CA2126761C - Photochemotherapeutic method using 5-aminolevulinic acid and precursors thereof - Google Patents

Abstract

A method of detecting and treating tissues and cells that preferentially accumulate a porphyrin following exposure to a biosynthetic precursor of the porphyrin, such as malignant and non-malignant tissue abnormalities and lesions of the skin; conjunctiva; respiratory, digestive and vaginal mucosa; endometrium and urothelium; nervous system, and for ablating endometrial tissue. As part of bone marrow rescuing autologous transplantation procedures, blood cells and bone marrow cells containing abnormal cells that preferentially accumulate porphyrin from biosynthetic precursors such as 5-aminolevulinic acid, can be returned to the patient after ex vivo contact with the precursor and photoactivation of the accumulated porphyrin. Alternatively, the patient can be treated with the precursor prior to removal of blood cells or bone marrow cells for ex vivo photoactivation.

Description

ACID AND PRECURSORS THEREOF

Background of the Invention This invention relates to the detection and treatment of certain malignant and non-malignant tissue abnormalities. More particularly, this invention relates to the use of fluorescence to detect photochemotherapy to treat certain malignant and non-malignant tissue abnormalities. The invention also relates to the detection and treatment of abnormalities in body fluids or suspensions containing abnormal cells by induced fluorescence and photochemotherapy respectively. The invention further relates to treatment of both normal and abnormal endometrial tissue and of leukemic and lymphoma cells by photochemotherapy.

Tissue abnormalities involving the skin usually are detected and assessed by a combination of visual inspection and palpation. In certain clinical situations the sensitivity of the visual inspection can be enhanced by the use of non-white light (either ultraviolet or a narrow band in the visible), or by the prior application of a contrast-enhancing agent such as dilute acetic acid or certain stains. Tissue abnormalities that involve surfaces that cannot be palpated (such as the bronchi or the urinary bladder) may be visualized via an appropriate scope.
Some specialized scopes can detect induced fluorescence. If the abnormality in question is associated with a difference in either the extent or the pattern of tissue vascularization, such an instrument may be used to determine the limits of the area involved by the abnormality, for example, by visualizing an injected bolus of fluorescein as it passes through the vasculature of both the lesion and the adjacent normal tissue.

In addition, fluorescence-detecting scopes are being used experimentally to identify areas of tissue that show strong porphyrin fluorescence following the intravenous injection of exogenous porphyrins such as hematoporphyrin IX
(HpIX), hematoporphyrin derivative (HpD), Photofri II or "dihematoporphyrin ether". Although, such porphyrins tend to accumulate semi-preferentially in malignant tissues, they also accumulate in tissues that are regenerating following an injury or in the rapidly growing tissues of an embryo or fetus. Normal liver, spleen, and kidney also tend to accumulate these porphyrins. Using such compounds and fluorescence-detecting scopes, areas of malignant tissue too small to be identified by standard forms of visual inspection have been identified in the bronchi and in the urinary bladder.

Unfortunately, following parenteral administration of the aforementioned exogenous porphyrins, clinically significant (photosensitizing) amounts of porphyrin persist in the skin for at least two weeks, occasionally for more than two months. This means that patients must avoid exposure to sunlight (either direct, or through window glass) for an inconveniently long period of time post-injection. Understandably, patient compliance often is poor, and accidental phototoxic "sunburn" is a common occurrence in the weeks following a diagnostic or therapeutic injection of porphyrins. Persistent photosensitivity is the major hazard associated with this technique, and is the main reason why it is not used more widely.

The standard or conventional forms of treatment for cancer comprise surgery, radiotherapy and chemotherapy. However, other forms of treatment are also known, including photochemotherapy or photodynamic therapy (PDT). PDT
is currently being used, on an experimental basis, to treat several different types of cancer as well as certain non-malignant lesions such as psoriasis. The patient is given a photo-activatable drug that has some degree of specificity for the tissue being treated. A tissue volume that includes the target tissue is then exposed to photoactivating light so as to destroy the target tissue while causing only mild and reversible damage to the other tissues in the same treatment volume.

There are two main types of photochemotherapeutic agents in clinical use at present. The first type, methoxypsoralens, are given systemically and are activated by ultraviolet light. Localized exposure of psoralen-containing tissues to ultraviolet light induces a localized photochemical reaction that causes the drug to bind covalently to the DNA of living cells, thus destroying their proliferative potential. The second type, porphyrins, are also given parenterally (typically by intravenous injection), although occasionally they are given either topically or by intralesional injection. They can be activated by visible (red) light. The localized exposure of porphyrin-containing tissues to such light ordinarily does not induce a chemical reaction between cell components and the porphyrin molecules.
Instead, the porphyrins act as catalysts by trapping the energy of the photoactivating light and then passing it on to molecules of oxygen, which in turn are raised to an excited state that is capable of oxidizing adjacent molecules or structures.
Cell death is not caused primarily by damage to the DNA, but by damage to essential membrane structures. Photochemotherapy is used at present for the treatment of certain types of cancer and non-malignant lesions, such as psoriasis.
Although, the goal of such treatment is sometimes cure (mainly for basal cell carcinomas), usually the goal is palliation through local control when none of the standard forms of therapy are considered likely to offer a significant degree of benefit to the patient.

Methoxypsoralen (PUVA) therapy is used mainly for the treatment of psoriasis, but sometimes it is also used to treat very superficial cancers that involve the skin (mainly mycosis fungoides). However, there are two serious problems with such treatments. First, the procedure is known to be carcinogenic in humans. Second, the photoactivating ultraviolet light is absorbed so strongly by most tissues that the depth at which malignant tissue can be killed is limited to a few millimeters below the illuminated surface. These problems severely limit the usefulness of the methoxypsoralens for photochemotherapy.

As noted above, the porphyrins most commonly used for photochemotherapy are hematoporphyrin IX (HpIX), hematoporphyrin derivative (HpD), and Photofri II, a semi-purified form of HpD. As noted above, when porphyrins are used as photosensitizers, cell death results from damage to cell membranes. Consequently, malignant transformation is not a serious problem.

Moreover, as the visible (red) light that is used to photoactivate porphyrins penetrates tissue much more deeply than does the ultraviolet light that must be used to photoactivate methoxypsoralens, the depth at which porphyrin-treated tissue can be killed is substantially greater. Also, as certain types of porphyrins show a significant tendency to accumulate preferentially in malignant tissues, it is sometimes possible to destroy such tissues without causing clinically significant damage to adjacent normal tissues.

As noted above, the main problem with the systemic use of prior art porphyrins (e.g. HpIX, HpD and Photofri II) is that photosensitizing concentrations persist in the skin for several weeks to several months following their administration. Consequently, severe accidental phototoxic skin reactions may occur unless the patient avoids exposure to sunlight until the concentration of the photosensitizer in the skin has been reduced to a harmless level. At present, the problem of photosensitivity following the administration of porphyrins is handled by advising the patient to avoid any form of exposure to sunlight (or to very bright artificial lights) for a period of at least two weeks post-injection, and to initiate subsequent exposure to sunlight very cautiously. Not all patients comply with these instructions, as it is often quite inconvenient to do so. In addition, the use of a sunscreen with a high blocking factor is generally recommended with warning that this will only reduce the hazard somewhat, not eliminate it completely. In a few cases, patients whose photosensitization persisted for more than a month post-treatment have been given large daily doses several months in an attempt to prevent accidental phototoxic damage. Finally, attempts have been made to reduce phototoxicity by applying the photosensitizer topically to a limited area.

However, another type of problem is frequently encountered when HpIX
or HpD are applied topically in DMSO (dimethylsulfoxide), Azone, or some other vehicle intended to enhance their diffusion through tissue. The porphyrins tend to become immobilized wherever they happened to be when the DMSO or Azon becomes diluted by normal tissue fluids to such an extent that the porphyrins can no longer diffuse through the tissue (or even remain in solution).
Consequently, the topical application of porphyrins often is associated with a loss of specificity for malignant tissues, and normal tissues near the site of application may develop persistent photosensitization from the localized concentration of porphyrin.

Object of Invention It is an object of the present invention to provide a method for the detection of certain types of malignant and non-malignant cell and tissue abnormalities by induced fluorescence.

It is another object of the present invention to provide a photodynamic (photosensitizing) treatment method using an agent which can be administrated either systemically or topically and which is not in itself a photosensitizer but which induces the synthesis and/or accumulation of protoporphyrin IX (PpIX) in vivo.

It is another object to provide a method of endometrial tissue ablation for the treatment of dysfunctional uterine bleeding, endometriosis, endometrial cancer, and iron deficiency anaemia caused by excessive bleeding at periods.
Another object is to provide a contraceptive method, a method of sterilization or near sterilization, a method of eliminating unwanted monthly periods, and a method of early termination of pregnancy.

Yet another object is to provide a method of detecting or treating by photochemotherapy leukemic and lymphoma cells that preferentially accumulate protoporphyrin IX from metabolic precursors.

Summarv of the Invention In one aspect of this invention there is provided a method for treating dysfunctional uterine bleeding, iron deficiency anaemia due to excessive menstrual bleeding, a method of sterilization or near sterilization, a method of contraception, and a method for early termination of pregnancy, in which an effective amount of a biosynthetic precursor of protoporphyrin IX ("PpIX") is administered to a patient in need of treatment so as to induce synthesis of PpIX
in endometrial tissue and exposing said tissue to light having a wavelength within in the biosynthetic pathway for heme is administered to a patient in need of treatment so as to induce synthesis of protoporphyrin IX (PpIX) in endometrial tissue and exposing said tissue to light having a wavelength within the photoactivating action spectrum of PpIX to thereby induce photoactivation in said tissue.

In another aspect of this invention, there is provided a method of detecting and treating leukemic and lymphoma cells and certain metastatic carcinomas by contacting such cells with one or more biosynthetic precursors of a protoporphyrin in vivo or ex vivo, and exposing such treated cells with light of a photoactivating wavelength. Where such a procedure is part of a bone marrow or peripheral blood purging autotransplantation procedure, bone marrow thus treated ex vivo ar returned to the patient.

In yet another aspect of this invention there is provided a composition comprising a biosynthetic precursor of PpIX in a pharmaceutically acceptable vehicle in appropriate dosage units for treating malignant and non-malignant tissue abnormalities and lesions of the skin, mucosa, exocrine glands and ducts, gonads, thymus, spleen, lymph, blood and the nervous system.

In preferred aspects of this invention, a natural biosynthetic precursor of PpIX, namely, 5-amino-4-oxo-pentanoic acid, otherwise known as 5-aminolevulinic acid ("ALA"), is employed to generate PpIX in situ, and a preferred wavelength of the photoactivating light is in the range of 350-750 nm, more preferably red light of 620 to 680 nm.

Brief Description of Drawinss Figure 1 is a histogram illustrating ALA-induced PpIX fluorescence in various leukemic and lymphoma cell lines.

Figure 2 is a histogram illustrating ALA-induced PpIX fluorescence in hematopoietic tissues of normal and leukemic (P388) mice.

Figure 3 is a graph illustrating the kinetics of ALA-induced PpIX
fluorescence in mouse leukemic cells (P388).

Figure 4 is a graph illustrating a characteristic PpIX fluorescence emission spectrum in P388 leukemia cells following incubation with ALA.

Figure 5 are graphs illustrating ALA-induced PpIX photodynamic killing of K562 leukemia cells in a radioactive chromium release cytotoxicity assay. (A) mM ALA in Krebs Ringer; or (B) control. Chromium release (cell death) was related to the dose of photoactivating light.

Figure 6 is a histogram illustrating ALA-induced PpIX photodynamic killing of P388 leukemia cells as assessed by a fluorometric cytotoxicity assay. A
loss of ability of the cells to cleave fluorescein intracellularly equates with cytotoxicity.

Figure 7 is a graph illustrating survival rates when mice were given a marrow-sterilizing dose of ionizing radiation (950 rads) followed by the intraperitoneal injection of a mixture of syngeneic normal spleen cells and mouse leukemia cells that had been preincubated in vitro with ALA, then exposed to 72 J/cm2 of photoactivating light (600-700nm).

Detailed Description of the Invention Protoporphyrin IX (PpIX), a naturally occurring photosensitizer, is the immediate precursor of heme in the heme biosynthetic pathway. Although all nucleated cells have at least a minimal capacity to synthesize PpIX, certain types of cells and tissues can synthesize relatively large quantities of this porphyrin.

Under normal conditions, the synthesis of PpIX in such tissues is under feedback control such that the cells produce it at a rate just sufficient to match their need for heme; feedback control is centered on ALA-synthesis which is the rate-limiting step in the process. This rate limiting step can be by-passed by providing exogenous ALA, porphobilinogen, or other immediate precursors of PpIX.

We have found that certain cells, tissues and organs will, following parenteral, oral or topical exposure to ALA, accumulate wuch large amounts of PpIX that they exhibit significant PpIX fluorescence and become photosensitive.
The PpIX appears to be synthesized in situ. The oral and parenteral routes lead to the induction of clinically useful concentrations of PpIX in certain benign and malignant tissues throughout the body.

As noted above, only certain types of cells and tissues synthesize and then accumulate clinically useful amounts of PpIX when provided with an excess of ALA. We have discovered that carcinomas and other lesions of the skin, mucosa (respiratory, digestive, and vaginal), endometrium and urothelium, certain other types of solid tumors and certain types of leukemic and lymphoma cells preferentially accumulate PpIX when the tissues or cells are contacted with appropriate concentrations of ALA. Sites of treatment therefore could include lesions or cellular abnormalities involving (i) skin and conjunctiva; (ii) the lining of the mouth, pharynx, esophagus, stomach, intestines and intestinal appendages, rectum, and anal canal; (iii) the lining of the nasal passages, nasal sinuses, nasopharynx, trachea, bronchi, and bronchioles; (iv) the lining of the ureters, urinary bladder, and urethra; (v) the lining of the vagina, uterine cervix, and uterus; (vi) the parietal and visceral pleura; (vii) the lining of the peritoneal and pelvic cavities, and the surface of the organs contained within those cavities; (viii) the dura mater and meninges; (ix) any tissues or suspensions of body fluids containing abnormal cells, including blood and bone marrow, that can be made accessible to photoactivating light either ex vivo, in vitro, at time of surgery, or via an optical fibre inserted through a needle; (x) all exocrine glands and associated ducts, including: mammary glands, sebaceous glands, ceruminous glands, sweat glands, and lacrimal glands; mucus-secreting glands of the digestive, urogenital, and respiratory systems; salivary glands; liver, bile ducts, and gall bladder;
pancreas (exocrine component); gastric and intestinal glands; prostate;
Cowper's, Bartholin's and similar glands. It is also contemplated that cell abnormalities in the gonads (testes and ovaries), thymus, spleen, lymph nodes, bone marrow, lymph and blood may also be treated according to the invention. Tumors of the nervous system or connective tissues (sarcomas) may also be treated according to this invention.

Treatment of non-malignant lesions such as genital warts and psoriasis and other indications of the endometrium, such as contraception, vaginal bleeding, abortion and sterilization is also contemplated. In effect, an alternative to hysterectomy is within the scope of this invention.

As used herein the term "skin" includes:

(A) the covering of the external surface of most of the body, commonly termed the skin.

(B) the covering of the external genitalia:

- labia majora, labia minora, clitoris, and associated structures - glans penis, prepuce, and associated structures (C) the covering of the zone of transition between skin and the mucosa of the digestive system:

- anal verge - vermillion border of the lips (D) the lining of the external auditory meatus, and the covering of the external surface of the tympanic membrane (E) all exocrine glands and associated ducts that are located at least partially within an epidermal surface described above, or within the underlying dermis.

The term "mucosa" includes:

(A) the lining of the whole of the respiratory tract:
- nasal passages and nasal sinuses - nasal pharynx and associated structures - larynx, oral pharynx and laryngeal pharynx, vocal cords, and associated structures - trachea, bronchi, and bronchioles (B) the lining of the whole of the digestive tract:
- oral cavity and tongue - esophagus - stomach - small intestine - large intestine, caecum, and appendix - sigmoid colon and rectum - anal canal (C) the lining of the whole of the urogenital tract:
- urethra, bladder, and ureters - renal pelvis and renal calyces - vagina, uterine cervix, uterus, and Fallopian tubes - vas deferens, seminal vesicles, ejaculatory duct, ampulla of vas, epididymis, and associated structures (D) the conjunctiva and the lining of the tear ducts.

(E) all exocrine glands and associated ducts that are located at least partially within one of the mucosal surfaces described above, or within the underlying submucosa.

The wavelength of the photoactivating light is of some importance, as it has been shown that between 1 and 10 percent of incident red light (600-700 nm) can pass through a slab of human tissue 1 cm thick, whereas only 0.001 percent or less of blue light (about 400 nm) can pass through the same thickness of human tissue.

The photosensitizer will, therefore, be more successful if it absorbs red light.
PpIX does strongly absorb red light. The present approach has several advantages over the prior art. First PpIX has a much shorter half-life (of the order of 2 hours) in normal tissues (human and mouse, at least) than does HpIX, HpD or Photofrin II (half-life approximately 1 week). This greatly reduces the danger of accidental phototoxic skin reactions in the days following treatment.
Second, the AI.A can be applied topically to certain types of lesions. Third, ALA
can be applied to cells (e.g., bone marrow pluripotent hematopoietic stem cells) ex vivo, and the treated cells returned to the patient as part of a bone marrow purging autologous transplantation procedure. These alternative procedures improve the specificity of the treatment, reduce the danger of accidental phototoxic reactions to a very low level, and greatly reduce the amount of both ALA and PpIX to which the entire body would be exposed if an equally effective dose of ALA were to be given systemically.

Both ALA and PpIX are normal products of intermediary metabolism, and are handled quite readily by the biochemical machinery of the body. However, as very large doses of ALA (just as with large doses of HpIX or HpD) are associated with a transient decrease in motor nerve conduction velocity, it is desirable to reduce the dose of ALA to the minimum that is still effective. Topical application requires much less ALA than systemic administration.

PpIX is rapidly inactivated by the photoactivating light. Following exposure of tissues containing PpIX to a therapeutic dose of photoactivating light, there is a substantial decrease in photosensitization of the tissues within the treatment volume. Consequently, if PpIX production is induced by the topical application of ALA to specific lesions, the patient can be exposed to sunlight immediately post-treatment without danger of serious photoxicity. Also, the dosimetry of the photoactivating light is greatly simplified.

As noted above, ALA is an effective inducer of PpIX when given by mouth, topical application, or parenterally. In contrast, HpIX, HpD and Photofr'u II are effective in most situations only when given by injection. The versatility of ALA enhances its acceptability for routine use by the medical profession. In addition, the normal and abnormal tissues that can be photosensitized by the administration of ALA are somewhat than those that can be photosensitized by the administration of HpIX, HpD or Photofriii II. Consequently, ALA may be useful in clinical situations in which the other photosensitizers are not.

We have discovered that exogenous ALA does not induce significant accumulation of PpIX (as determined by fluorescence) in normal cells of the blood, bone marrow, spleen, or lymph nodes, although it does so in cells of the thymus. However, as noted above and as will be detailed in the examples below, we have discovered that exogenous ALA can induce the accumulation of fluorescing- and photosensitizing concentrations of PpIX in certain types of leukemia and lymphoma cells, but not in others, as well as in certain types of metastatic carcinomas. Importantly, we have discovered that, exogenous ALA
induces much less accumulation of PpIX in pluripotent hematopoietic stem cells than in leukemic cells. Incubation of ALA with bone marrow cell suspensions containing a mixture of pluripotent hematopoietic stem cells and leukemic cells, followed by photoactivation, results in preferential phototoxic destruction of the leukemic cells. ALA-induced PpIX can thus be of value in the treatment of some types of leukemias and lymphomas, especially when used for the selective destruction of malignant cells in bone marrow prior to autotransplantation of the treated marrow (bone marrow rescue) after whole body chemotherapy or irradiation.

For the leukemia/lymphoma cells studies to be detailed in the examples below, the cells lines K562, RPMI-7666 and Daodi human leukemias, Raji, El-4 and P815 murine leukemia cells were obtained from Dr. H.F. Pross, Queen's University, Kingston, Ontario and were cultured in RPMI-1640 growth medium supplemented with 10% FCS. P388 murine leukemia cells can be maintained as an ascites tumor in the (B6D')F' mouse hybrid and transferred mouse to mouse.

For PpIX detection by fluorescence, cells are incubated with various concentrations of ALA (0-10 mM) in Krebs-Ringer bicarbonate buffer for various time periods (1-13 hours). Preferred is 5mM ALA for 6 hours at body temperature and 5% CO2. For in vivo experiments, normal and P388 leukemic mice can be injected with ALA at a dosage of about 250 mg in saline per kg body weight, and about 5 hours thereafter the animals can be killed and tissues excised for examination. For example, hematopoietic tissues can be gently homogenized in Krebs-Ringer buffer to produce cell suspensions. ALA-induced PpIX

fluorescence in cell lines and thus-produced cell suspensions can be measured in, for example, a Coulter Elite flow cytometer (488 nm excitation with a 595 nm long pass emission filter).

In order to assess the cytotoxic effects of exposure to ALA followed by photosensitization of the thus-produced PpIX, two methods may be used. In the chromium release assay, cells are pre-loaded with radioactive chromium (51Cr) then incubated with AI.A and photoactivated (600-700 nm). At various time points after irradiation, cells may be sedimented and the supernatant fluids tested for radioactive chromium in a gamma counter (e.g., Beckman Gamma 5500 counter). The release of chromium is directly proportional to cell toxicity.
In a fluorometric microculture cytotoxicity assay, cells are incubated with ALA, exposed to a single dose of photoactivating light (72 J/cm2), then incubated with fluorescein diacetate. Retention fluorescein is determined by fluorescence (excitation at 488 nM and emission at 538 nm) using flow cytometry equipment (Coulter). An inability to retain the fluorescein is reflective of cell membrane damage.

For photosensitization assays of stem cells, recipient animals can be given a large dose of ionizing radiation (e.g., 950 rad for mice) to destroy pluripotent bematopoietic stem cells. These animals can then be injected with leukemic mouse cells (for example, 106 P388 cells) or normal spleen cells (for example, cells) intraperitoneally, the cells having been prior-treated with ALA and photosensitized with 72 J/cm2 of light. Controls are injected with leukemic or spleen cells that have not been exposed to light. Resistance of the mice to the development of leukemia is then followed.

Thus, the present technique represents a significant advance in photodynamic therapeutic capability.

The following examples are provided merely to exemplify several embodiments of the invention, and are not to be construed as limiting in any way the scope of the invention that is set forth in the specification and in the appended claims.

Example 1 ALA was injected into rats at doses ranging from 1 to 50 mg directly into one horn of the didelphic uterus to minimize systemic photosensitization. The contralateral horn was injected with saline alone so that a paired comparison could be made. At a site 0.5 cm above the uterine bifurcation, ALA (Sigma Chemical Company, St. Louis, MO) was injected into the right uterine horn using a 1 ml tuberculin syringe with a 26 gauge needle (Bectin Dickinson and Company, Rutherford, NJ). The rats were allowed to recover and the uterus was removed 3 hours after ALA injection and the tissues were processed for either fluorescent microscopy or spectrophotofluorometry.

In other studies, 51 rats were divided into three different groups of 17 rats.
The animals were anesthetized with ether and a 3 cm incision was made through the anterior abdominal wall 1 cm rostral to the symphysis pubis. ALA dosages of 4, 8, or 16 mg in 0.1 ml saline were administered into one horn and an equivalent volume of saline was injected into the contralateral horn. The abdomen was closed and the rats were allowed to recover from the anaesthesia. Three hours later, 9 rats from the 4 or 16 mg ALA treated group, and 8 rats from the 8 mg ALA treated group were anaesthetized with ether. The sutures were removed and the incision was opened and extended 3 cm along the midline. The intestines were pushed away with a saline soaked gauze so that both uterine horns could be exposed for 30 minutes to red light from a 500 watt CBA halogen lamp (Kodak, Carousel 860 projector, Rochester, NY) equipped with a red filter (Hoya R-60, Tokyo, Japan) positioned 15 cm from the tissue (approximately 150 joules per cm2). The uterine horns were kept moistened with saline. The abdomen was then closed and the rats were allowed to recover. 10 days later all rats (including those not exposed to light) were bred to a fertile male. Mating was confirmed by the presence of either a sperm plug or the presence of sperm in vaginal smears.
Another control group consisted of 4 rats in whom a unilateral pregnancy was achieved by ligating one horn at its distal end prior to breeding. 10-15 days after breeding, rats were killed by decapitation. The abdomen was opened to confirm pregnancy and to determine the number and location of fetuses. Both uterine horns were harvested and preserved in 10% formalin. The nonpregnant uterine horns were dissected and histologically processed. The results are noted in Table 1 below.

Table 1 Fertility Assessment 10-20 Days after ALA

ALA Dose 4 4 8 8 16 16 (MG) Light NO YES NO YES NO YES
Exposure (30 minutes) N/Group 8 9 9 8 8 9 Preg./ 7/8 9/9 9/9 8/8 8/8 9/9 Saline Preg./ALA 8/8 1/9 6/9 0/8 5/8 0/9 Administration of 4 mg of ALA without light had no effect on fertility.
Pregnancy occurred in 8 of 8 uterine horns treated with ALA and 7 of 8 uterine horns treated with saline. In contrast, rats exposed to light following the treatment of 4 mg ALA exhibited compromised fertility. Only 1 pregnancy occurred in 9 uterine horns treated with ALA whereas fetuses were present in 9 of 9 uterine horns treated with saline. Somewhat different results occurred in rats treated with either 8 or 16 mg of ALA. In absence of light, fetuses occurred in all uterine horns treated with saline (n = 17) and 6 of 9 uterine horns treated with 8 mg of ALA and 5 of 8 uterine horns treated with 16 mg ALA. When the uterus was exposed to light following treatment of 8 mg ALA or 16 mg ALA or saline, all pregnancies were restricted to the saline-treated side. No pregnancies occurred in the ALA treated side.

212fi7b1 Example 2 Long Term Photodynamic Endometrial Ablation Rats were divided into 2 groups (6 and 7 rats/group and injected with 4 or 8 mg ALA. Example lwas repeated with the exception that all rats were exposed to light and the time from ALA administration to breeding was extended from 10-20 days to 60-70 days. All other procedures were identical to Example 1.

Breeding 60-70 days after photodynamic treatment with 4 mg ALA resulted in no implantations in the uterine horns treated with ALA (n = 6) whereas fetuses were found in all control uterine horns treated with saline (n = 6).
These results confirmed the long term endometrial ablative effect of PDT. In the groups of rats (n = 7) treated with 8 mg ALA 2 of 7 became pregnant in ALA
treated uterine horns compared with 7 of 7 pregnancies in the saline treated horns.

In order to show normal uterine histology of a nonpregnant uterine horn contralateral to a pregnant uterine horn one uterine horn was ligated at its distal end prior to breeding. At gestation of 10-15 days nonpregnant uterine horns were harvested and histologically processed. The uterine mucosa was lined with columnar epithelium and there was hypertrophic infolding of endometrial tissue with tortuous glands. In contrast, prior photodynamic treatment with ALA
consistently resulted in an atrophic endometrium despite the hormonal stimulus of the contralateral pregnancy.

Example 3 In vitro assessment of human endometrial fluorescence after treatment with ALA

Slices (one-half mm) of human uterine tissue were prepared so as to include both myometrium and endometrium. Tissues were incubated at 37'C in a CO2 incubator with 0,1,10 or 100 mM ALA for 2 hours. Slides were prepared and covered and the fluorescence emission spectrum was determined using a spectrophotofluorimeter (Princeton Instruments Inc., Princeton, NJ). The fibre optic head was positioned 1 cm from the tissue surface. No fluorescence was observed in the control (0 mM) sample or in any of the myometrial samples.
Sharp fluorescent peaks at a wavelength of 640 nM were observed in the 1, 10 and 100 mM ALA treated samples of endometrial tissue. 10 and 100 mM ALA
samples yielded peak fluorescence (calculated by subtracting background fluorescence from the zenith) at the two hour incubation level. Fluorescence after a 5 hour incubation was slightly lower.

Example 4 The procedures of Example 3 were repeated with 1, 2, 3, 4 and 5 hour incubation periods using a level of 1 mM of ALA. No significant fluorescence was observed in the myometrial samples or in the endometrial samples incubated for 2 hours. Peak fluorescence was observed in the endometrial samples incubated for 4 hours.

Example 5 Endometrial Fluorescence in Vivo following Topical Application of ALA in the Non-human Primate ALA (50 mg) was injected into the uterine lumen of an adult, healthy, female rhesus monkey following exposure of the uterus at laparotomy. A
hysterectomy was performed 3 hours later and cross sectional slices incorporating endometrial and myometrial tissue were taken from the uterine specimen. These slices were subjected to fluoroscopic examination as described above.
Fluorescence was observed throughout the endometrium of all slices. No fluorescence was observed in the myometrium.

The above examples clearly illustrate that endometrial ablation in a range of animal species, including humans, by photodynamic therapy using ALA can be achieved with little or no damage to the underlying myometrial tissues. This offers a possible alternative to hysterectomy and may be used as a method of contraception and/or a method for aborting an early pregnancy.

Example 6 Treatment of leukemias and lymphomas This study was designed to evaluate the possibility of using ALA-induced production of PpIX in the treatment of leukemias and lymphomas, with special emphasis on the possibility of destroying malignant cells in bone marrow without destroying the pluripotent hemopoietic stem cells that are essential for repopulation following marrow transplantation.

Flow cytometry of various leukemia and lymphoma cell lines after incubation with 5-6 mM ALA in Krebs Ringer bicarbonate buffer for 6 hours at 37'C indicated that fluorescing concentrations of PpIX accumulated in certain types of cells (murine lines P815, EL-4, P388, and human lines, Raji and K562), but much less in other (human lines RPMI 7666 and Daudi) (Figure 1).

Normal and P388 leukemic mice were given a large dose of ALA (250 mg ALA per kg of body weight) by intraperitoneal injection. By 5 hours thereafter, significant PpIX fluorescence (assayed by flow cytometry) was observed in no normal hematopoietic tissue tested; PpIX accumulation was, however, great in P388 leukemia cells (Figure 2). Murine line P388 and human line K562 were used to determine optimal conditions for the induction of PpIX fluorescence in vitro by ALA. Sufficient PpIX accumulated under such optimal conditions sufficent to cause cell death if the cells were exposed to photoactivating light (chromium release and fluorescein release assays in vitro).

In the experiment whose results are shown in Figure 3, time and dose parameters for incubation of P388 cells with ALA were determined. Relative fluorescence intensity as measured by flow cytometry demonstrated that maximum accumulation of PpIX occurred at about 8 hours at an initial ALA dose of 6 mM.

As shown in Figure 4, estimation of PpIX production by fluorescence measurements was optimum using a wave length of about 638 nm.
Determination of the percent of damaged K562 leukemic cells (chromium release cytotoxicity assay) following 6 hours of exposure to (A) 5 mM ALA in Krebs Ringer bicarbonate buffer or (B) buffer alone, and then photoactivation, showed that cell damage is dependent upon the light dose and the time post-irradiation (Figure 5). Maximum photodynamic killing occurred within 4 to 18 hours post-irradiation at an ALA dose of 5 mM for 6 hours of incubation.
ALA-induced PpIX photodynamic killing of P388 leukemic cells were determined at an in vitro fluorometric assay using fluorescein, following exposure of cells to 5 mM ALA in Krebs Ringer buffer for 6 hours, then irradiation with J/cm2 of 600-700 nm light. Such treated cells were unable to retain intracellular fluorescein, relative to untreated control cells, cells treated only with light, and cells treated only with ALA, demonstrating cytotoxicity (Figure 6).

Mice were given a dose of ionizing radiation (950 rad) sufficient to destroy bone marrow pluripotent hematopoietic cells. Such animals survived, and did not develop leukemia when P388 leukemia cells and normal spleen cells were injected intraperitoneally, provided that the leukemia cells had been pretreated with 5 mM
ALA for 6 hours and then exposed to 72 J/cm' of light at 600-700 nm prior to injection (Figure 7). If the leukemia cells were treated with ALA alone (no photoactivation) prior to injection, all mice died of leukemia within 14 days.

Claims (29)

1. Use of a precursor of protoporphyrin IX in the biosynthetic pathway for heme for treatment of at least one of leukemia and lymphoma in a subject, wherein the precursor is for use with blood cells, bone marrow cells, or blood and bone marrow cells, wherein the precursor is for use with light within the photoactivating spectrum of protoporphyrin IX, and wherein the cells comprise leukemia cells, lymphoma cells, or leukemia and lymphoma cells.

2. Use according to claim 1, wherein the use with blood cells, bone marrow cells, or blood and bone marrow cells is associated with at least one of synthesis and accumulation of protoporphyrin IX in blood cells, bone marrow cells, or blood and bone marrow cells.

3. Use according to claim 1 or 2, wherein the use with blood cells, bone marrow cells, or blood and bone marrow cells is ex vivo.

4. Use according to claim 1 or 2, wherein the use with light within the photoactivating spectrum of protoporphyrin IX is ex vivo.

5. Use according to any one of claims 1 to 4, wherein the precursor is 5-aminolevulinic acid.

6. Use according to any one of claims 1 to 5, wherein the light is of a wavelength that induces protoporphyrin IX in the cells to fluoresce.

7. Use according to claim 6, wherein the light has a wavelength from about 350 to 750 nm.

8. Use according to claim 6, wherein the light has a wavelength from about 600 to 700 nm.

9. Use according to claim 6, wherein the light has a wavelength from about 620 to 680 nm.

10. Use of a precursor of protoporphyrin IX in the biosynthetic pathway for heme, for the manufacture of a medicament for treatment of at least one of leukemia and lymphoma, wherein the precursor is for use with blood cells, bone marrow cells, or blood and bone marrow cells, wherein the precursor is for use with light within the photoactivating spectrum of protoporphyrin IX, and wherein the cells comprise leukemia cells, lymphoma cells, or leukemia and lymphoma cells.

11. Use according to claim 10, wherein the use with blood cells, bone marrow cells, or blood and bone marrow cells is associated with at least one of synthesis and accumulation of protoporphyrin IX in blood cells, bone marrow cells, or blood and bone marrow cells.

12. Use according to claim 10 or 11, wherein the use with blood cells, bone marrow cells, or blood and bone marrow cells is ex vivo.

13. Use according to claim 10 or 11, wherein the use with light within the photoactivating spectrum of protoporphyrin IX is ex vivo.

14. Use according to any one of claims 10 to 13, wherein the precursor is 5-aminolevulinic acid.

15. Use according to any one of claims 10 to 14, wherein the light is of a wavelength that induces protoporphyrin IX in the cells to fluoresce.

16. Use according to claim 15, wherein the light has a wavelength from about 350 to 750 nm.

17. Use according to claim 15, wherein the light has a wavelength from about 600 to 700 nm.

18. Use according to claim 15, wherein the light has a wavelength from about 620 to 680 nm.

19. Use of a precursor of protoporphyrin IX in the biosynthetic pathway for heme for detecting at least one of leukemia and lymphoma in a subject, wherein the precursor is for use with blood cells, bone marrow cells, or blood and bone marrow cells, wherein the precursor is for use with light within the photoactivating spectrum of protoporphyrin IX, and wherein protoporphyrin IX is detected in the cells as an indication of at least one of leukemia and lymphoma in the subject.

20. Use according to claim 19, wherein the use with blood cells, bone marrow cells, or blood and bone marrow cells is associated with at least one of synthesis and accumulation of protoporphyrin IX in blood cells, bone marrow cells, or blood and bone marrow cells.

21. Use according to claim 19 or 20, wherein the use with blood cells, bone marrow cells, or blood and bone marrow cells is ex vivo.

22. Use according to claim 19 or 20, wherein the use with light within the photoactivating spectrum of protoporphyrin IX is ex vivo.

23. Use according to any one of claims 19 to 22, wherein the cells comprise leukemia cells, lymphoma cells, or leukemia and lymphoma cells.

24. Use according to any one of claims 19 to 23, wherein the precursor is 5-aminolevulinic acid.

25. Use according to any one of claims 19 to 24, wherein the light is of a wavelength that induces protoporphyrin IX in the cells to fluoresce.

26. Use according to claim 25, wherein the light has a wavelength from about 350 to 750 nm.

27. Use according to claim 25, wherein the light has a wavelength from about 600 to 700 nm.

28. Use according to claim 25, wherein the light has a wavelength from about 620 to 680 nm.

29. Use according to claim 25, wherein detecting protoporphyrin IX comprises detecting fluorescence of protoporphyrin IX.