CA2229662A1 - Endometrial ablation using photodynamic therapy with green porphyrins - Google Patents
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Abstract
The invention describes Photodynamic therapy using green porphyrins for endometrial ablation. This method is useful to treat endometrial disorders such as dysfunctional uterine bleeding, menorrhagia, endometriosis and endometrial neoplasia. Other applications of the method are for sterilization and termination of early pregnancy.
Description
W O g7~U6797 PCTAUS96/12828 ENDOMETRIAL ABLATION USING PHOTODYNAMIC THERAPY
WITH GREEN PORPHYRINS
This invention was made with support in part by funds from certain United States government agencies. The government has certain rights in this invention.
Technical Field The invention is in the field ol 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.
15 Background Art Dysfunctional uterine bleeding, menorrhagia and endometriosis affflict 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 20 addition to physical, social and psychological effects.
Endometrial ablation has long been seen as a possible alternative 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 25 residual epithelium, partial ablation could provide an alternative to surgical abortion.
Routinely performed minimal invasive surgical treatments forendometrial destruction in humans are hysteroscopic Nd:YAG laser ablation and ~ electrocautery. These methods hold certain risks, such as perforation, 30 bleeding, cervical stenosis, fluid overload and air embolism. Moreover, general anesthesia and at least 24 hours hospitalization are required.
W O 97/06797 PCT~US96/12828 One contemplated approach to endometrial ablation is the use of photodynamic therapy (PDT). 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 5 photosensitizing drug that is retained in the target tissue. When light of theappropriate wavelength and suffficient energy interacts with the sensitizer, highly reactive oxygen intermediates are generated. These intermediates, primarily singlet molecular oxygen, cause irreversible tissue damage and necrosis. The main side effect related to systemic drug delivery is photosensitization of the 10 skin. In order to avoid severe erythema, the patient must avoid direct sunlight and prolonged contact with bright artificial light for several weeks.
In contrast to laser ablation of the endometrium, photodynamic therapy requires relatively low power light to activate the photochemical effects, offers selective therapy if the sensitizer is accumulated by the target tissue and no 15 anesthetics or hospitalization are required. 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 20 manipulating the hormonal state. PDT may therefore offer a simple, cost effective and safe alternative to more radical surgical procedures for treatmentof endometrial disorders, sterilization and abortion.
Animal models for photodynamic endometrial destruction using hematoporphyrin derivative (HPD), Photofrin ll and 5-Aminolevulinic acid (ALA) 25 show promising results. Manyak ef al. (Fertility and Sterility 1989; 52: 140-145) showed effficacy of photodynamic therapy of a rabbit endometriosis model.
Schneider et al. (Colposcopy Gynecol Laser Surg. 1988; 4:73-85 and Colposcopy and Gynecologic Laser Surgery 1988; 4:67-69) observed both selective uptake of intravenous HPD by rat endometrium and photodynamic 30 endometrial destruction. Bhatta ef al. (Am J. Obstet Gynecol 1992; 167:1856-1863) found a predominately interstitial fluorescence in the rabbit endometrium WCI 97~<!167~7 PCT/US96~12~28 after systemic Photofrin gR administration and achieved endometrial destruction after intrauterine light irradiation. Yang et a/. (Am J Obstet Gynecol 1993;
168:995-1001) treated female rats with various doses of ALA in the uterine horn followed by exposure to red light. This treatment profoundly decreased the rate 5 of implantation in the ALA-treated uterine horns of rats bred 10 or 60 days after treatment. Histologic examination revealed that the treated uterine horns were completely dlevoid of endometrium.
There is a drawback associated with most of the hematoporphyrin-related photosensitizers currently used in animal models of endometrial 10 ablation. The wavelength of light required for activation of these photosensitizers is in the range of 630 nm, an energy which is readily absorbed by hemoglobin and other natural chromophores found in blood and other tissues. Therefore relatively large amounts of the photosensitizer and irradiation must be administered, potentially resulting in damage to non-target 15 tissue. It would be desirable to administer photosensitizers which would mediate the effects of radiation at lower dosage, thus avoiding the problems of hypersensitivity exhibited nonspecifically throughout the subject organism.
Hydro-monobenzoporphrins, or "green porphyrins" (Gp), designed to absorb light at higher wavelengths, meet this requirement. The green 20 porphyrins have been described in dletail 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.
25 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 andl the geometry of the human uterus may impose problems in light distribution. Several studies suggest that the endometrial surface regenerates 30 from the residual epithelium of the gland stumps where stem cells are present (Ferenczy, A., Am J. Obstet Gynecol 1976; 124:64-74). In humans, the glanduiar crypts of the basal endometrial layer, from which new endometrial ceils 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.
5 Therefore, 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 10 nm. Hence, green porphyrins having an absorption maxima in the range of 670-780 nm can facilitate more efficient treatment at greater tissue depths thanother hematoporphyrin derivatives with absorption maxima at lower wavelengths.
Benzoporphyrin derivatives (BPD) are members of the green porphyrin 15 family. One particular BPD, benzoporphyrin derivative monoacid ring A (BPD-MA) is 10-70 fold more toxic toward various cell lines than hematoporphyrin derivative. The safety of BPD-MA, the preferred green porphyrin of the invention, 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 1.25-20 10 mg/kg. The majority of the i.v. injected dose was cleared from the bodyduring the first 24 hours (Richter A.M. et al., Photochem. Photobiol 1990;
52:495-500 and J. Photochem. Photobiol. B. Biol. 1990; 5:231-244) and more than 60% of the injected dose was excreted with the feces. The amount of active BPD-MA is reduced to about 40% at 24 hours post injection from 100%
25 at 3 hours post injection. Thus, skin photosensitivity occurs with BPD-MA only transiently, with minimal reactivity after 24 hours in in vivo models. Toxicity of liposomal BPD-MA has been extensively studied in rats and dogs. BPD doses up to 10 mg/kg and 5 mg/kg respectively i.v. daily for 2 weeks demonstrated no mortality or apparent clinical signs of toxicity. (Wolford S. R., Pearl River30 Studies No. 92020 1993; 1-425, No.92052,1993; 1-318 and No. 93004, 1994) WO 971067g7 PCT~US96/12828 Preliminary studies with systemic application of BPD-MA in humans are ongoing. They indicate that photodynamic therapy using intravenously administered BPD-MA has been effective in clearing both melaslalic skin cancers and basal cell carcinoma. Systemic toxicity of BPD-MA (0.15-0.5 5 mg/kg i.v.) has not been observed. The major incidence of adverse events are associated with local skin reactions following the photodynamic treatment of malignanVpsoriatic skin tissue and surrounding normal skin. The period of photosensitivity has been shown to be relatively short, with 3 days or less at doses of 0.25 mg/kg BPD-MA or less and up to 6 days after the highest dose 10 of BPD-MA (0.5 mg/kg) used in the study. The same study measured plasma elimination half-life values of BPD-MA ranging from approximately 5 to 6 hours.
It is expected that intra-uterine delivery of a green porphyrin will make the risk of skin photosensitivity negligible.
The green porphyrins offer advantages in their selectivity for 15 neovasculature such as the endometrium. The present applicants have further determined that 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 20 for endometrial ablation, without re-epithelialization in long-term follow-up. In addition, the absorption peak of benzoporphyrin derivative at 690 nm may offer a deeper penetration depth of light in the human endometrium.
Disclosure of the Invention The invention is directed to treatment of certain conditions of the endometrium using photodynamic rnethods 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.
In another aspect, the invention is directed to a method for steriiization, 5 which method comprises administering to a subject an amount of a green porphyrin that will localize in the endometrium; and irradiating the endometriumwith light absorbed by the green porphyrin. Topical administration is preferred.In still another aspect, the invention is directed to a method to terminate early pregnancy, which method comprises administering to a subject, preferably 10 topically, an amount of green porphyrin that will localize in the endometriumand 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.
In still another aspect, the invention is directed to a formulation, for 15 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.
Brief Description of the Drawings 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 25 time point).
Figure 3 shows light microscopic images of untreated (A, C) and treated (B, D) rabbit uterine horns 4 weeks after photodynamic therapy. Low magnification (X 16) and high magnification ( X 100) images are shown in panels A, B, and C, D, respectively.
W o 97/06797 PCTnUS96rIZ828 i Figure 4 shows scanning electron micrographs (X 5000) of control (A) and treated rabbit endometrium 1 week (B) and 4 weeks (C) after photodynamic therapy.
Modes of carrying out the Invention In general, the green porphyrin is of a formula shown in Figure 1 or a mixture thereof. Referring to Figure 1, in preferred embodiments each of R1 and R2 is independently selected from the group consisting of carbalkoxyl (2-6C), alkyl (1-6C), arylsulfonyl (6-10C), cyano and -CoNR5Co wherein R5 is aryl (6-10C) or alkyl (1-6C); each i~3 is independently carboxyl, carboxyalkyl (2-6C) or a salt, amide, ester or acylhydrazone thereof or is alkyl (1-6C); R4 is CH=CH2 or -CH(oR4 )CH3 wherein R4 is H, or alkyl (1-6C) optionally substituted with a hydrophilic substituent. Especially preferred also are green porphyrins of the formula shown in Figures 1-3 or 1-4 or mixtures thereof.
More preferred are embodiments are those wherein the green porphyrin is of the formula shown in Figure 1~3 or 1~ or a mixture thereof and wherein each of R' and R2 is independently carbalkoxyl (2-6C); one R3 is carboxyalkyl (2-6C) and the other R3 is an ester of a carboxyalkyl (2-6C) substituent; and R4 is CH=CH2 or-CH(OH)CH3.
Still more preferred are embodiments wherein green porphyrin is of the formula shown in Figure 1-3 or 1-4 and wherein R1 and R2 are methoxycarbonyl; one R3 is -CH2CH2COOCH3 and the other R3 is CH2CH2COOH; and R4 is CH=CH2; i.e., BPD-MA or BPD-MB, more preferably BPD-MA.
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 30 with lipoproteins and are easily packaged in liposomes. Compositions of greenporphyrins 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 5 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. These 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 or10 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. Of course, 15 these compositions may also contain minor amourits of nontoxic, auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth. One preferred formulation is liposomally formulated Benzoporphyrin Derivative Monoacid Ring A in dextran 70 (HyskonR, Pharmacia Inc., Piscataway, NJ) HyskonR is viscous, hydrophilic, branched polysaccharide 20 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, 25 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 lower doses than complete endometrial ablation. Typically, the dose of green porphyrin used is 30 within the range of from about 0.04 to about 20 mglkg, preferably from about 0.04-2.0 mg/kg. This range is merely suggestive, as the number of variables W O 97/06797 PCTrUS96/12828 in regard to an individual treatment regime is large and considerable excursionsfrom these recommended values are expected.
It should be noted that the various parameters used for effective, selective photodynamic therapy in the invention are interrelated. Therefore, the5 dose should also be adjusted with respect to other parameters, for example, fluence, irradiance, duration of the li~ht used in photodynamic therapy, and time interval between administration of the dose and the therapeutic irradiation. Allof these parameters should be adjusted to produce significant damage to endometrial or neoplastic tissue without significant damage to the surrounding 1 0 tissue.
In a preferred method of the invention, 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.
After the photosensitizing green porphyrin has been administered, 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 20 penetration enhancing agents ha~e 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.
In a rabbit model, liposomally formulated Benzoporphyrin Derivative Monoacid 25 Ring A (BPD-MA) induced highest glandular and stromal fluorescence of the endometrium after 1.5 hours. Light administration at this time interval resultedin lasting endometrial destruction verified by histology after 4 weeks.
The endometrium is exposed to light at the wavelength of maximum - absorbence of the green porphyrin, usually between about 670 and 780 nm.
30 A wavelength in this range is especially preferred for enhanced penetration.
Shorter wavelengths can also be used, if desired, for convenience. In many W O 97/06797 PCTrUS96/12828 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 5 preferably varies from about 10-200 Jouies/cm2. The irradiance typically varies from about 100-900 mW/cm2, with the range between about 100-600 mW/cm2 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 10 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 15 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 20 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 25 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 1995; 10:221-226 and Steiner et al., Geburtsh. U. Frauenheilk. 1995; 55:
Artikel 243), the entire contents of which are incorporated herein by reference.Complications, such as hemorrhage and photosensitivity in non-target tissues, 30 are not noted with the invention method in rabbit and rat models. Thus, photodynamic therapy with a green porphyrin appears to be a simpler, safer W Og71067g7 PCTnUS96~1Z8Z8 alternative than surgery for treating disorders of the endometrium, sterilization and termination of early pregnancy.
The following examples are to illustrate, but not to limit, the invention.
E~ample 1 Treatment of rabbit endometrium with benzoporphvrin derivative and photodynamic therapy Eighteen mature female New Zealand White rabbi~s weighing 3.6-4.3 kg were anesthetized with an intramuscular injection of ketamine and xylazine (2:1), 0.75 ml/kg and isoflurane was added during the surgical intervention.
Liposomally formulated benzoporphyrin derivative MA (BPD-MA, QLT
PhotoTherapeutics, Inc., Vancouver, BC, Canada) was stored in the dark at 4~C
and protected from light at all times. Shortly before administration, BPD-MA
was equilibrated to room temperature and reconstituted to 2 mg/ml in dextran 15 70 (Hyskon, Pharmacia, Inc., Piscataway, NJ). One milliliter was injected into the left uterine horn 3-5 mm distal l:o 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 20 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, 30 MA) was used to verify the wavelength. Because of the length of the rabbit uterine horn is 10-15 cm, multiple (four to five) segmental irradiation was W O 97/06797 PCT~US96/12828 required. A total of 195 mW was launched into the fiber (65 mW/cm fiber tip) during 20 minutes, resulting in a variable tissue dose which, depending on geometry, ranged from 40-80 J/cm2.
No adverse side effects, such as hemorrhagia or photosensitivity were 5 found in non-target tissue during 4 weeks of observation.
Example 2 Analysis of BPD-MA Pharmacokinetics in Rabbit Uteri Following Photodynamic TheraPy For specimen retrieval, the rabbits were first anesthetized with isoflurane 10 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 15 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 12 hours following administration of 2 mg/mL BPD-MA-Hyskon. Three animals 20 were studied for each time point. Frozen sections of 6,u thickness were made in low diffuse light (Cryostat Microtome; AO Reichert, Buffalo, NY). Low light level tissue fluorescence was performed with a slow-scan, thermoelectrically cooled CCD camera system (Model ST-180; Princeton Instruments, Trenton, NJ) coupled to a Zeiss Axiovert 10 inverted fluorescence microscope (Carl Zeiss 25 Inc., Oberkochen, Germany). A 10X objective Zeiss Plan neofluar (numerical aperture 0.3) was used to visualize bright-field and fluorescence images of the frozen sections. A 100-W mercury arc lamp coupled to a mechanical shutter (UniBlitz model Tl32; Vincent Associates, Rochester, NY) and filtered through a 405 nm, broad-bandpass filter provided excitation light. A dichroic mirror (FT30 420) refiected the excitation light onto the sample and transmitted the fluorescence emission through a 635 nm broad-bandpass filter onto the detector.
W O 97/~6797 PCTAUS96~282g Instrument control, image acquisition, and processing were performed with a Maclntosh llfx computer (Apple Computers, Cupertino, CA) and IPlab - software (Signal Analytics Corp., Vienna, VA). Minimal sample photobleaching was achieved by synchronizing the mercury lamp and CCD-camera shutters.
5 To esLi,nale light distribution in the tissue slices, background images were acquired from blank slides with identical characteristics. All fluorescence images were normalized by the following algorithm to correct for nonuniform illumination:
Normalized fluorescence image =
mean (back~round - dark noise) X image (fluorescence - dark noise) image (background - dark noise) in which mean (background - dark noise) is the mean gray-scale value for the 15 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 20 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 25 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, strornal, and circular muscular (myometrium) tissue at four time points (1.5, 3, 6, and 12 hours). Glandular fluorescence wassignificantly 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 5 appreciable contrast evident at 12 hours. The difference between glandular and stromal or myometrial fluorescence decreased with time (P = 0.01 and P
= 0.02, respectively). Glandular and stromal drug concentrations were highest at 1.5 hours, the earliest time point tested.
Fluorescence microscopy data revealed a significantly higher 10 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 15 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 20 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.
W O 97/06797 PCT~US96~1Z828 Example 3 Analvsis of Structural Changes in the Rabbit Uterus Following Photodynamic Therapy with BPD-MA
Following intrauterine photodynamic therapy in the rabbits, the samples 5 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 dehydrat~d in graded ethyl alcohol, cleared in Histo-clear (National Diagnostics, Manville, NJ), inrilllaled with paraffin using a tissue processor (Model 155MP; Fisher Scientific, Pittsburgh, PA), and embedded in 10 paraffln. Sections were cut at 6 rn, 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 15 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 Elecl:ronic Instrument Co., Mahwah, NJ).
Structural changes in the endometrium following photodynamic therapy were evaluated in the right (control) and left (treated) uterine horns of the same 20 rabbit using both light and scanning electron microscopy. Figure 3 shows the light-microscopy results for IOX and 50X magnifications. Scanning electron microscopy images (5000X) are presented in Figure 4.
Low magnification light microscopy images of untreated (Figure 3A) and treated (Figure 3B) uterine horns showed destruction of epithelial and stromal 25 endometrial structures 4 weeks after photodynamic therapy. The lumen was obliterated and replaced by stroma resembling scar tissue. Processes of recanalization were not evident. The bordering circular layer of the myometrium (right side in picture) was loosened and invaded by connective - tissue. Because of spillage through the perforated uterine wall during and after 30 intrauterine drug injection, the description of damage in the longitudinal myometrial layer and serosa may lead to wrong conclusions. Minimal re-epithelialization was observed in a minority of sections (not shown), perhaps due to uneven light distribution. High-magnification images (Figures 3C and 3D) revealed dramatic changes in the endometrial structure, with complete absence of the endometrial epithelium. In addition, the post-irradiation stromal5 components were abundant in the extracellular matrix, with obviously decreased v~scul~rization. One week after irradiation, the treated area demonstrated acute changes such as hemorrhagia, moderate vessel damage, and white blood cell i, IrilLl ~lion (not shown). For studies following photodynamic therapy, 1 and 4 weeks were selected as time points for long-10 term follow-up because regeneration after mechanical destruction of the endo-metrium in rabbits is complete in 3 days. Generally, substantial, persistent destruction of the endometrium was effected (Figures 4B and C). However, regional variations in re-epithelialization indicate that maintaining the optical dose at or above the photodynamic threshold is crucial for irreversible damage.
15 Equal drug distribution within the uterine cavity, illumination of short duration to avoid photodegradation, drug washout, and homogeneous light distribution may also contribute to successful and complete endometrial destruction.
Scanning electron micrographs of the untreated uterine horn (Figure 4A) showed ciliated cells surrounded by nonciliated microvillus cells. In contrast, the 20 treated horns 1 week (Figure 4B) and 4 weeks (4C) following photodynamic therapy exhibited complete loss of luminal columnar epithelium and glandular openings. The surface was replaced by a collagen network resembling scar tissue.
ExamPle 4 Photodynamic TheraPv of the Rat Endometrium Usinq BPD-MA
Endometrial ablation experiments using BPD-MA were conducted in Sprague Dawley rats employing the methods described in examples 1 through 3. BPD-MA (2 mg and 4 mg) was diluted in 1 mL Hyskon TM (Dextran 70).
30 These two drug concentrations were injected topically (I.U.) in rats (volume of 0.15 mL). Fluorescence microscopic studies have demonstrated that there is no significant difference in uptake and distribution between these two drug concentrations. Low light level tissue fluorescence imaging revealed a distinct positive endometrium to myometrium ratio within the first 12 hours. Relative fluorescence was highest in the endometrial glands and lowest in the 5 myometrium. After 12 hours the intensity of fluorescence leveled off in all compartments and values of the glands approached those of other layers.
PDT (2 mg/mL BPD-MA an:l 80J/cm2 intra-uterine light) resulted in endometrial destruction at one week. No regeneration of endometrial tissue was observed at 4 weeks. None of the animals showed any alterations in light 10 treated skin areas at any time.
In reproductive performance assays, only 0.44 implantations per rat occurred in the treated area of the left horn after PDT. In contrast, 8.9 implantations per rat were found in the corresponding area of the untreated right horn.
Example 5 Topical APPlication of BPD-MA to Human Endometrium To analyze the distribution olF topically applied BPD-MA in the human uterus, patients scheduled for hysterectomy are recruited for inclusion in the 20 study. Three mg liposomally formulated Benzoporphyrin Derivative Monoacid Ring A is dissolved just before use under sterile conditions in 1.5 mL dextran 70 (32% WN) in dextrose (10% WN; HyskonR, Pharmacia Inc., Piscataway, NJ). This amount is equal to 0.05 mg/kg (for 60 kg body weight) and is well below any sysl:emic topical dose administered to any of the previous human or 25 animal studies and no side effects are anticipated.
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 30 is cleansed with providone-iodine. A Sholkoff balloon hysterosalpingography catheter (Cook, Bloomington, IN) with an outer diameter of 2 mm is inserted W O 97/06797 PCTrUS96/12828 into the cervical canal. 1.5 mL of 2 mg/mL BPD-MA/HyskonR 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 5 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 i"s~illaLion and operation.
Example 6 Analvsis of BPD-MA Distribution in the Human Uterus Followina, Topical APPlication of BPD-MA
Immediately after hysterectomy 2-3 areas of the uterine wall are 15 obtained. The samples represent the corpus and fundus of the uterus, each 4-6 mm thick, 10 mm wide and show all layers of the uterine wall (endometrium, myometrium, serosa). Specimens are immediately placed in molds containing embedding medium for frozen sections (Tissues Tek, O.C.T.
media, Miles, Elkhart, IN), snap frozen on dry ice and stored at -70 o C in a 20 light-impermeable container. All specimens are handled in the dark. Tissues are sectioned in low diffuse light (Cryostar microtome, AO Reichert, Buffalo, NY) to obtain 5-micron-thick slices for fluorescence analysis taken from severallocations, approximately 3 mm apart.
The main specimen (>99% of the uterus) is handled in the operating 25 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
30 100-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. These features permit selection of optimal excitation and emission spectral regions for visualizing samples. In addition, tissue autofluorescence is isolated from drug fluorescence by thermoelectrically cooled, slow-scan CCD (charge-coupled device) camera (Princeton 5 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 10 about 1 second for most frozen section fluorescent images. In order to estimate light distribution, 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. All images are acquired and automatically stored in a 1-Gb 15 capacity rewritable optical drive system. Processing and camera control are performed by a Maclntosh llfx computer with appropriate software (Signal Analytics Corp., Vienna, Virginia. Specimens are divided into anatomical layers for comparative analysis, i.e., endiometrial glands, endometrial stroma and myometrium.
WITH GREEN PORPHYRINS
This invention was made with support in part by funds from certain United States government agencies. The government has certain rights in this invention.
Technical Field The invention is in the field ol 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.
15 Background Art Dysfunctional uterine bleeding, menorrhagia and endometriosis affflict 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 20 addition to physical, social and psychological effects.
Endometrial ablation has long been seen as a possible alternative 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 25 residual epithelium, partial ablation could provide an alternative to surgical abortion.
Routinely performed minimal invasive surgical treatments forendometrial destruction in humans are hysteroscopic Nd:YAG laser ablation and ~ electrocautery. These methods hold certain risks, such as perforation, 30 bleeding, cervical stenosis, fluid overload and air embolism. Moreover, general anesthesia and at least 24 hours hospitalization are required.
W O 97/06797 PCT~US96/12828 One contemplated approach to endometrial ablation is the use of photodynamic therapy (PDT). 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 5 photosensitizing drug that is retained in the target tissue. When light of theappropriate wavelength and suffficient energy interacts with the sensitizer, highly reactive oxygen intermediates are generated. These intermediates, primarily singlet molecular oxygen, cause irreversible tissue damage and necrosis. The main side effect related to systemic drug delivery is photosensitization of the 10 skin. In order to avoid severe erythema, the patient must avoid direct sunlight and prolonged contact with bright artificial light for several weeks.
In contrast to laser ablation of the endometrium, photodynamic therapy requires relatively low power light to activate the photochemical effects, offers selective therapy if the sensitizer is accumulated by the target tissue and no 15 anesthetics or hospitalization are required. 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 20 manipulating the hormonal state. PDT may therefore offer a simple, cost effective and safe alternative to more radical surgical procedures for treatmentof endometrial disorders, sterilization and abortion.
Animal models for photodynamic endometrial destruction using hematoporphyrin derivative (HPD), Photofrin ll and 5-Aminolevulinic acid (ALA) 25 show promising results. Manyak ef al. (Fertility and Sterility 1989; 52: 140-145) showed effficacy of photodynamic therapy of a rabbit endometriosis model.
Schneider et al. (Colposcopy Gynecol Laser Surg. 1988; 4:73-85 and Colposcopy and Gynecologic Laser Surgery 1988; 4:67-69) observed both selective uptake of intravenous HPD by rat endometrium and photodynamic 30 endometrial destruction. Bhatta ef al. (Am J. Obstet Gynecol 1992; 167:1856-1863) found a predominately interstitial fluorescence in the rabbit endometrium WCI 97~<!167~7 PCT/US96~12~28 after systemic Photofrin gR administration and achieved endometrial destruction after intrauterine light irradiation. Yang et a/. (Am J Obstet Gynecol 1993;
168:995-1001) treated female rats with various doses of ALA in the uterine horn followed by exposure to red light. This treatment profoundly decreased the rate 5 of implantation in the ALA-treated uterine horns of rats bred 10 or 60 days after treatment. Histologic examination revealed that the treated uterine horns were completely dlevoid of endometrium.
There is a drawback associated with most of the hematoporphyrin-related photosensitizers currently used in animal models of endometrial 10 ablation. The wavelength of light required for activation of these photosensitizers is in the range of 630 nm, an energy which is readily absorbed by hemoglobin and other natural chromophores found in blood and other tissues. Therefore relatively large amounts of the photosensitizer and irradiation must be administered, potentially resulting in damage to non-target 15 tissue. It would be desirable to administer photosensitizers which would mediate the effects of radiation at lower dosage, thus avoiding the problems of hypersensitivity exhibited nonspecifically throughout the subject organism.
Hydro-monobenzoporphrins, or "green porphyrins" (Gp), designed to absorb light at higher wavelengths, meet this requirement. The green 20 porphyrins have been described in dletail 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.
25 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 andl the geometry of the human uterus may impose problems in light distribution. Several studies suggest that the endometrial surface regenerates 30 from the residual epithelium of the gland stumps where stem cells are present (Ferenczy, A., Am J. Obstet Gynecol 1976; 124:64-74). In humans, the glanduiar crypts of the basal endometrial layer, from which new endometrial ceils 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.
5 Therefore, 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 10 nm. Hence, green porphyrins having an absorption maxima in the range of 670-780 nm can facilitate more efficient treatment at greater tissue depths thanother hematoporphyrin derivatives with absorption maxima at lower wavelengths.
Benzoporphyrin derivatives (BPD) are members of the green porphyrin 15 family. One particular BPD, benzoporphyrin derivative monoacid ring A (BPD-MA) is 10-70 fold more toxic toward various cell lines than hematoporphyrin derivative. The safety of BPD-MA, the preferred green porphyrin of the invention, 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 1.25-20 10 mg/kg. The majority of the i.v. injected dose was cleared from the bodyduring the first 24 hours (Richter A.M. et al., Photochem. Photobiol 1990;
52:495-500 and J. Photochem. Photobiol. B. Biol. 1990; 5:231-244) and more than 60% of the injected dose was excreted with the feces. The amount of active BPD-MA is reduced to about 40% at 24 hours post injection from 100%
25 at 3 hours post injection. Thus, skin photosensitivity occurs with BPD-MA only transiently, with minimal reactivity after 24 hours in in vivo models. Toxicity of liposomal BPD-MA has been extensively studied in rats and dogs. BPD doses up to 10 mg/kg and 5 mg/kg respectively i.v. daily for 2 weeks demonstrated no mortality or apparent clinical signs of toxicity. (Wolford S. R., Pearl River30 Studies No. 92020 1993; 1-425, No.92052,1993; 1-318 and No. 93004, 1994) WO 971067g7 PCT~US96/12828 Preliminary studies with systemic application of BPD-MA in humans are ongoing. They indicate that photodynamic therapy using intravenously administered BPD-MA has been effective in clearing both melaslalic skin cancers and basal cell carcinoma. Systemic toxicity of BPD-MA (0.15-0.5 5 mg/kg i.v.) has not been observed. The major incidence of adverse events are associated with local skin reactions following the photodynamic treatment of malignanVpsoriatic skin tissue and surrounding normal skin. The period of photosensitivity has been shown to be relatively short, with 3 days or less at doses of 0.25 mg/kg BPD-MA or less and up to 6 days after the highest dose 10 of BPD-MA (0.5 mg/kg) used in the study. The same study measured plasma elimination half-life values of BPD-MA ranging from approximately 5 to 6 hours.
It is expected that intra-uterine delivery of a green porphyrin will make the risk of skin photosensitivity negligible.
The green porphyrins offer advantages in their selectivity for 15 neovasculature such as the endometrium. The present applicants have further determined that 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 20 for endometrial ablation, without re-epithelialization in long-term follow-up. In addition, the absorption peak of benzoporphyrin derivative at 690 nm may offer a deeper penetration depth of light in the human endometrium.
Disclosure of the Invention The invention is directed to treatment of certain conditions of the endometrium using photodynamic rnethods 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.
In another aspect, the invention is directed to a method for steriiization, 5 which method comprises administering to a subject an amount of a green porphyrin that will localize in the endometrium; and irradiating the endometriumwith light absorbed by the green porphyrin. Topical administration is preferred.In still another aspect, the invention is directed to a method to terminate early pregnancy, which method comprises administering to a subject, preferably 10 topically, an amount of green porphyrin that will localize in the endometriumand 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.
In still another aspect, the invention is directed to a formulation, for 15 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.
Brief Description of the Drawings 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 25 time point).
Figure 3 shows light microscopic images of untreated (A, C) and treated (B, D) rabbit uterine horns 4 weeks after photodynamic therapy. Low magnification (X 16) and high magnification ( X 100) images are shown in panels A, B, and C, D, respectively.
W o 97/06797 PCTnUS96rIZ828 i Figure 4 shows scanning electron micrographs (X 5000) of control (A) and treated rabbit endometrium 1 week (B) and 4 weeks (C) after photodynamic therapy.
Modes of carrying out the Invention In general, the green porphyrin is of a formula shown in Figure 1 or a mixture thereof. Referring to Figure 1, in preferred embodiments each of R1 and R2 is independently selected from the group consisting of carbalkoxyl (2-6C), alkyl (1-6C), arylsulfonyl (6-10C), cyano and -CoNR5Co wherein R5 is aryl (6-10C) or alkyl (1-6C); each i~3 is independently carboxyl, carboxyalkyl (2-6C) or a salt, amide, ester or acylhydrazone thereof or is alkyl (1-6C); R4 is CH=CH2 or -CH(oR4 )CH3 wherein R4 is H, or alkyl (1-6C) optionally substituted with a hydrophilic substituent. Especially preferred also are green porphyrins of the formula shown in Figures 1-3 or 1-4 or mixtures thereof.
More preferred are embodiments are those wherein the green porphyrin is of the formula shown in Figure 1~3 or 1~ or a mixture thereof and wherein each of R' and R2 is independently carbalkoxyl (2-6C); one R3 is carboxyalkyl (2-6C) and the other R3 is an ester of a carboxyalkyl (2-6C) substituent; and R4 is CH=CH2 or-CH(OH)CH3.
Still more preferred are embodiments wherein green porphyrin is of the formula shown in Figure 1-3 or 1-4 and wherein R1 and R2 are methoxycarbonyl; one R3 is -CH2CH2COOCH3 and the other R3 is CH2CH2COOH; and R4 is CH=CH2; i.e., BPD-MA or BPD-MB, more preferably BPD-MA.
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 30 with lipoproteins and are easily packaged in liposomes. Compositions of greenporphyrins 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 5 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. These 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 or10 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. Of course, 15 these compositions may also contain minor amourits of nontoxic, auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth. One preferred formulation is liposomally formulated Benzoporphyrin Derivative Monoacid Ring A in dextran 70 (HyskonR, Pharmacia Inc., Piscataway, NJ) HyskonR is viscous, hydrophilic, branched polysaccharide 20 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, 25 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 lower doses than complete endometrial ablation. Typically, the dose of green porphyrin used is 30 within the range of from about 0.04 to about 20 mglkg, preferably from about 0.04-2.0 mg/kg. This range is merely suggestive, as the number of variables W O 97/06797 PCTrUS96/12828 in regard to an individual treatment regime is large and considerable excursionsfrom these recommended values are expected.
It should be noted that the various parameters used for effective, selective photodynamic therapy in the invention are interrelated. Therefore, the5 dose should also be adjusted with respect to other parameters, for example, fluence, irradiance, duration of the li~ht used in photodynamic therapy, and time interval between administration of the dose and the therapeutic irradiation. Allof these parameters should be adjusted to produce significant damage to endometrial or neoplastic tissue without significant damage to the surrounding 1 0 tissue.
In a preferred method of the invention, 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.
After the photosensitizing green porphyrin has been administered, 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 20 penetration enhancing agents ha~e 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.
In a rabbit model, liposomally formulated Benzoporphyrin Derivative Monoacid 25 Ring A (BPD-MA) induced highest glandular and stromal fluorescence of the endometrium after 1.5 hours. Light administration at this time interval resultedin lasting endometrial destruction verified by histology after 4 weeks.
The endometrium is exposed to light at the wavelength of maximum - absorbence of the green porphyrin, usually between about 670 and 780 nm.
30 A wavelength in this range is especially preferred for enhanced penetration.
Shorter wavelengths can also be used, if desired, for convenience. In many W O 97/06797 PCTrUS96/12828 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 5 preferably varies from about 10-200 Jouies/cm2. The irradiance typically varies from about 100-900 mW/cm2, with the range between about 100-600 mW/cm2 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 10 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 15 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 20 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 25 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 1995; 10:221-226 and Steiner et al., Geburtsh. U. Frauenheilk. 1995; 55:
Artikel 243), the entire contents of which are incorporated herein by reference.Complications, such as hemorrhage and photosensitivity in non-target tissues, 30 are not noted with the invention method in rabbit and rat models. Thus, photodynamic therapy with a green porphyrin appears to be a simpler, safer W Og71067g7 PCTnUS96~1Z8Z8 alternative than surgery for treating disorders of the endometrium, sterilization and termination of early pregnancy.
The following examples are to illustrate, but not to limit, the invention.
E~ample 1 Treatment of rabbit endometrium with benzoporphvrin derivative and photodynamic therapy Eighteen mature female New Zealand White rabbi~s weighing 3.6-4.3 kg were anesthetized with an intramuscular injection of ketamine and xylazine (2:1), 0.75 ml/kg and isoflurane was added during the surgical intervention.
Liposomally formulated benzoporphyrin derivative MA (BPD-MA, QLT
PhotoTherapeutics, Inc., Vancouver, BC, Canada) was stored in the dark at 4~C
and protected from light at all times. Shortly before administration, BPD-MA
was equilibrated to room temperature and reconstituted to 2 mg/ml in dextran 15 70 (Hyskon, Pharmacia, Inc., Piscataway, NJ). One milliliter was injected into the left uterine horn 3-5 mm distal l:o 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 20 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, 30 MA) was used to verify the wavelength. Because of the length of the rabbit uterine horn is 10-15 cm, multiple (four to five) segmental irradiation was W O 97/06797 PCT~US96/12828 required. A total of 195 mW was launched into the fiber (65 mW/cm fiber tip) during 20 minutes, resulting in a variable tissue dose which, depending on geometry, ranged from 40-80 J/cm2.
No adverse side effects, such as hemorrhagia or photosensitivity were 5 found in non-target tissue during 4 weeks of observation.
Example 2 Analysis of BPD-MA Pharmacokinetics in Rabbit Uteri Following Photodynamic TheraPy For specimen retrieval, the rabbits were first anesthetized with isoflurane 10 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 15 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 12 hours following administration of 2 mg/mL BPD-MA-Hyskon. Three animals 20 were studied for each time point. Frozen sections of 6,u thickness were made in low diffuse light (Cryostat Microtome; AO Reichert, Buffalo, NY). Low light level tissue fluorescence was performed with a slow-scan, thermoelectrically cooled CCD camera system (Model ST-180; Princeton Instruments, Trenton, NJ) coupled to a Zeiss Axiovert 10 inverted fluorescence microscope (Carl Zeiss 25 Inc., Oberkochen, Germany). A 10X objective Zeiss Plan neofluar (numerical aperture 0.3) was used to visualize bright-field and fluorescence images of the frozen sections. A 100-W mercury arc lamp coupled to a mechanical shutter (UniBlitz model Tl32; Vincent Associates, Rochester, NY) and filtered through a 405 nm, broad-bandpass filter provided excitation light. A dichroic mirror (FT30 420) refiected the excitation light onto the sample and transmitted the fluorescence emission through a 635 nm broad-bandpass filter onto the detector.
W O 97/~6797 PCTAUS96~282g Instrument control, image acquisition, and processing were performed with a Maclntosh llfx computer (Apple Computers, Cupertino, CA) and IPlab - software (Signal Analytics Corp., Vienna, VA). Minimal sample photobleaching was achieved by synchronizing the mercury lamp and CCD-camera shutters.
5 To esLi,nale light distribution in the tissue slices, background images were acquired from blank slides with identical characteristics. All fluorescence images were normalized by the following algorithm to correct for nonuniform illumination:
Normalized fluorescence image =
mean (back~round - dark noise) X image (fluorescence - dark noise) image (background - dark noise) in which mean (background - dark noise) is the mean gray-scale value for the 15 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 20 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 25 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, strornal, and circular muscular (myometrium) tissue at four time points (1.5, 3, 6, and 12 hours). Glandular fluorescence wassignificantly 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 5 appreciable contrast evident at 12 hours. The difference between glandular and stromal or myometrial fluorescence decreased with time (P = 0.01 and P
= 0.02, respectively). Glandular and stromal drug concentrations were highest at 1.5 hours, the earliest time point tested.
Fluorescence microscopy data revealed a significantly higher 10 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 15 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 20 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.
W O 97/06797 PCT~US96~1Z828 Example 3 Analvsis of Structural Changes in the Rabbit Uterus Following Photodynamic Therapy with BPD-MA
Following intrauterine photodynamic therapy in the rabbits, the samples 5 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 dehydrat~d in graded ethyl alcohol, cleared in Histo-clear (National Diagnostics, Manville, NJ), inrilllaled with paraffin using a tissue processor (Model 155MP; Fisher Scientific, Pittsburgh, PA), and embedded in 10 paraffln. Sections were cut at 6 rn, 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 15 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 Elecl:ronic Instrument Co., Mahwah, NJ).
Structural changes in the endometrium following photodynamic therapy were evaluated in the right (control) and left (treated) uterine horns of the same 20 rabbit using both light and scanning electron microscopy. Figure 3 shows the light-microscopy results for IOX and 50X magnifications. Scanning electron microscopy images (5000X) are presented in Figure 4.
Low magnification light microscopy images of untreated (Figure 3A) and treated (Figure 3B) uterine horns showed destruction of epithelial and stromal 25 endometrial structures 4 weeks after photodynamic therapy. The lumen was obliterated and replaced by stroma resembling scar tissue. Processes of recanalization were not evident. The bordering circular layer of the myometrium (right side in picture) was loosened and invaded by connective - tissue. Because of spillage through the perforated uterine wall during and after 30 intrauterine drug injection, the description of damage in the longitudinal myometrial layer and serosa may lead to wrong conclusions. Minimal re-epithelialization was observed in a minority of sections (not shown), perhaps due to uneven light distribution. High-magnification images (Figures 3C and 3D) revealed dramatic changes in the endometrial structure, with complete absence of the endometrial epithelium. In addition, the post-irradiation stromal5 components were abundant in the extracellular matrix, with obviously decreased v~scul~rization. One week after irradiation, the treated area demonstrated acute changes such as hemorrhagia, moderate vessel damage, and white blood cell i, IrilLl ~lion (not shown). For studies following photodynamic therapy, 1 and 4 weeks were selected as time points for long-10 term follow-up because regeneration after mechanical destruction of the endo-metrium in rabbits is complete in 3 days. Generally, substantial, persistent destruction of the endometrium was effected (Figures 4B and C). However, regional variations in re-epithelialization indicate that maintaining the optical dose at or above the photodynamic threshold is crucial for irreversible damage.
15 Equal drug distribution within the uterine cavity, illumination of short duration to avoid photodegradation, drug washout, and homogeneous light distribution may also contribute to successful and complete endometrial destruction.
Scanning electron micrographs of the untreated uterine horn (Figure 4A) showed ciliated cells surrounded by nonciliated microvillus cells. In contrast, the 20 treated horns 1 week (Figure 4B) and 4 weeks (4C) following photodynamic therapy exhibited complete loss of luminal columnar epithelium and glandular openings. The surface was replaced by a collagen network resembling scar tissue.
ExamPle 4 Photodynamic TheraPv of the Rat Endometrium Usinq BPD-MA
Endometrial ablation experiments using BPD-MA were conducted in Sprague Dawley rats employing the methods described in examples 1 through 3. BPD-MA (2 mg and 4 mg) was diluted in 1 mL Hyskon TM (Dextran 70).
30 These two drug concentrations were injected topically (I.U.) in rats (volume of 0.15 mL). Fluorescence microscopic studies have demonstrated that there is no significant difference in uptake and distribution between these two drug concentrations. Low light level tissue fluorescence imaging revealed a distinct positive endometrium to myometrium ratio within the first 12 hours. Relative fluorescence was highest in the endometrial glands and lowest in the 5 myometrium. After 12 hours the intensity of fluorescence leveled off in all compartments and values of the glands approached those of other layers.
PDT (2 mg/mL BPD-MA an:l 80J/cm2 intra-uterine light) resulted in endometrial destruction at one week. No regeneration of endometrial tissue was observed at 4 weeks. None of the animals showed any alterations in light 10 treated skin areas at any time.
In reproductive performance assays, only 0.44 implantations per rat occurred in the treated area of the left horn after PDT. In contrast, 8.9 implantations per rat were found in the corresponding area of the untreated right horn.
Example 5 Topical APPlication of BPD-MA to Human Endometrium To analyze the distribution olF topically applied BPD-MA in the human uterus, patients scheduled for hysterectomy are recruited for inclusion in the 20 study. Three mg liposomally formulated Benzoporphyrin Derivative Monoacid Ring A is dissolved just before use under sterile conditions in 1.5 mL dextran 70 (32% WN) in dextrose (10% WN; HyskonR, Pharmacia Inc., Piscataway, NJ). This amount is equal to 0.05 mg/kg (for 60 kg body weight) and is well below any sysl:emic topical dose administered to any of the previous human or 25 animal studies and no side effects are anticipated.
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 30 is cleansed with providone-iodine. A Sholkoff balloon hysterosalpingography catheter (Cook, Bloomington, IN) with an outer diameter of 2 mm is inserted W O 97/06797 PCTrUS96/12828 into the cervical canal. 1.5 mL of 2 mg/mL BPD-MA/HyskonR 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 5 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 i"s~illaLion and operation.
Example 6 Analvsis of BPD-MA Distribution in the Human Uterus Followina, Topical APPlication of BPD-MA
Immediately after hysterectomy 2-3 areas of the uterine wall are 15 obtained. The samples represent the corpus and fundus of the uterus, each 4-6 mm thick, 10 mm wide and show all layers of the uterine wall (endometrium, myometrium, serosa). Specimens are immediately placed in molds containing embedding medium for frozen sections (Tissues Tek, O.C.T.
media, Miles, Elkhart, IN), snap frozen on dry ice and stored at -70 o C in a 20 light-impermeable container. All specimens are handled in the dark. Tissues are sectioned in low diffuse light (Cryostar microtome, AO Reichert, Buffalo, NY) to obtain 5-micron-thick slices for fluorescence analysis taken from severallocations, approximately 3 mm apart.
The main specimen (>99% of the uterus) is handled in the operating 25 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
30 100-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. These features permit selection of optimal excitation and emission spectral regions for visualizing samples. In addition, tissue autofluorescence is isolated from drug fluorescence by thermoelectrically cooled, slow-scan CCD (charge-coupled device) camera (Princeton 5 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 10 about 1 second for most frozen section fluorescent images. In order to estimate light distribution, 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. All images are acquired and automatically stored in a 1-Gb 15 capacity rewritable optical drive system. Processing and camera control are performed by a Maclntosh llfx computer with appropriate software (Signal Analytics Corp., Vienna, Virginia. Specimens are divided into anatomical layers for comparative analysis, i.e., endiometrial glands, endometrial stroma and myometrium.
Claims (23)
1. A method to treat disorders of the endometrium or for sterilization, which method comprises:
administering to a subject an amount of green porphyrin sufficient to permit an effective amount to localize in said endometrium;
permitting sufficient time to elapse to allow an effective amount of said green porphyrin to localize in said endometrium; and irradiating endometrium with light absorbed by the green porphyrin.
administering to a subject an amount of green porphyrin sufficient to permit an effective amount to localize in said endometrium;
permitting sufficient time to elapse to allow an effective amount of said green porphyrin to localize in said endometrium; and irradiating endometrium with light absorbed by the green porphyrin.
2. The method of claim 1, wherein said green porphyrin is topically applied to the endometrium.
3. The method of claim 1 wherein said endometrial disorder is dysfunctional uterine bleeding.
4. The method of claim 1 wherein said endometrial disorder is endometriosis.
5. The method of claim 1 wherein said endometrial disorder is menorrhagia.
6. The method of claim 1 wherein said endometrial disorder is a neoplasm.
7. The method of claim 1 wherein the green porphyrin is of the formula 1-6 shown in Figure 1, wherein R1, R2, R3 and R4 are non-interfering substituents.
8. The method of claim 7 wherein said R1 and R2 are carbomethoxy and carboethoxy.
9. The method of claim 7 wherein each R3 is -CH2CH2COOH or a salt, amide, ester or acyl hydrazone thereof.
10. The method of claim 7 wherein said green porphyrin is of the formula shown in Figure 1-3 or 1-4 or a mixture thereof and wherein each of R4 is a non-interfering substituent.
11. The method of claim 10 wherein said green porphyrin is selected from the group consisting of BPD-DA, BPD-DB, BPD-MA and BPD-MB.
12. The method of claim 11 wherein said green porphyrin is BPD-MA.
13. A method to terminate early pregnancy which method comprises:
administering to a pregnant subject an amount of green porphyrin sufficient to permit an effective amount to localize in endometrium;
permitting sufficient time to elapse to allow an effective amount of said green porphyrin to localize in said endometrium; and irradiating said endometrium with light absorbed by the green porphyrin to result in partial ablation and re-epitheliazation of the endometrium.
administering to a pregnant subject an amount of green porphyrin sufficient to permit an effective amount to localize in endometrium;
permitting sufficient time to elapse to allow an effective amount of said green porphyrin to localize in said endometrium; and irradiating said endometrium with light absorbed by the green porphyrin to result in partial ablation and re-epitheliazation of the endometrium.
14. The method of claim 13, wherein said green porphyrin is topically applied to the endometrium.
15. The method of claim 13 wherein the green porphyrin is of the formula 1-6 shown in Figure 1, wherein R1, R2, R3 and R4 are non-interfering substituents.
16. The method of claim 15 wherein said wherein R1 and R2 are carbomethoxy and carboethoxy.
17. The method of claim 15 wherein each R3 is -CH2CH2COOH
or a salt, amide, ester or acyl hydrazone thereof.
or a salt, amide, ester or acyl hydrazone thereof.
18. The method of claim 15 wherein said green porphyrin is of the formula shown in Figure 1-3 or 1-4 or a mixture thereof and wherein each of R4 is a non-interfering substituent.
19. The method of claim 18 wherein said green porphyrin is selected from the group consisting of BPD-DA, BPD-DB, BPD-MA and BPD-MB.
20. The method of claim 19 wherein said green porphyrin is BPD-MA.
21. A composition for topical application of a green porphyrin to the endometrium comprising a topical carrier and a therapeutically effective amount of green porphyrin, wherein said topical carrier consists essentially of a viscous solution or gel, and optionally, a penetration enhancing agent.
22. The composition of claim 21 where in said viscous solution is dextran 70.
23. The composition of claim 21 wherein said penetration enhancing agent is Azone or DMSO.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US227695P | 1995-08-14 | 1995-08-14 | |
| US60/002,276 | 1995-08-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2229662A1 true CA2229662A1 (en) | 1997-02-27 |
Family
ID=21700029
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002229662A Abandoned CA2229662A1 (en) | 1995-08-14 | 1996-08-06 | Endometrial ablation using photodynamic therapy with green porphyrins |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0844876A4 (en) |
| JP (1) | JP2000502044A (en) |
| AR (1) | AR004181A1 (en) |
| AU (1) | AU6692396A (en) |
| CA (1) | CA2229662A1 (en) |
| WO (1) | WO1997006797A1 (en) |
| ZA (1) | ZA966833B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2821831T3 (en) * | 2009-06-11 | 2021-04-27 | Photocure Asa | Semi-Solid Pharmaceutical Compositions and Products Comprising 5-Aminolevulinic Acid Esters |
| RU2714599C1 (en) * | 2018-12-26 | 2020-02-18 | Министерство науки и высшего образования Российской Федерации Федеральное государственное бюджетное учреждение здравоохранения Центральная клиническая больница Российской академии наук (ЦКБ РАН) | Method of infertility treatment in patients with chronic endometritis |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5283255A (en) * | 1987-01-20 | 1994-02-01 | The University Of British Columbia | Wavelength-specific cytotoxic agents |
| US5422093A (en) * | 1989-07-28 | 1995-06-06 | Queen's University | Photochemotherapeutic method using 5-aminolevulinic acid and precursors thereof |
| US5234940A (en) * | 1989-07-28 | 1993-08-10 | Queen's University | Photochemotherapeutic method using 5-aminolevulinic acid and precursors thereof |
| US5330741A (en) * | 1992-02-24 | 1994-07-19 | The Regents Of The University Of California | Long-wavelength water soluble chlorin photosensitizers useful for photodynamic therapy and diagnosis of tumors |
-
1996
- 1996-08-06 JP JP9509347A patent/JP2000502044A/en 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
- 1996-08-06 CA CA002229662A patent/CA2229662A1/en not_active Abandoned
- 1996-08-06 AU AU66923/96A patent/AU6692396A/en not_active Abandoned
- 1996-08-13 ZA ZA9606833A patent/ZA966833B/en unknown
- 1996-08-14 AR ARP960103988A patent/AR004181A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP0844876A4 (en) | 1999-12-01 |
| EP0844876A1 (en) | 1998-06-03 |
| WO1997006797A1 (en) | 1997-02-27 |
| JP2000502044A (en) | 2000-02-22 |
| AU6692396A (en) | 1997-03-12 |
| AR004181A1 (en) | 1998-11-04 |
| ZA966833B (en) | 1997-02-27 |
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