of destruction and the extent of endometrial regeneration is a complex function of the optical fluence delivered to the endometrial-myometrial interface (at a depth of about 4-6 mm) and the tissue photodynamic threshold. Optimum dosages of drug and light can be established through clinical trials.
• the present inventor has also published the results of endometrial ablation using photodynamic therapy with BPD-MA in rabbits and rats (Wyss et al. Obstet. Gynecol. 1994; 84:409-414, Wyss et al., Human Reproduction
• One milliliter was injected into the left uterine horn 3-5 mm distal to the uterine bifurcation through a lower abdominal midline incision.
• a 2-ml syringe with a 20-gauge needle was used.
• the abdomen was closed by a three layer suture (resorbable threads).
• Temperature, pulse, and respiration were monitored during and after the anesthetic until the animal was ambulatory and able to eat and drink.
• Photodynamic therapy was performed on 6 rabbits following a second laparotomy 1.5 hours after drug administration.
• Light from an argon pumped dye laser operating at 690 nm (Spectra Physics, Mountain View, CA) was delivered to the uterine cavity via a 400 mm diameter quartz optical fiber terminated with a 3.0 cm long cylindrical diffusing tip (Model 4420-A02: PDT Systems, Buellton, CA).
• the fiber was placed through a perforation in the middle part of the uterine horn.
• the distance between the fiber and the lumen wall varied between 0-1.5 mm depending on the anatomy of the uterine cavity.
• a clinical Hartridge reversion spectroscope (Ealing Electro-Optics, South Natick, MA) was used to verify the wavelength.
• Example 2 Analysis of BPD-MA Pharmacokinetics in Rabbit Uteri Following Photodynamic Therapy
• the rabbits were first anesthetized with isoflurane and then euthanized by intracardiac injection of 1.5 mL Euth-6 (Western Medical Supply, Arcadia, CA). Uteri were retrieved via laparotomy immediately following euthanasia.
• the specimens were sectioned into four blocks of 3-4 mm each without rinsing the uterine lumen, and placed in molds containing embedding medium for frozen sections (OCT Media; Miles, Elkhart, IN). The blocks were rapidly frozen on dry ice and stored at -70°C in the dark. Specimens retrieved for histology were fixed in 10% formaldehyde.
• Benzoporphyrin derivative pharmacokinetics were evaluated by analyzing frozen sections (fluorescence microscopy) from rabbits sacrificed 1.5, 3, 6 and
• Normalized fluorescence image mean (background - dark noise) X image (fluorescence - dark noise) image (background - dark noise)
• mean background - dark noise
• mean gray-scale value for the dark noise corrected background image.
• the rabbit uteri were divided into different anatomical layers for comparative analysis: endometrial glands, endometrial stroma, and the circular muscle (myometrium).
• Fluorescence measurements were transformed using a logarithmic transformation to reduce the variability. Multiple fluorescence measurements from individual rabbits were averaged. At each time point (1.5, 3, 6, and 12 hours), the average of three animals was calculated (total 12 animals). The overall differences in fluorescence between glandular, stromal, and circular muscular tissue were compared using repeated-measures analysis of variance. Contrast tests were also examined between glandular fluorescence and fluorescence of stromal and circular tissue, and the interactive effect of fluorescence with time. The standard error used in Figure 2 was obtained by computing a pooled estimate of standard deviation from the 12 fluorescence means and dividing by the square root of 3 as each mean is composed of three fluorescence values.
• Figure 2 shows the average fluorescence, corresponding to BPD-MA concentrations, for glandular, stromal, and circular muscular (myometrium) tissue at four time points (1.5, 3, 6, and 12 hours).
• Glandular fluorescence was significantly higher than stromal and myometrial (P ⁇ 0.0001 , analysis of vari ⁇ ance).
• the difference in drug concentration in the various tissues was significantly higher at 1.5 and 3 hours than at 6 and 12 hours, with no appreciable contrast evident at 12 hours.
• Fluorescence microscopy data revealed a significantly higher accumulation of BPD-MA in epithelial structures of the endometrium compared to the stroma.
• the high concentration of BPD-MA in epithelium is probably due to differences in intercellular distribution and diffusion through cellular membranes and, fortuitously, may provide selectivity during photodynamic therapy.
• Drug concentration was lowest in the surrounding myometrium compared to other layers. The low drug concentration and myometrial thickness may protect the intra-abdominal organs from photochemical effects during photodynamic therapy.
• Benzoporphyrin derivative-Hyskon exhibited an early concentration peak about 1.5 hours after topical application, followed by a rapid decrease.
• the great difference between benzoporphyrin derivative concentrations in the uterine structures at 1.5 hours decreased with time and was not evident after 12 hours. Therefore, timing of the light application is crucial to optimize photodynamic efficacy.
• These pharmacokinetic results indicate an optimal interval 1.5 hours following topical BPD-MA application.
• the benzoporphyrin derivative-Hyskon pharmacokinetic data are similar to those with benzoporphyrin derivative in water. Although there is no conclusive evidence that Hyskon influences drug distribution, there is no indication that it adversely affects uptake and fluorescence in the endometrium.
• Example 3 Analysis of Structural Changes in the Rabbit Uterus Following Photodynamic Therapy with BPD-MA Following intrauterine photodynamic therapy in the rabbits, the samples for histology (light microscopy and scanning electron microscopy) were fixed in 10% formalin in phosphate buffer at room temperature for 24 hours. Light microscopy samples were dehydrated in graded ethyl alcohol, cleared in Histo- clear (National Diagnostics, Manville, NJ), infiltrated with paraffin using a tissue processor (Model I55MP; Fisher Scientific, Pittsburgh, PA), and embedded in paraffin. Sections were cut at 6 m, deparaffinized, and stained with either hematoxylin and eosin or sirius red 3BA.
• Scanning electron microscopy specimens were fixed as mentioned above and further processed in 10% osmium tetroxide, dehydrated in graded acetone, critical point dried (Ladd Critical Point Dryer; Ladd Research Industries, Inc., Burlington, VT), and sputter-coated with gold (Pelco PAC-1 evaporating system; Ted Pella, Inc., Redding, CA). Micrographs were then taken using a scanning electron microscope (SEM 515; Philips Electronic Instrument Co., Mahwah, NJ).
• BPD-MA (2 mg and 4 mg) was diluted in 1 mL Hyskon TM (Dextran 70). These two drug concentrations were injected topically (I.U.) in rats (volume of
• Example 5 Topical Application of BPD-MA to Human Endometrium
• dextran 70 32% W/V
• Topical application of the BPD-MA solution is performed in lithotomy position at one of the following time points: Immediately before surgery (i.e. 30-40 min. prior to actual removal of the organ) and 2 or 6 hours prior to the scheduled hysterectomy. A standard bivalve speculum is placed. The cervix is cleansed with providone-iodine. A Sholkoff balloon hysterosalpingography catheter (Cook, Bloomington, IN) with an outer diameter of 2 mm is inserted into the cervical canal. 1.5 mL of 2 mg/mL BPD-MA/Hyskon R solution is injected slowly into the uterine cavity. The time span for injection is 30 seconds and accomplished at a uniform flow rate via slow manual push. No dilatation of the cervix is performed. If the catheter does not easily pass the cervical canal no drug is injected.
• Systemic drug uptake is monitored by 3 blood samples of 5 cc taken before drug administration, at the time of hysterectomy and 1 hour after surgery. For the longer (6 hours) time interval, another sample is added between drug instillation and operation.
• the samples represent the corpus and fundus of the uterus, each
• the main specimen (>99% of the uterus) is handled in the operating room for further analysis as indicated. Handling of the specimen does not hamper the quality of pathologic evacuation of the specimen.
• the samples are analyzed by low-light level tissue fluorescence imaging.
• the fluorescent Imaging System consists of a Zeiss Anxiovert 10 inverted microscope which can be configured to visualize fluorescent images of tissue frozen sections.
• a 100-W- mercury lamp is coupled to a filter wheel to provide excitation in a variety of spectral regions.
• the emission is similarly isolated by a filter wheel in the emission path.
• tissue autofluorescence is isolated from drug fluorescence by thermoelectrically cooled, slow-scan CCD (charge-coupled device) camera (Princeton Instruments, Trenton, NJ) interfaced to a computer. Camera resolution is determined over 2.2 X 105 pixels with 16 bit per pixel dynamic range.
• a UniBlitz shutter and driver (model T132) are used to synchronize the CCD- camera with the excitation source in order to minimize sample photobleaching. Due to the exceptional sensitivity of the system, typical exposure times are about 1 second for most frozen section fluorescent images.
• background images are acquired from blank slides with identical parameters (i.e., filters, exposure times). All fluorescent and background images are corrected for dark noise contributed during the exposure time.

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• 1996
  • 1996-08-06 JP JP9509347A patent/JP2000502044A/ja active Pending
  • 1996-08-06 WO PCT/US1996/012828 patent/WO1997006797A1/en not_active Application Discontinuation
  • 1996-08-06 EP EP96926917A patent/EP0844876A4/en not_active Withdrawn
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