WO2004082626A2

WO2004082626A2 - Aromatase inhibitor diagnosis and therapy

Info

Publication number: WO2004082626A2
Application number: PCT/US2004/008082
Authority: WO
Inventors: Michel Gensini; Tony Huang
Original assignee: Ethicon, Inc.
Priority date: 2003-03-18
Filing date: 2004-03-17
Publication date: 2004-09-30
Also published as: EP1603535A2; WO2004082626A3; EP1603535A4

Abstract

The present invention provides pharmaceutical compositions which are useful for imaging and/or treating endometriosis. The compositions are useful for the treatment of reproductive age women, as well as post-menopausal women suffering from this disorder. The pharmaceutical composition is a liposome composition that includes a liposome carrier component that contains on its surface a moiety targeted for an endometriotic implant or leaky blood vessels within the implant. The inventive liposome composition includes an aromatase inhibitor and/or a radiolabeled complex encapsulated by the liposome component. A label capable of external detection can be included so as to permit the detection of an endometrial implant and/or diagnosis of the presence of endornetriosis in a patient. The label can be bound to the aromatase inhibitor. Alternatively, when the label is separate from the inhibitor, or when the inhibitor is not present, a radiolabeled complex can be included.

Description

AROMATASE INHIBITOR DIAGNOSIS AND THERAPY

FIELD OF THE INVENTION

The present invention is directed to pharmaceutical compositions useful for imaging and/or treating endometriosis. More particularly, the present invention relates to endometriotic implant-targeted delivery systems useful in the detection of and/or treatment of endometriosis.

BACKGROUND OF RELATED TECHNOLOGY

Endometriosis is a disorder which is characterized by the presence, growth and progression of endometrial glands and stroma outside the uterine cavity. This disorder is linked to both pelvic pain and infertility. For example, it is estimated that 25-50% of infertile women have endometriosis. It is further estimated that 70% of women with endometriosis are infertile. Moreover, the prevalence of endometriosis of reproductive age women is believed to be as high as 10% as described by Aral and Gates, Journal of the American Medical Association 250: 2327-2331 (1983).

Estrogen in endometriotic lesions comprises both estrone (a weak estrogen) and estradiol (a potent estrogen). The figure schematically depicts a biosynthetic pathway for estrogen formation in an endometriotic implant. Estrogen is an important factor known to stimulate the growth of endometriosis. It is the estradiol that primarily affects an endometriotic lesion. In a reproductive age woman, circulating estradiol is secreted directly from the ovary in a cyclic fashion. In the early follicular phase and post-menopause, extra- ovarian tissues, such as adipose and skin tissue (for e.g., skin fibroblasts), are the most important sources for circulating estradiol. Estradiol is also produced locally in the endometriotic implant in both reproductive age women, as well as post-menopausal women.

Referring to Figure 1, the most important precursor of estrogen is androstenedione, which mainly originates from the adrenal gland and ovaries and is converted to estrone through the action of the enzyme aromatase, also referred to as CYP 450 19. Notably, the estrone produced is only very weakly estrogenic and can be subsequently reduced to estradiol in peripheral tissues and the endometriotic implant, through the actions of 17-β- hydroxysteroid dehydrogenase-type 1 (17-β-HSD-type 1) as described by Zeitoun, et al., Molecular Endocrinology 13: 239-253 (1999). Estradiol can be inactivated by conversion to estrone in epithelial cells of the eutopic endometrium. This inactivation is catalyzed by another 17-β-HSD isozyme, 17-β-HSD-type 2. However, in endometriotic tissues, the potent estrogen estradiol is not oxidized because of a lack of 17-β-HSD-type 2, leading to increased levels of estradiol. Moreover, estradiol and cytokines, such as IL-lβ and TNF-α, which are increased in endometriosis due to inflammation, induce cylco-oxygenase-2 (COX-2), leading to elevated levels of PGE₂ in the endometriotic tissue. See Huang, et al., American Society for Reproductive Medicine 5: (Abstract) (1996). Over-expression of aromatase is stimulated by PGE₂, CREB, and transcription factor SFi in endometriotic stromal cells as described by Noble, et al., Endocrinology and Metabolism 82: 600-606 (1997).

It has recently been demonstrated that significant levels of aromatase activity and mRNA are present in the stromal cells of pelvic endometrial implants, whereas aromatase expression was minimal in the eutopic endometrium of women with endometriosis. See, for example, Noble, et al., Endocrinology and Metabolism 81: 174-179 (1996) and Endocrinology and Metabolism 82: 600-606 (1997).

The aberrant expression of aromatase and its stimulation by PGE₂ in endometriotic tissues results in local production of estrogen, which induces PGE₂ synthesis and establishes a positive feedback loop for continuous estrogen formation in endometriosis. Moreover, the lack of 17-β-HSD-type 2 in endometriotic tissues gives rise to increased local concentrations of estradiol, the potent estrogen. Elevated estradiol, in turn, promotes the growth of endometriotic tissue and, in addition, promotes local PGE₂ synthesis in stromal cells. Since PGE₂ is the most potent inducer of aromatase in endometriosis, this completes the positive feedback cycle that favors increased levels of estradiol in endometriosis.

Approaches currently in use to treat endometriosis are not optimal. These approaches include a laparoscopic approach, oral contraceptives, tamoxifene, steroid hormones, proteins and antibodies, cytokines, IL-6 like polynucleotides and polypeptides and COX-2 inhibitors. A disadvantage of the laparoscopic approach is that it fails to detect endometriotic implants which are hidden behind organs and/or transparent, and/or located outside the abdominal region. Moreover, tamoxifene, steroid hormones, and cytokines such as IL-6, TNF-alpha and enzymes like COX-2, are not specific to endometriosis, and some steroid hormones are metabolized before reaching their target. Furthermore, endometriosis treatment with oral contraceptives is only successful in a limited percentage of women.

In addition to the above-described approaches, it is known to use stand-alone non- steroidal imidazole drags that specifically inhibit the enzymatic function of cytochromes, such as aromatase and, therefore, decrease/annihilate the production of an excess of the potent estrogen estradiol. As described above, aromatase is up-regulated in endometriotic implants. Therefore, it is of benefit to provide inhibitors of aromatase activity. Blocking the catalytic action of the aromatase prevents the aberrant production of estradiol, characteristic of endometriotic implants. These non-steroidal drags appear to successfully complex the Fe²⁺ ion of the CYP 450 19 heme group. The stabilization of the ferrous ion could, therefore, prevent the demethylation and reductive action of the targeted enzyme. After administration of stand-alone non-steroidal competitive aromatase inhibitors such as anastrozole, or letrozole in post-menopausal women, aromatase activity has been shown to be significantly reduced in the peripheral-tissues and in endometriotic implants, giving rise to markedly diminished estradiol availability for endometriosis from both sources. Moreover, the positive feedback loop involving PGE₂ stimulation of local aromatase expression is also interrupted. The end result is that there is a significant lowering of the concentrations of estradiol in endometriotic tissues.

However, there are several major disadvantages of these stand-alone non-steroidal aromatase inhibitors. To begin with, they lack specificity for endometriotic tissues and thus, in addition to affecting the level of estradiol produced locally in the endometriotic implant, they also affect circulating estradiol production. Therefore, the stand-alone aromatase inhibitors also block the ovarian functions, thereby creating an artificial menopause and contributing to significant bone loss. This treatment is therefore usually limited to one year. This makes these drags, as presently administered, useful only as temporary treatment for treating women who are of a non-reproductive age due to the inability of the drug to discriminate between the endometriotic implant and other tissues, such as the ovaries. Moreover, these drugs are not specific in that they and are also known to bind to other cytochromes other than CYP 450 19, and would be affecting the biochemistry of these other cytochromes. In addition, although these imidazoles are non-steroidal and would, therefore, be expected to be more stable in the body than steroidal aromatase inhibitors, they also eventually undergo a metabolization process. It is, therefore, critical to preserve the biological activity of the drug by shielding it.

Liposomes are spherical vesicles prepared from either natural or synthetic phospholipids or cholesterol. These vesicles can be composed of either one (unilamellar liposomes) or several (oligo-or multilamellar liposomes) lipid by-layers surrounding internal aqueous volumes. It is known to use liposomes as carriers for drags. For example, Kwon, et al. in Journal of Controlled Release 48: 195-201 (1997) and U.S. Patent No. 6,365,179, assigned to Alza Corporation, each describe liposomes useful as carriers for the hydrophilic anticancer drug doxorubicin, which is entrapped within the internal aqueous space of the liposome. Liposomes allow the parenteral administration of insoluble or poorly soluble drags. Moreover, toxic side effects on organs or cells can be reduced or eliminated by using a liposomal drug delivery method. In addition, fast drag elimination or metabolism can be impeded by shielding the drug in a liposome. See, for example, Schwendener, et al., Biochem. Biophys. Ada, 1026: 69-79 (1990) and Schwendener, Chimia 46: 69-77 (1992).

Research is currently being carried out to develop radiolabeled antibodies or fragments thereof which are specific to endometriosis. For example, U.S. Patent Nos. 5,776,095 and 5,776,093 each disclose the use of radiolabeled antibody or antibody fragments specific for endometriotic tissues for endometriosis detection and/or therapy. They further disclose that labeled antibodies and antibody fragments specific for a targeted tissue or organ may be conjugated to a drug. A disadvantage of this approach is that it is extremely difficult to bind only one radioactive atom to proteins, antibodies or polypeptides in a time compatible with its half-life, which is one of the necessary conditions for performing a meaningful quantitative evaluation of endometriosis with a radiolabeled compound. Moreover, from a therapy standpoint, this approach would let the organs surrounding the endometriotic implants to be irradiated, as well.

Thus, there is a need for therapeutic agents which are specific to endometriosis, would be useful for the treatment of reproductive age women, as well as post-menopausal women suffering from this disorder, and which would not suffer from the disadvantages described above. The advantage of specifically targeting endometriotic tissue is that the agent would not completely eliminate endogenous estrogen production, and thus would not alter the function of other estrogen-responsive tissues. With the high specificity of a new compound for endometriotic tissues, the long term and/or repeated therapy programs would be possible, when necessary.

There is also a need in the art for a non-invasive diagnostic method which could be used to generate a 3 -dimensional mapping of the body and the organs where endometriotic cells are present. It would further be desirable to provide a diagnostic method to collect valuable information on the quantitative diagnosis (size and shape) of endometriotic implants. Presently, diagnosis of endometriosis is only performed in qualitative or semi-quantitative fashions. The problems with the qualitative way (the patient consults because of pain - usually pelvic pain) are that the diagnosis is difficult, might be inaccurate and the presence, size and location of endometriotic implants are unknown. The problems with the semi- quantitative way (i.e. laparoscopic procedure) are that only implants in the abdominal region can be seen. Therefore, extra-abdominal lesions will be missed, and even within the abdominal region, some endometriotic implants might be hidden or difficult to see (transparent lesions). Thus, there is a need in the art for a diagnostic method that would allow a fully quantitative diagnosis in terms of size, shape, and location, of the endometriotic implants over the entire body. Preferably, a useful diagnostic agent would be one that, because of its high specificity for endometriotic tissues, would maintain the normal functions of the ovaries, thus allowing not only post-menopausal women, but also women within reproductive age to be treated for endometriosis, while preventing artificial menopause and sudden decrease of the bone mass.

SUMMARY OF THE INVENTION

The present invention provides a composition useful in the treatment and/or detection of endometriotic implants. The composition includes a liposome carrier component; a moiety targeted for an endometriotic implant or leaky blood vessels within the implant, the moiety being attached to the liposome carrier component; and an aromatase inhibitor and/or a radiolabeled complex encapsulated by the liposome carrier component. Radiolabeled complexes for use in the compositions and methods of the present invention are complexing/chelating agents that are complexed with a radioactive nuclide, preferably a positron- or γ-emitter. Complexing/chelating agents can include, but are not limited to, oxine, ethylene diaminetetraacetic acid and diethylenetriaminepentaacetic acid.

The invention further provides a pharmaceutical composition that is targeted for endometriotic implants. The pharmaceutical composition includes a liposome carrier component having an external phospholipid layer and an internal phospholipid layer; a moiety targeted for an endometriotic implant or leaky blood vessels within the implant, the moiety being chemically bound to the external phospholipid layer; and an aromatase- inhibiting imidazole drag and/or a radiolabeled complex, the drug and/or complex being encapsulated by the liposome component.

Also provided is a drag composition useful in the treatment and/or detection of diseases associated with the upregulation of aromatase. The drug composition includes a liposome carrier component; a moiety targeted for cells or tissues which are growth- stimulated by the upregulation of aromatase, the moiety being attached to the liposome carrier component; and an aromatase inhibitor and/or a radiolabeled complex encapsulated by the liposome carrier component.

The present invention is further directed toward methods for the treatment of endometriosis. For example, the invention provides a method for treating endometriosis by administering to a patient a drug delivery system. The system includes a liposome carrier component; a moiety targeted for an endometriotic implant or leaky blood vessels within the implant, the moiety being attached to the liposome carrier component; and an aromatase- inhibiting imidazole encapsulated by the liposome carrier component. The administered delivery system can further include a radiolabel capable of external detection. For example, a radiolabel can be chemically bound to the aromatase inhibitor. Alternatively, the label can be separate from the aromatase inhibitor. For example, a radiolabeled complex can be encapsulated along with the inhibitor.

Also encompassed by the present invention is a method for detecting an endometrial implant and/or diagnosing the presence of endometriosis in a patient. The method includes the step of administering to a patient a drug delivery system, wherein the system includes a liposome carrier component; a moiety targeted for an endometriotic implant or leaky blood vessel within the implant, the moiety being attached to the liposome earner component; and a radiolabeled aromatase-inhibiting imidazole drug and/or radiolabeled complex, the labeled drag and/or radiolabeled complex being encapsulated by the liposome component. The radioactive nuclide is preferably a gamma- or a positron-emitter. The method further includes the step of monitoring the presence of the liposome component in the endometrial implant by detecting the labeled drug and/or labeled complex. The present invention also provides a method of modulating and/or inhibiting the metabolism of steroid hormones by aromatase. The method includes administering to a mammal a modulating or inhibiting amount of a composition that includes: a liposome carrier component; a moiety targeted for a tissue or cell in which the metabolism by aromatase occurs, the moiety being attached to the liposome carrier component; and an aromatase inhibitor encapsulated by the liposome carrier component. The administered composition can further include a radiolabel capable of external detection. For example, the aromatase inhibitor can be chemically bound to a radionuclide, or where the label is separate from the inhibitor, a radiolabeled complex can be encapsulated along with the inhibitor.

Further provided is a method of preparing a drug delivery composition that includes the steps of: providing a liposome component having an external phospholipid layer and an internal phospholipid layer; attaching a moiety to the external layer which is targeted for an endometriotic implant or leaky blood vessels within the implant to form a targeted liposome component; and combining the liposome component with an aromatase inhibitor and/or a radiolabeled complex under suitable conditions for the inhibitor and/or complex to diffuse inside the liposome component and become encapsulated therein.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 schematically depicts a biosynthetic pathway for estrogen formation in an endometriotic implant.

Figure 2 schematically depicts the metabolism of androstenedione to estradiol.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms "endometriotic tissue", "endometriotic implant", "endometriosis", and the like refer to ectopic endometrium-like tissues outside the uterine cavity.

The term "endometrium" refers to the eutopic or intra-uterine endometrial tissue in its normal location.

The term "radiolabeled complex" and the like refers to a complexing agent or chelating agent that is complexed with a radioactive nuclide. As discussed above, current drug therapies exhibit several problems since the treatment is only temporary (usually up to one year), and is usually limited to post- menopausal women or pre-menopausal women not desiring pregnancy. This is because they do not specifically target endometriotic tissue, and thus have the disadvantage of altering the function of other estrogen-responsive tissues, such as the ovaries. The present invention solves a need in the art by providing liposome compositions that are targeted for endometriotic tissues. These inventive compositions are useful for the treatment of reproductive age women, as well as post-menopausal women suffering from endometriosis. Given the specificity of the inventive liposome compositions for endometriotic tissues, long- term and/or repeated therapy programs are possible, when necessary.

The present invention solves a further need in the art by providing a diagnostic method which can be used to generate a three-dimensional mapping of the body and selected organs where endometriotic cells are present. The diagnostic methods of the present invention allow the clinician to collect valuable information on the quantitative diagnosis (precise location, number, size and shape) of endometriotic implants. As described above, currently used diagnostic methods are, at best, semi-quantitative. In general, such prior methods rely on laparoscopic procedures, where only implants in the abdominal region can be seen. With the diagnostic methods of the present invention, extra-abdominal lesions, and even hidden or transparent lesions may be detected.

In the present invention, liposomes are loaded with an aromatase inhibitor and/or a radiolabeled complex. Liposomes are useful as drug carriers for in vivo drug delivery. Liposome-encapsulated drugs have several advantages. For example, encapsulation within a relatively impermeable bilayer membrane protects the drag from the environment.

Liposomes can be taken up by cells without overt cytotoxic effects, which thus enhance cellular uptake of the encapsulated material. In addition, encapsulation can alter pharmokinetics. Finally, liposomes are natural, biodegradable and non-toxic in small quantities.

In preferred embodiments, the aromatase inhibitor is an aromatase-inhibiting imidazole. Preferably, the imidazole is selected from the following: vorozole ( Johnson & Johnson), letrozole (Novartis), itraconazole (Johnson and Johnson) and anastrozole (Zeneca). The imidazole is present in suitable amounts to block the reductive action of aromatase. In desired embodiments, the imidazole is present in amounts of about 1 x 10^"6 mg to about 1,000 mg to block the reductive action of aromatase.

As described above, significant levels of aromatase activity and mRNA have been shown to be present in endometriotic implants, whereas aromatase expression is minimal in the eutopic endometrium of women with or without endometriosis. It is the aberrant expression of aromatase and its stimulation by PGE₂ in endometriotic tissues which leads to continuous estrogen formation in endometriosis. Since estrogen is an important factor known to stimulate the growth of endometriosis, it would be of benefit to provide inhibitors of aromatase activity so as to block the catalytic action of the aromatase and prevent the aberrant production of estrogen, characteristic of endometriotic implants.

The liposome compositions of the present invention are targeted for endometriotic tissues (including the ones containing leaky vessels) and thus, when the liposomes are loaded with an aromatase-inhibiting imidazole, they affect the level of estradiol produced locally in the endometriotic implant, while not affecting circulation estradiol production. By not blocking the normal ovarian functions, a woman of reproductive age avoids artificial menopause and further avoids significant bone loss. The discrimination between the endometriotic implant and other tissues, such as ovaries, is accomplished by providing the external surface of the liposome component with a moiety (directly or indirectly attached) which is either targeted for the endometriotic implant or to leaky or open-ended blood vessels within the implant. The liposome compositions can further include a radiolabel capable of external detection. The label can be chemically bound to the aromatase inhibitor. In alternative embodiments, where the label is separate from the aromatase inhibitor, or when the aromatase inhibitor is not present, a radionuclide may be complexed with a complexing/chelating agent and the resulting radiolabeled complex can be encapsulated within the liposome.

In one embodiment of the present invention, the moiety present on the outside surface of the liposome is a protein or protein fragment which is specific for the endometriotic implant. The attached moiety may be a polymer, peptide, polypeptide, protein, or glycoprotein. In one preferred embodiment, the attached moiety is an antibody Fab fragment. The attached moiety may be specific for a cytokine, cell, or enzyme which is present in increased amounts in the implant as compared to normal tissues. In a further embodiment, the attached moiety is specific for a cytokine, cell, or enzyme present in increased amounts in response to inflammation in the implant. For example, the attached moiety may be specific for an eosinophil peroxidase-binding component described in WO 00/59547. Moreover, the attached moiety may be specific for a cytokine such as IL-l-β or tumor necrosis factor (TNF)-α, each of which is present in increased amounts in response to inflammation in the implant. In a further embodiment, the attached moiety may be specific for a matrix metalloproteinase enzyme, such as matrix metalloproteinase-7 or matrix metalloproteinase- 11, which may be present in increased amounts in response to inflammation in the implant. Such embodiments are described in further detail below. The attached moiety may bind a marker produced by, or associated with, an endometriotic cell. A liposome carrier component including an attached protein or protein fragment that is targeted for an endometriotic implant is desirably present in suitable amounts to obtain the specific binding to target cells.

In another aspect of the present invention, the moiety attached to the external layer of the lipid bilayer is targeted for leaky blood vessels within the implant. In one embodiment, this moiety is a hydrophilic polymer chain. It is noted that these hydrophilic polymer chains may be attached either directly or indirectly to the external phospholipid layer. Moreover, these hydrophilic polymer chains may be releasable. This will be described in further detail below. Desirably, the hydrophilic polymer chain is polyethylene glycol. Liposomes comprising a surface coating of these attached hydrophilic polymer chains are capable of exiting from the blood stream to the endometriotic implant from leaky or open-ended blood vessels within the implant. In one embodiment, an attached protein or protein fragment, such as an antibody fragment, which is specific for the endometriotic implant may be attached to the distal ends of the polyethylene glycol chains.

The liposome carrier component including the attached moiety is preferably present in amounts of about 1 to about 1,500 nM. More desirably, the liposome carrier component including the attached moiety is present in amounts of about 10 to about 150 nM.

In one embodiment, the inventive liposome composition may further include a label which is capable of external detection. The label can be chemically bound to the aromatase inhibitor. In other embodiments, where the label is separate from the aromatase inhibitor, or when the aromatase inhibitor is not present, the label can be complexed with a complexing/chelating agent and the resulting labeled complex can be encapsulated within the liposome. For example, during detection/diagnosis, the aromatase inhibitor need not be present. In one desired embodiment, the label is a radioactive nuclide which allows one to detect endometriotic implants throughout the body, and/or monitor treatment of the endometriotic implants following administration of the inventive liposome composition. The radioactive nuclide may be a positron-emitter or a γ-emitter. Examples of suitable positron- emitters include, but are not limited to, ^UC, ¹⁸F, ^7δBr, ⁷⁷Br and ⁸⁹Zr. Useful γ-emitters include, but are not limited to, ⁶⁷Ga or ϊn.

Detection of an endometrial implant and/or diagnosis of the presence of endometriosis in a patient may be accomplished by monitoring the labeled imidazole drug or radioactively labeled complex by such methods as positron emission tomography (PET) or single photon emission computed tomography (SPECT, γ-camera), wherein the radioactive nuclide is a positron- or γ-emitter, respectively. In one embodiment, the radioactively-labeled drug is quantitatively measured following intravenous administration of the drag delivery system of the present invention. Preferably, the radioactively labeled drag is administered in amounts of about 1 10^" mg to about 1 x 10^" mg. Transdeπnal administration is also well within the contemplation of the present invention.

It is noted that it is well within the contemplation of the present invention that the inventive drug composition may be useful in the treatment and/or detection of any disease which is associated with the upregulation of aromatase. For example, aromatase (cytochrome P-450) is the enzyme responsible for the metabolism of three steroids: androstenedione to estrone; testosterone to estradiol; and 16-α-hydroxyandrostenedione to 16-α-hydroxyestrone. The amount of aromatase available to perform a given metabolism can be altered with an aromatase inhibitor. For example, with reference to Figure 2, a variation in the concentration of aromatase could influence the level of testosterone and, therefore, the level of estradiol, which is one of its by-products.

Aromatase is widely expressed in many tissues and cell-types. For example, aromatase is expressed in ovaries, testes, adipocytes, fatty tissue, breasts, brain, skin, placenta, bone, heart, prostate, endometriotic implants, stromal cells, Leydig cells, and adrenals. See, for example, Santen, R., Steroids Vol. 50 (1987); Simpson, et al., J. Steroid Biochem. Molec. Biol. 43: 923-930 (1992); Perkins, L. and Payne, A., Endocrinology, 123: 2675-2682 (1988); and Stuerenburg, H., et al. NEL 18: 203-213 (1998).

Regarding the metabolism of testosterone to estradiol by aromatase, there are several diseases/disorders which could potentially be influenced by the administration of an aromatase-inhibiting liposome composition according to the present invention. For example, diseases/ disorders which are influenced by the metabolism of testosterone to estrogen, such as estradiol, include fatigue (lack of testosterone), prostatic hypertrophy and prostate cancer (low ratio of testosterone to estradiol), heart disease, high blood pressure, or a decrease in coronary artery elasticity (which are each, at least in part, the result of a lack of testosterone), loss of libido and sexual functions (lack of testosterone and low ratio of testosterone to estradiol), male osteoporosis (lack of testosterone), male gynecomastia (lack of testosterone and low ratio of testosterone to estradiol), and diabetes associated with insulin production.

For example, normal aging results in a gradual weakening of the heart, even in the absence of significant coronary artery disease. Testosterone is a muscle-building hormone, and there are many testosterone-receptor sites in the heart. There are an ever-increasing number of studies indicating an association between high testosterone and low cardiovascular disease rates in men. Therefore, by providing an aromatase-inhibiting liposome composition according to the present invention, the present inventors anticipate that higher levels of testosterone are likely to be achieved, leading to lower cardiovascular disease rates.

In another example, estrogen has been identified as the primary culprit in the development of benign prostate hypertrophy (BPH). Estrogen has been shown to cause a proliferation of epithelial cells in the prostate. This is corroborated by the fact that as men develop benign prostate enlargement, their levels of free-testosterone plummet, while their estrogen levels remain the same or are rising. Therefore, the aromatase-inhibiting liposome compositions of the present invention may be useful for restoring testosterone to its former level in patients suffering from BPH.

Moreover, clinical studies using testosterone injections, creams, or patches have often failed to provide a long-lasting libido-enhancing effect in aging men. This is because testosterone can be converted to estrogen, which is then taken up by testosterone receptor sites in cells throughout the body. When an estrogen molecule occupies a testosterone receptor site on a cell membrane, it blocks the ability of serum testosterone to induce a healthy hormonal signal. Since aromatase metabolizes testosterone to estrogen, the present inventors anticipate that the inventive liposome compositions containing an aromatase inhibitor are likely to be useful in enhancing the libido in aging men.

Furthermore, men with severe male infertility have significantly lower testosterone than fertile control reference subjects, resulting in a decreased testosterone-to-estradiol ratio. See, for example, Pavlovich, et al., The Journal of Urology 165: 837-841 (2001). Therefore, it is likely that infertile men having decreased serum testosterone-to-estradiol ratios will benefit from the aromatase-inhibiting compositions of the present invention.

Moreover, increased serum testosterone-to-estradiol ratios have been associated with a possible increased risk of osteoporosis. See, for example, an editorial by Swerdloff, R. and Wang, C. in Annals of Internal Medicine, Vol. 133, No. 12: 1002-1004 (2000).

An association between aromatase and gynecomastia has also been established as described by Braugnstein, G. in Endocrine-Related Cancer 6: 315-324 (1999). Enhancement of aromatization of antigens to estrogens appears to be important in the pathogeneses of gynecomastia associated with obesity, aging, puberty, liver disease, thyrotoxicosis, 17- oxosteroid reductase deficiency, RTinefelter's Syndrome, and neoplasms of the testes, adrenals and liver. Therefore, aromatase inhibition through the administration of the inventive compositions would likely be useful in treating gynecomastia.

Furthermore, recent studies have been establishing a connection between estrogen exposure and hormone-related diseases including breast, uterine and prostate cancer, as described by Bosland, M., J., Natl Cancer Inst Monogr 27: 39-66 (2000); Coffey, D., Urology 57 (4 suppl 1): 31-38 (2001); Hill, M. et al., Physiol Res 49 (suppl 1): S 113-118 (2000); and Latil, A. et al., Cancer Res 61 (5): 1919-1926 (2001). In fact, stand-alone aromatase inhibitors are presently being used in the treatment of breast cancer. As described above, a disadvantage of stand-alone inhibitors is that they can send a reproductive age woman into premature menopause. In contrast, the aromatase-inhibiting liposome compositions of the present invention may be directly targeted to particular cells or tissue in need of treatment as a result of the attached moieties on the external phospholid layer of the liposomes. In desired embodiments of the invention, the moiety attached to the liposome carrier component is a moiety targeted for a tissue or cell in which the metabolism by aromatase occurs. For example, in one embodiment, the tissue or cell is selected from the following: brain, testes, adipose, breasts, skin, bone, heart, prostate, endometriotic implant, ovary, placenta, stromal, Leydig cells, adrenals, and combinations thereof. These tissue or cells may be characterized by aberrant activity or expression of aromatase. In one embodiment, the metabolism of a steroid hormone, such as androstenedione or testosterone, by aromatase may be modulated or inhibited by administering to the mammal a modulating or inhibiting amount of the inventive liposome composition. See, for example, Figure 2. The moiety attached to the liposome carrier component (for targeting the tissue or cell in which metabolism by aromatase occurs) may be a peptide, polypeptide, protein, or glycoprotein. In one desired embodiment, the moiety is an antibody fragment.

In one embodiment, the aromatase-inhibiting liposome compositions are capable of targeting a cell or tissue which is growth-stimulated by the upregulation of aromatase. Such liposome compositions are likely to be useful in the treatment of breast cancer or other gynecological cancers. It is known that eosinophil peroxidase (EPO) is expressed in many lymphomas, breast cancers and gynecological cancers. Therefore, aromatase-inhibiting liposome compositions including an externally bound moiety capable of specifically binding to EPO are likely to be useful in the treatment of these disorders.

Moreover, as described above, prostate cancer is a disorder that could likely be influenced by the administration of an aromatase-inliibiting liposome composition according to the present invention. It is known that inflammatory cytokines, such as IL-1 and TNF-α, are present in the prostate cancer tumor microenvironment. Therefore, antibodies to these cytokines may be useful as moieties for targeting the tumor.

Preferably, the aromatase inhibitor that is encapsulated within the liposome is an aromatase-inhibiting imidazole useful for modulating and or inhibiting the metabolism of steroid hormones by aromatase. Diagnosis and therapy of various disorders/diseases affected by the metabolism of steroid hormones by aromatase may be accomplished by further including within the liposome composition a label which is capable of external detection. For example, the label can be chemically bound to the encapsulated aromatase inhibitor. Alternatively, where the label is not associated with aromatase, or when aromatase is not present, the label can be complexed with a complexing/chelating agent and the resultant labeled complex can be encapsulated within the liposome.

Aromatase Inhibitors Preferred aromatase inhibitors for purposes of the present invention include third generation aromatase inhibitors such as vorozole (Rivizor^®, Johnson & Johnson), anastrozole (Arimidex^®, Zeneca), letrozole (Femara^®, Novartis). These third generation non-steroidal aromatase inhibitors appear to be superior to previous generations of aromatase inhibitors in terms of potency and selectivity. However, it is further contemplated that first generation or second generation aromatase inhibitors may also be useful in the present invention. For example, first generation aromatase inhibitors include aminoglutethimide (Brodie, A. et al., Endocrinology 100: 1684-1695 (1997). Another first generation aromatase inhibitor is testololactone. Suitable second generation aromatase inhibitors include formestane (see Coombes, et al., Lancet, 2: 1237-1239 (1984), fadrazole and roglethimide (see Foster, et al., Journal of Medicinal Chemistry 28: 200-204 (1985); Beretta, et al., Annals of Oncology 1 : 421 (1990); Dowsett, et al., Clinical Endocrinology 32: 623-634 (1990); Lonning, et al., British Journal of Cancer 63: 789-793 (1991); and Trunet, et al., Journal of Clinical Endocrinology and Metabolism 74: 571-576 (1992). Suitable third generation aromatase inhibitors include anastrozole, letrozole and vorozole. See Bhatnagar, et al., Journal of Steroid Biochemistry and Molecular Biology 37: 1021-1027 (1990); Dowsett, Journal of

Steroid Biochemistry and Molecular Biology 37: 1037-1041 (1990); Lipton, et al., Cancer 65: 1279-1285 (1990); Johnstone, et al, Cancer Research, 54: 5875-5881 (1994); Plourde, et al., Breast Cancer Research and Treatment, 30: 103-111 (1994); Geisler, et al., British Journal of Cancer 74: 1286-1291 (1996); and Trunet, et al., Acta Oncologica 34: 15-18 (1996).

Table 1 below provides an overview of aromatase inhibitors which are suitable for use in the liposome compositions of the present invention. Potency and specificity of aromatase inhibition in Table 1 is compared with aminoglutethimide, which has been shown to be an effective inhibitor of several enzymes of the cytochrome P-450 family. This inhibition also included aromatase. Another useful aromatase inhibitor is mesterolone (Proviron ) which is available from Schering-Plough Corporation. This drag is an anabolic steroid and is a known aromatase inhibitor. TABLE 1

Liposome Components and Their Preparation

The liposome components may be prepared by a variety of techniques, such as those detailed in Szoka, et al., Biochem. Biophys. Acta 601: 559-571 (1980). Multilamellar vesicles (MLVs) can be formed by simple liquid-film hydration techniques. Briefly, a mixture of liposome-forming lipids of the type listed below are dissolved in a suitable organic solvent and subjected lo evaporation in a vessel to form a thin film, which is then covered by an aqueous medium. The liquid film hydrates to form MLVs, typically with sizes between about 0.1 to 10 microns.

Suitable liposome components of the present invention are composed primarily of vesicle-forming lipids. Such a vesicle-forming lipid is one which (a) can form spontaneously in bilayer vesicles in water, as exemplified by the phospholipids, or (b) can be stably incorporated into the lipid bilayer. The vesicle-forming lipids of this type are preferably ones which have two hydrocarbon chains, typically acyl chains, and a head group, either polar or non-polar. Some preferred diacyl-chain lipids for use in the present invention include diacyl glycerol, phosphatidylethanolamine (PE), diacyl aminopropane diols, such as distearyl aminopropanediol (DS) and phosphatidylglycerol (PG).

The vesicle-forming lipid is selected to achieve a specified degree of fluidity or rigidity, to control the stability of the liposome in the serum and to control the rate of the release of the entrapped agent in the liposome. The rigidity of the liposome, as determined by the vesicle-forming lipid, may also play a role in the fusion of the liposome to a targeted cell.

In one embodiment of the invention, the liposomes are prepared with a relatively rigid lipid to impart rigidity to the lipid bilayer. In a preferred embodiment, the vesicle-forming lipid is distearyl phospatidyl choline (DSPC).

In another embodiment of the invention, the lipids forming the bilayer vesicle, i.e., liposome, are effective to impart a positive liposome-surface charge. Such lipids include those typically referred to as cationic lipids, which have a lipophilic moiety such as sterol, an acyl or diacyl chain, and where the lipid has an overall net positive charge. Exemplary cationic lipids include l,2-dioleyloxy-3-(trimethylamino) propane (DOTAP); N-[l-(2,3,- ditetradecyloxy) propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N-[l- (2,3,-dioleyloxy) propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE); N-[l- (2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3βpsf-(N',N'- dimethylaminoethane) carbamoly] cholesterol (DC-Choi); and dimethyldioctadecylammonium (DD AB) .

The cationic vesicle-forming lipid may also be a neutral lipid, such as dioleoylphosphatidyl ethanolamine (DOPE) or an amphipathic lipid, such as a phospholipid, derivatized with a cationic lipid, such as polylysine or other polyamine lipids. For example, the neutral lipid (DOPE) can be derivatized with polylysine to form a cationic lipid.

Methods for preparing suitable liposome components for the present invention are provided in U.S. Patent Publication No. 2002/0172711 Al and in U.S. Patent No. 4,182,330. The entire contents of each of these is herein incorporated by reference.

Ligand Moieties for Attachment to the Liposome Carrier Component

The present invention provides liposome compositions which can be targeted for an endometriotic implant. The liposome carrier component includes an external phospholipid layer and an internal phospholipid layer. The targeted moiety is preferably bound to the external phospholipid layer. In one embodiment, the moiety is a peptide, polypeptide, protein or glycoprotein. For example, the moiety may be an antibody Fab fragment specific for the endometriotic implant. The moiety may be specific for endometriotic cells within the implant. For example, the moiety may bind a marker produced by or associated with an endometriotic cell. In another embodiment, the moiety is specific for a cytokine, cell, or enzyme which is present in increased amounts in the endometriotic implant as compared to normal tissue. In another embodiment, the moiety is specific for a cytokine, cell or enzyme present in increased amounts in response to inflammation in the implant.

It is further noted that a moiety bound to the external phospholipid layer of the liposome carrier component may be targeted for leaky blood vessels within the endometriotic implant. For example, the moiety may be a hydrophilic polymer chain. Desirably, the hydrophilic polymer chain is a polyethylene glycol chain, as will be described in further detail below. In one preferred embodiment, a protein or protein fragment, such as an antibody Fab fragment specific for the endometriotic implant, is attached to the distal ends of the polyethylene glycol chains.

As described above, a moiety which specifically targets a cytokine, cell or enzyme in the endometriotic implant would be useful in the present invention. For example, it is known that TNF-α is produced following the initial irmmmologic response subsequent to the inflammation process characteristic of endometriosis lesions. Therefore, an antibody fragment specific for TNF-α (such as Centocor's Remicade) would be useful.

Regarding the targeting of enzymes in the endometriotic implant, it is known that eosinophil peroxidase (EPO) (an intracellular enzyme that is released from eosinophils as they degranulate) is expressed in human endometriosis specimens. EPO is also present in normal endometrium where degranulation occurs just prior and during menstruation. Therefore, liposomes which include a moiety capable of specifically binding to EPO would be useful in targeting endometriosis tissue, although they might lack specificity if used just prior to and during menstruation. A useful moiety for targeting EPO is described in International Publication No. WO 00/59547.

Moreover, U.S. Patent No. 5,618,680 describes the use of a monoclonal anti-HLA-

ABC antibody as being useful in the detection and diagnosis of endometriosis. This ligand is specific to MHC-Class I antigens. This patent discloses that a woman with endometriosis has a different expression of the major histo-compatibility complex (MHC) Class I antigens (especially HLA-A, B and C surface antigens) in or on their endometrial cells when compared with the expression of the same antigens of endometrial cells of a healthy woman. The presence of a different expression of these antigens on endometrial cells is therefore predictive of endometriosis. Therefore, it is well within the contemplation of the present invention that ligands specific to MHC-Class I antigens, especially HLA-A, B and C antigens, would be useful as moieties attached to the external phospholipid layer of the liposome components present in the inventive compositions. The entire contents of U.S. Patent No. 5,618,680 are herein incorporated by reference.

Furthermore, U.S. Patent No. 5,891,644 describes an antibody specific for an isolated chemotactic factor from patients with endometriosis. In particular, the chemotactic factor is a soluble peptide having a molecular weight of about 27 kD, chemotactic to neutrophils and macrophages, and is naturally occurring in the peritoneal fluid of mammals with minimal to moderate endometriosis. Suitable methods for isolating and purifying this chemotactic factor for use as an antigen in generating a suitable antibody for the present invention are described in U.S. Patent No. 5,891,644, the entire contents of which are herein incorporated by reference.

Moreover, it is known that certain matrix metalloproteinases are present in increased amounts in the endometriotic implant in response to inflammation. See, for example, Gynecol. Obstet. Inves. 48, Suppl. 1: 2-13 (1999). In one embodiment, the metalloproteinase which is present in the endometriotic implant in increased amounts, as compared to normal endometrium, is matrix metalloproteinase-7 or matrix metalloproteinase- 11. Therefore, a liposome carrier component including an attached moiety targeting a metalloproteinase present in increased amounts in the endometriotic implant in response to inflammation would be useful in the present invention.

Most preferably, suitable antibodies for the liposome compositions of the present invention are human antigen-binding antibody fragments and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (scFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable regions alone, or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments which further include any combination of variable regions with a hinge region, CHI, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, chimeric, murine (e.g., mouse and rat), donkey, rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, "human" antibodies include antibodies that have the amino acid sequence of a human immunoglobulin and include antibodies that are isolated from human immunoglobulin libraries or from animals transgehic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described in U.S. Patent No. 5,939,598 to Kucherlapati, et al.

The antibodies useful for the present invention may be monospecific, bispecific, trispecific, or of greater multi-specificity. For example, multi-specific antibodies may be specific for different epitopes of a cytokine, cell, or enzyme which may be present in increased amounts in the implant as compared to normal tissues. Alternatively, an antibody may be specific for both an epitope of a cell in the endometriotic implant, as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.

Bispecific antibodies, designed with dual antigenic specificities and prepared by chemically linking two different monoclonal antibodies or by fusing two hybridoma cell lines to produce a hybrid-hybridoma, are known. These are described by Brennan, M. et al. in Science 229: 81-83 (1985); by Paulus, H. in Behring Inst. Mitt. 78: 118-132 (1985); by Rammensee, H.G. et al., Eur. in J. Immunol. 17: 433-436 (1987); by Segal, D. et al. in Princess Takamatsu Symp. 19: 323-331 (1988); by Kranz, D. et al. in J. Hβmatothβr, 4: 403- 408 (1995); and by Morimoto, K. and Inouve, K., J. Immunol. Methods, 224: 43-50 (1999).

An "antibody" in accordance with the present specification is defined broadly as a protein that binds specifically to an epitope. The antibody may be polyclonal or monoclonal. Antibodies further include recombinant polyclonal or monoclonal Fab fragments prepared in accordance with the method of Huse, et al., Science 246: 1275-1281 (1989) and Coligan, J.E. et al. (Eds.) Current Protocols in Immunology, Wiley Intersciences, NY, (1999).

The antibodies for use with the present invention may include derivatives that are modified, i.e. by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotipic response. For example, the antibody derivatives may include antibodies that have been modified by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by techniques which are known such as, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the antibody derivative may contain one or more non-classical amino acids.

The targeted antibodies for use in the liposome compositions of the present invention may be generated by any suitable method known in the art. For example, polyclonal antibodies may be isolated from mammals that have been inoculated with the targeted cell marker, cytokine, or enzyme or a functional analog of any of these in accordance with known methods such as those described in Coligan, J.E., et al. (Eds.), Current Protocols in Immunology, Wiley Intersciences, NY, (1999). For example, a cytokine, cell, or enzyme can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce production of sera containing polyclonal antibodies specific for the antigen.

Various adjuvants may be used to increase the immunological response, depending on the host species, and include, but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, key hole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as bacille Calmette-Guerin (BCG) and corynebacteriumparvum. Such adjuvants are well known in the art.

Monoclonal antibodies may be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstein in Nature 256: 495-497 (1975) and by Campbell in "Monoclonal Antibody Technologyi The Production and Characterization of Rodent and Human Hybridomas" in Burdon et al. (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1985); and Coligan, J.E., et al. (Eds.), Current Protocols in Immunology, Wiley Intersciences, New York, (1999); as well as the recombinant DNA method described by Huse et al., Science 246: 1275-1281 (1989).

In order to produce monoclonal antibodies, a host mammal is inoculated with an antigen, known to be present in the endometriotic implant as described above, and then boosted. Spleens are collected from inoculated mammals a few days after the final boost. Cell suspensions from the spleens are fused with a tumor cell in accordance with the general method described by Kohler and Milstein in Nature 256: 495-497 (1975). See also Campbell, "Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas" in Burdon et al. (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1985) and Coligan, J.E., et al. (Eds.), Current Protocols in Immunology, Wiley Intersciences, New York, (1999). In order to be useful, an antigen must contain sufficient amino acid residues to define the epitope of the molecule being detected. If the antigen is too short to be immunogenic, it may be conjugated to a carrier molecule. Some suitable carrier molecules include keyhold limpet hemocyanin and bovine seram albumen. Conjugation may be carried out by methods known in the art. See, for example, Coligan, J.E., et al. (Eds.), Current Protocols in Immunology, Chapter 9, Wiley Intersciences, New York, (1999). One such method is to combine a cysteine residue of the antigen with a cysteine residue on the carrier molecule.

Hydrophilic Polymer Chain Moieties For Attachment to the Liposome Component

In one embodiment of the present invention, the liposome composition has an outer surface coating of hydrophilic polymer chains. For example, suitable liposome compositions comprising an outer surface coating of hydrophilic polymer chains are described in U.S. Patent Publication No. US-2002/0172711 Al . These hydrophilic polymer chains may be releasable. Preferably, the liposomes are designed to have an extended blood circulation time.

It is noted that the hydrophilic polymer chains may be either directly or indirectly linked to the polar head group of a vesicle-forming lipid. For example, the hydrophilic polymer chains may be connected to the liposome lipids, or to hydrophobic chains connected to liposome lipids, desirably by chemically releasable bonds - that is, covalent chemical bonds that can be released by a suitable cleaving agent, such as a reducing agent, a reduced or elevated pH, a hydrolytic enzyme, or a photolytic stimulus. The hydrophilic chains preferably have a surface density sufficient to create a molecular barrier which is effective to substantially prevent the interaction of a serum proteins with the liposome surface. As such, the hydrophilic chain coating is effective to extend the circulation time of the liposomes in the blood-stream for periods of up to several hours to several days. Such an extended circulation time allows the inventive liposome compositions to exit the blood stream to the endometriotic implant from leaky and open-ended blood vessels within the implant. The hydrophilic polymer chains are preferably composed of polymer chains of polyethylene glycol, polyvinyl pyrrolidone, polyvinyl methylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacralimide, polymethacralimide, polydimethylacrylamide, polyhydroxypropyl metharacalate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, or polyaspartamide. These polymer chains have a preferred molecular weight of between about 500-10,000 daltons. In preferred embodiments, the hydrophilic polymer chains are polyethylene glycol (PEG) chains. In one embodiment, these hydrophilic polymer chains may be releasably attached to the liposome via a reducible chemical linkage. After administering the liposome composition to the subject, a reducing agent, such as cysteine, glutathione or ascorbate, may subsequently be administered to the subject to release the hydrophilic polymer chains. Although not wishing to be bound by any one theory, liposomes which have releasable PEG chains appear to be retained to a greater degree in the target tissue as compared to liposomes which do not have releasable PEG chains. Release of the PEG chain exposes the positive liposome surface charges of cationic liposomes, enhancing binding to the negative cell membranes and improving retention of the liposomes in the tissues.

Preferably, the hydrophilic chains are present in the outer lipid layer of the liposomes in an amount corresponding to about 1-20 mole percent of the liposome surface lipids, with lower molecular weight polymers, e.g., 500 daltons, being present at a higher density, e.g., 20 mole percent, and higher molecular weight polymers, e.g., 10,000 dalton chains, being present at a lower density, e.g., 1-5 mole percent.

In one embodiment of the invention, the hydrophilic polymer chains may include a ligand which can specifically bind to the target endometriotic tissue. For example, an antibody Fab fragment may be attached to the distal ends of PEG chains on the surface of the liposomes. As disclosed in U.S. Publication No. US 2002/0172611 Al, an unshielded ligand may be attached to the hydrophilic polymer coating so as to effect ligand-specific binding to a molecule on the target cell surface prior to chemical release of the hydrophilic polymer coating.

It is known that liposomes having PEG chains attached to the outer surface thereof have prolonged circulation time in the blood stream. They can effectively evade the immune system, which would otherwise attack the liposome soon after injection causing rupture of the liposome and premature release of the therapeutic agent entrapped inside. By increasing the blood circulation time, the therapeutic agent entrapped in the liposome stays within the liposome until it reaches the target endometriotic tissue. It has been shown previously that therapeutic agents delivered via liposomes concentrate in tissues with leaky vasculatures. For example, high drug accumulation has been shown to occur in human prostate carcinoma Xenograft after administration of liposome- encapsulated doxorubicin. Such tumor tissues are characterized by "leaky" or open-ended blood vessels as described by Vaage, J., et al. Cancer 73: 5 (1994). By virtue of their size, liposomes containing a hydrophilic polymer coating on their outer surface do not escape from normal blood vessels. However, many diseases, including endometriosis, are characterized by "leaky" or open-ended blood vessels. Therefore, the present inventors contemplate that liposomes would exit from the blood stream and build up in these leaky or open-ended blood vessel area within the endometriotic implant.

Attachment of a Moiety to the Liposome Component Suitable means for preparing a coating of hydrophilic polymer chains on the liposomes are provided in U.S. Patent Publication No. 2002/0172711 Al and in the Example Section below.

As described above, in one embodiment of the liposome compositions of the present invention, a liposome that includes a hydrophilic polymer coating, such as PEG, may further include a ligand for targeting the liposomes to a selected cell type, enzyme, or cytokine within the endometriotic implant. Preferably, the ligand is an antibody Fab fragment that is bound to the liposome by covalent attachment to the free distal end of a lipid-anchored hydrophilic polymer chain, such as PEG.

In one embodiment of the invention, the hydrophilic polymer chain is PEG, and several methods for attachment of the ligands to the distal end of PEG chains have been described. See. for example, Allen, et. al., Biochim. Biophys. Acta. 1237:99-108 (1995); Zalipsky, Bioconj. Chem. 4: 296-299 (1993); Zalipsky, et al., React. Polym. 22: 243-258 (1994); Zalipsky, Bioconj. Chem. 6:150-165 (1995 A); and Zalipsky, Adv. Drug Delivery Rev. 16: 157-182 (1995 B). In these methods, the inert terminal methoxy group of methoxy PEG (mPEG) is replaced with a reactive functionality suitable for conjugation reactions, such as an amino or hydrazide group. The end functionalized PEG is attached to a lipid, typically distearyl phosphatidylethanolamine (DSPE). The functionalized PEG-DSPE derivatives are employed in liposome formation and the desired ligand is attached to the reactive end of the PEG chain before or after liposome formation.

In addition to liposome components wherein the ligand is conjugated to the distal end of a hydrophilic polymer chain, it is noted that a ligand such as a Fab antibody fragment may be directly bound to the surface of the liposomes by attachment to surface lipid components. In one embodiment, such liposomes would further include hydrophilic polymer chains which are preferably present in the outer lipid layer of the liposomes and proximate to the Fab antibody fragments. Thus, in this instance, the Fab antibody fragment would be initially shielded by the hydrophilic surface coating from interaction with the target cells. Provided the hydrophilic polymer chains are releasable, the Fab fragment would be shielded until such time as after the removal of the hydrophilic polymers by suitable means, such as reducing agents.

A ligand, such as an antibody Fab fragment may be coupled to the polar head group of a vesicle-forming lipid and various methods have been described for attaching ligands to lipids. For example, suitable methods are described in U.S. Publication No. 2002/0172811 Al and in the Example Section below. Moreover, detailed methods of antibody modification and coupling to liposomes are described by Schwendener, et al. in Biochim, Biophys, Acta 1026: 69 (1990). In one method, the affinity moiety may be coupled to a lipid by a coupling reaction so as to form an affinity-moiety-lipid conjugate. This conjugate may then be added to a solution of lipids for formation of the liposomes. In another method, a vesicle-forming lipid which has been activated for covalent attachment of an affinity moiety, such as a Fab fragment, is employed in liposome formation. The formed liposomes may then be exposed to the affinity moiety to achieve attachment of the affinity moiety to the activated lipids.

A variety of methods are available for preparing a conjugate composed of an affinity moiety and a vesicle-forming lipid. For example, water-soluble, amine-containing affinity moieties can be covalently attached to lipids, such as phosphatidylethanolamine (PE), by reacting the amine-containing moiety with a lipid which has been derivatized to contain an activated ester of N-hydroxysuccinimide.

As another example, biomolecules, and in particular large biomolecules such as proteins (an example of which is an antibody), can be coupled to lipids according to reported methods. One method involves Schiff-base formation between an aldehyde group on a lipid, typically a phospholipid, and a primary amino acid on the affinity moiety. The aldehyde group is preferably formed by periodate oxidation of the lipid. The coupling reaction, after removal of the oxidant, is carried out in the presence of a reducing agent, such as dithiothreitol, as described by Heath, et. al., Biochim. Biophys. Acta 640 (1): 66-81 (1981). Typical aldehyde-lipid precursors suitable in the method include lactosylceramide, trihexosylceramine, galacto cerebroside, phosphatidylglycerol, phosphatidylinositol and gangliosides.

A second general coupling method is applicable to thiol-containing affinity moieties, and involves formation of a disulfide or thioether bond between a lipid and the affinity moiety. In the disulfide reaction, a lipid amine, such as phosphatidyl-ethanolamine, is modified to contain a pyridylditho derivative which can react with an exposed thiol group in the affinity moiety. Reaction conditions for such a method can be found in Martin et. al., Biochemistry, 20: 4229-4238 (1981). The thioether coupling method, described by Martin et. al., J. Biol. Chem. 257 (1982) 286-288 (1982), is carried out by forming a sulfhydryl-reactive phospholipid, such as N-(4)P-maleimidophenyl(butyryl)phosphatidylethanolamine, and reacting the lipid with the thiol-containing affinity moiety.

Another method for reacting an affinity moiety with a lipid involves reacting the affinity moiety with a lipid which has been derivatized to contain an activated ester of N- hydroxysuccinimide. The reaction is typically carried out in the presence of a mild detergent, such as deoxycholate. Like the reactions described above, this coupling reaction is preferably performed prior to employing the lipid in liposome formation.

The above-described coupling techniques are exemplary and it will be appreciated that other suitable methods are known in the art and have been described, for example in U.S. Patent Nos. 4,605,630, 4,731,324, 4,429,008, 4,622,294 and 4,483,929.

Labels

In one embodiment of the present invention, the liposome compositions may include a label which is capable of external detection. As such, the inventive liposome compositions may be useful for monitoring the presence of the inventive composition in the endometriotic implants by detecting the label for diagnosis or treatment. In one embodiment, the label is associated with the aromatase inhibitor. For example, the label may be chemically or physically bound to the aromatase inhibitor. Alternatively, the label may be separate from the aromatase inhibitor, but proximate thereto. For example, the label can be on a radiolabeled complex that can be encapsulated along with the aromatase inhibitor. Moreover, when it is desirable to detect and/or diagnosis the disease without treatment, the radiolabeled complex can be encapsulated without the aromatase inhibitor. The label may be a fluorescent dye, contrast agent or radiopaque agent. In another embodiment, the label may be a radioactive nuclide. In one embodiment of the invention, the radioactive nuclide may be a positron-emitter or γ-emitter. Preferred positron-emitters are ⁿC, ¹⁸F, ⁷⁶Br, ⁷⁷Br or ⁸⁹Zr. Suitable γ-emitters include ⁶⁷Ga or ¹¹¹In.

The monitoring may be accomplished by positron emission tomography (PET) in cases where the label is a radioactive positron-emitter. In addition, monitoring may be accomplished by SPECT, γ-camera in cases where the label is a radioactive γ-emitter. Moreover, monitoring may be by fluorescent scanning in situations where the label is a fluorescent dye.

In preferred embodiments, the aromatase inhibitor is covalently bound (either directly or indirectly) to a radionuclide. In particular, one radioactive atom can be readily bound to the aromatase inhibitor in a time compatible with its half-life to provide a meaningful quantitative evaluation of endometriosis.

In alternative embodiments, where the label is separate from the aromatase inhibitor, or when the aromatase inhibitor is not present, it is contemplated that a radioactive nuclide may be complexed with ligands like diethylenetriaminepentaacetic acid (DTP A). Such complexes may be encapsulated along with the aromatase inhibitor. For example, as will be described in further detail in the examples below, liposomes can be prepared that contain DTP A, for the complexation of ^mIn. These liposomes may be prepared according to standard procedures, such as those described in U.S. Patent Publication 2002/0172711 Al, except that the dried lipid film is hydrated with an aqueous phase that includes the DPTA chelating agent and the aromatase inhibitor. In one embodiment, the fusogenic liposomes include the entrapped aromatase inhibitor and the chelating agent. Following sequential extrusion through a membrane to obtain liposomes of approximately 100 nm in size, non- entrapped molecules are removed. The liposomes may then be loaded with the radioactive nuclide. For example, ^ιπIn-oxine is commercially available (Amersham). This nuclide may be equilibrated with the liposome preparation so as to effect binding of the nuclide to the entrapped chelate. This is followed by removal of free radionuclide and free oxine so as to produce the desired, labeled liposome preparation.

Administration of the Inventive Liposome Composition

The liposome compositions of the present invention are designed for use in delivering an aromatase inhibitor to a target cell, enzyme, or cytokine at the endometriotic implant site or other targeted site. Once at this site, delivery of the therapeutic agent may be accomplished by fusion of the vesicles with the plasma membrane of cells within the endometriotic implant, releasing the agent into the cytoplasmic compartment of the cells.

In one embodiment, the liposome compositions of the present invention are administered intravenously. In another embodiment, the inventive liposome composition is administered via transdermal administration.

The amount and frequency of administration of the inventive composition will be regulated according to the judgment of the attending clinician (physician) considering such factors as age, condition and size of the patient, as well as severity of the endometriosis being treated. Moreover, it is well within the contemplation of the present invention that the inventive compositions may be useful for modulating and/or inhibiting the metabolism of steroid hormones by aromatase in patients suffering from disorders other than endometriosis as described above. Determination of the proper dosage regiment for a particular situation is within the scale of the art.

In one embodiment, the inventive composition is administered in a therapeutically effective amount. This amount is to be deteπnined by the attending clinician. In one embodiment, the aromatase inhibitor is administered in amounts of about 1x10^" mg to about 1x10 ⁺³mg.

It is noted that in embodiments wherein a specific ligand (such as an antibody fragment) is attached to the liposome carrier component, this attachment can be either a direct or indirect attachment. For example, as described above, the ligand may be covalently attached by suitable means to a lipid in the external phospholipid layer. In an alternative embodiment, a specific ligand may be attached to the distal ends of lipid-anchored hydrophilic polymer chains. In either instance, the specific moiety would be exposed for purposes of binding and targeting the endometriotic implant or other tissues in need of regulation of the modulation and/or inhibition of the metabolism of steroid hormones by aromatase.

In other embodiments of the present invention, targeting to selected cells or tissue of the endometriotic implant may be passive, i.e., through the normal bio-distribution of the liposomes after administration, without the requirement for ligands having a specific binding affinity for the implant. For example, it is well within the contemplation of the present invention that liposomes including hydrophilic polymer chains, such as PEG, which are known to have a prolonged circulation time within the blood, can accumulate, after IV administration, at the site of the endometriotic implant by exiting the blood stream through leaky or open-ended blood vessels within the endometriotic implant.

In one embodiment, when PEG liposomes have reached a selected target site, the liposomes are contacted at the target endometriotic cells with a chemical agent effective to release the hydrophilic PEG chains on the liposome surface. For example, as described in US Publication 2002/0172711 Al, the hydrophilic PEG chains may be linked to hydrophobic chains on the liposome surface (or directly to the liposome lipids) via disulfide linkages. In this embodiment, after intravenous administration of the liposome composition, the subject is treated by IV administration of a reducing agent. For example, the reducing agent cysteine may be added to reduce disulfide bonds in order to release releasable PEG from the liposomes. As described above, release of the PEG chains allows exposure of the positive liposome surface charges of cationic liposomes, which in turn can enhance binding to the negative cell membranes and improve retention of the liposomes in the target tissue.

It is also noted that in embodiments where the hydrophilic polymer chains are linked via disulfide linkages to hydrophobic chains on the liposome surface (see US Publication 2002/0172711 Al), treatment with a reducing agent can serve to expose the hydrophobic polymers on a liposome surface to the target cells, promoting fusion of the liposomes with the target cell surface. While not wishing to be bound by any one theory, it is believed that the hydrophobic segment, now in an aqueous environment, will seek a more favorable, e.g. hydrophobic environment, both in the liposome bilayer and in the adjacent target cell membrane. This makes the liposomes more susceptible to fusion with target cells.

EXAMPLES

EXAMPLE 1

DETERMINTNG THE BIODISTRIBUTION OF PEG LIPOSOMES CONTAINING AN ENTRAPPED RADIOLABELED LIGAND

A Stealth^® liposome is obtained by binding PEG on the lipid bi-layer of liposomes. PEG prevents liposomes from being quickly damaged by proteins after injection and, therefore, prolongs the circulation time in the blood. In particular, encapsulated therapeutic agents stay in the liposome until reaching target tissues. Encapsulated therapeutic agents included in such PEG liposome compositions concentrate in tissues with leaky or open-ended blood vessels. The present example was used to assess the biodistribution of PEG liposomes containing a chelated radionuclide.

Preparation of Cationic Liposomes Containing Entrapped ^ιπIn-DTPA

Cationic liposomes composed of the lipids dimethyldioxideeyl ammonium and cholesterol (DDAB:Chol) are prepared according to standard procedures by dissolving 10 μmole DDAB and 10 μmole Choi in an organic solvent containing primarily CHC1₃. The lipid solution is dried as a thin film by rotation under reduced pressure. The lipid film is subsequently hydrated by the addition of an aqueous phase comprised of 10 mM DTPA/0.15 M NaCl, pH 6.5, to form liposomes (at a total lipid concentration of 20 μ ole/ml) which were sized by sonication or by sequential extrusion through Nucleopore polycarbonate membranes with pore sizes of 0.4 μm, 0.2 μm, 0.1 μm and 0.05 μm to obtain liposomes of approximately 100-150 nm in size. Free DTPA is removed by purification on a Sephadex G- 50 column that has been pre-equilibrated with three volumes of saline (total 30 ml). Liposome fractions purified from free DTPA are collected.

The purified liposome composition is radiolabeled upon addition of ' ^l 'in-oxine

(Amersham) to the liposome. In particular, 50 μl of indium (1 μCi/μl) per ml of liposome is added, wherein the concentration of total lipids is about 10 mM. Incubation is for one hour at room temperature, which allows the radioactive complex to diffuse inside the liposome. The nuclide then binds preferably to DTPA due to its stronger affinity therefore, allowing the release of free oxine. Free indium is removed by passage through a 10 DG column that has been pre-equilibrated with 3 volumes of saline. The radiolabeled liposome composition is loaded onto the column at a volume of not more than about 3 ml and the purified liposome fractions are collected.

Insertion of PEG

Distearyl phosphatidylethanolamine (DSPE) is derivatized with PEG, as described by Kirpotin et. al., FEBS Lett. 388: 115-118 (1986), Zalipsky, Adv. Dwg. Delivery Revs. 16: 157 (1995); and Woodle et. al., Biochem. Biophys. Acta 1113:171 (1992). PEG-DSPE micelles are prepared from PEG-DSPE by dissolving 1 mM in water and sonicating.

Micelles of PEG-diothiopropionate (DTP)-DSPE, that is, PEG attached to the DSPE by a cleavable disulfide linkage, are prepared by dissolving 1 mM PEG-DTP-DSPE in water and sonicating.

Liposomes containing 2.5 mole % of PEG-DSPE are prepared by adding 140 μl of the PEG-DSPE micelle suspension (1 μmole lipid/ml) to 5.6 μmoles lipid of the cationic liposomes prepared as described above. The micelle-liposome suspension is incubated for 5 minutes at room temperature with gentle vortexing to achieve insertion of the PEG-DSPE into the cationic liposomes.

In Vivo Administration of PEG-Coated Cationic Liposomes Containing Entrapped ^ιnIn- DTPA The PEG-coated cationic liposomes including the entrapped, chelated radionuclide are administered to scid mice that have developed endometriosis (stage I-IV) obtained from Charles River Laboratories (Wilmington, MA). In particular, the Stealth^® liposomes are used for targeting leaky vessels present at different stages of endometriosis.

The liposome composition contained the entrapped radiolabeled ligand (i.e. ^{i n}hi- DTPA) is administered by tail vein injection. In particular, 20 mice are each injected with 10-20 μCi per mouse (preferably, 10-100 μCi). This corresponds to about 20 nmoles lipid in 100-200 μl saline. Mice are sacrificed after 24, 48 and 72 hours and scanned as described below.

Each of the sacrificed mice is scanned by Single Photon Emitter Computed

Tomography (SPECT) camera, i.e. γ-camera. Such cameras are made by manufacturers such as GE and Toshiba. Scanning is performed in order to assess biodistribution of the radiolabel within tissues. The mice are then dissected. In particular, select organs are obtained such as brain, kidneys, liver, etc., and all endometriotic implants. The amount of radiolabeled compound present in each of the organs is quantified to establish biodistribution. In particular, radioactivity can be determined in a Germanium lithium (GeLi) detector (available from EG&G Ortec or Canbera). Activity for each organ is quantified by comparison with a phantom which is created for each organ by methods well known in the art. After activity values are obtained using the phantom, these values are normalized to an organ, usually the one that has the highest activity. The specific activity and the percentage of accumulation are also calculated by methods well known in the art. This process is repeated for each time point and for each mouse. By establishing the biodistribution for each time point, a time point is selected which provides optimal distribution of PEG liposomes in the endometriotic implant, as a function of the type of implant (clear, yellow, red, etc.) and the stage of the disease (I to IV).

EXAMPLE 2

PEG LIPOSOMES WITH ENTRAPPED AROMATASE INHIBITOR

In this example, liposomes are prepared having entrapped aromatase inhibitor molecule and including a surface coating of PEG.

Preparation of PEG Liposomes with Entrapped F-Vorozole

Cationic liposomes composed of the lipids dimethyldioctadecyl ammonium and cholesterol (DDAB:Chol) are prepared according to the procedure described in Example 1, except that the dried lipid film was hydrated with an aqueous solution containing ¹⁸F- Vorozole to form fusogenic liposomes having entrapped, radiolabeled vorozole molecules. These liposomes (at a total lipid concentration of 20 μmole/ml) are subsequently sized by sonication or by sequential extrusion through Nucleopore polycarbonate membranes with pore sizes of 0.4 μm, 0.2 μm, 0.1 μm and 0.05 μm to obtain liposomes of 100-150 nm in size.

PEG is inserted into the external lipid layer of the liposomes by the same method as described in Example 1, except that the PEG liposomes are prepared by adding 140 μl of the PEG-DSPE micelle suspension (1 μmole lipid/ml) to 5.6 μmoles lipid of the cationic liposomes containing entrapped, radiolabeled vorozole. Diagnosis and Therapy

Fusogenic PEG liposomes containing radiolabeled vorozole are delivered to target cells for in vivo drag therapy and/or diagnosis. The drug is directly introduced intravenously by tail vein injection of the liposome composition in scid mice with endometriosis (50 μCi mouse). Liposomes containing the drag are incorporated into the genome of endometriotic cells and, preferably, are suitable for autologous replication within the cell.

Twenty mice are each injected with 50 μCi/mouse. Mice are sacrificed at time points that were determined in Example 1 to provide optimal distribution of PEG liposomes in the endometriotic implants following liposome injection, as a function of the type of implant (clear, yellow, red, etc.) and the stage of the disease. Each of the mice are scanned by Positron Emission Tomography (PET) to assess biodistribution of the radiolabel within tissues. Organs including brain, kidneys, liver, etc. and all endometriotic implants are analyzed for radioactivity. In particular, the PEG chains on the surface of the ¹⁸F- Vorozole liposomes causes the liposomes to accumulate in endometriotic tissues, allowing for inhibition of the over-expression of aromatase therein.

EXAMPLE 3

Fab/PEG LIPOSOMES CONTAINING ENTRAPPED ¹⁸F-Vorozole

A variety of methods are known for attaching an affinity moiety to the external phospholipid layer of the liposomes. Some of these methods have been described above. In the present example, the conjugate is first formed between an antibody Fab fragment and a vesicle-forming lipid. This conjugate is then added to PEG liposomes for formation of the Fab/PEG liposomes.

A water-soluble, amine-containing antibody fragment specific for TNF-α, (Alza, Menlo Park, CA and Centocor, Malvern, PA), is covalently attached to lipids, such as phosphatidylethanolamine (PE), by reacting the amine-containing moiety with a lipid which has been derivatized to contain an activated ester of N-hydroxysuccinimide. Micelles are prepared from this lipid conjugate by sonicating. This conjugate is then combined with the liposomes containing PEG chains on their surface by adding 140 μl of the micelle suspension of the lipid conjugate (1 μmole lipid per ml) to 5.6 μmole lipid of cationic PEG liposomes containing entrapped F-Vorozole, which is prepared according to the same procedure described in Example 2 above. The micelle-cationic liposome suspension is incubated for 5 minutes at room temperature with gentle vortexing to achieve insertion of the Fab-PE into the cationic PEG liposomes.

Immunoliposomes are separated from free antibody fragments by HPLC using methods well known in the art. Liposomal absorption is monitored and fractions of free antibody fragments and immunoliposomes are collected and pooled separately.

Diagnosis/Therapy with the purified immunoliposomes prepared in the present example is performed essentially as described in Example 2 above.

Claims

WHAT IS CLAIMED IS:

1. A composition useful in the treatment and/or detection of endometriotic implants comprising: (a) a liposome carrier component;

(b) a moiety targeted for an endometriotic implant or leaky blood vessels within said implant, said moiety being attached to said liposome carrier component; and

(c) an aromatase inhibitor and/or a radiolabeled complex encapsulated by said liposome carrier component.

2. The composition of claim 1 , wherein said moiety is a protein or protein fragment specific for said implant.

3. The composition of claim 1, wherein said moiety is a hydrophilic polymer chain.

4. The composition of claim 3, wherein said hydrophilic polymer chain is a polyetiiylene glycol chain.

5. The composition of claim 4, wherein a protein or protein fragment specific for said implant is attached to the distal ends of said polyethylene glycol chains.

6. The composition of claim 3, wherein said liposomes with said attached hydrophilic polymer chains are capable of exiting from the blood stream to said endometriotic implant from said leaky blood vessels.

7. The composition of claim 1 , wherein said aromatase inhibitor is an aromatasβ- inliibiting imidazole.

8. The composition of claim 1, further comprising a label chemically bound to said aromatase inhibitor, which label is capable of external detection.

9. The composition of claim 8, wherein said label is a radioactive nuclide.

10. The composition of claim 9, wherein said nuclide is a positron-emitter or γ-emitter.

11. The composition of claim 10, wherein said positron-emitter is selected from the group consisting of ⁿC ,¹⁸F, ⁷⁶Br, ⁷⁷Br and ⁸⁹Zr.

12. The composition of claim 10, wherein said γ-emitter is ⁶⁷Ga or ^ιπIn.

13. The composition of claim 1, wherein said complex includes a complexing/chelating agent selected from the group consisting of oxine, ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid.

14. The composition of claim 13, wherein said complex further includes a positron- emitter or γ-emitter.

15. The composition of claim 1 , wherein said liposome carrier component comprises an external phospholipid layer and an internal phospholipid layer.

16. The composition of claim 15, wherein said moiety is chemically bound to said external phospholipid layer.

17. The composition of claim 15, wherein said external and internal layers are of an ionicity sufficient to allow diffusion of said aromatase inhibitor inside said liposome component.

18. The composition of claim 2, wherein said attached moiety is an antibody Fab fragment.

19. The composition of claim 2, wherein said attached moiety is a peptide, polypeptide, protein, or glycoprotein.

20. The composition of claim 2, wherein said attached moiety is specific for endometriotic cells in said implant.

21. The composition of claim 2, wherein said attached moiety binds a marker produced by or associated with an endometriotic cell.

22. The composition of claim 2, wherein said attached moiety is specific for a cytokine, cell, or enzyme present in increased amounts in said implant as compared to normal tissues.

23. The composition of claim 2, wherein said attached moiety is specific for a cytokine, cell or enzyme present in increased amounts in response to inflammation in the implant.

24. The composition of claim 23, wherein said cell is an eosinophil.

25. The composition of claim 23, wherein said cytokine is IL-lβ or tumor necrosis factor-α.

26. The composition of claim 23, wherein said enzyme is matrix metalloproteinase-7 or matrix metalloproteinase- 11.

27. The composition of claim 1, wherein said aromatase inhibitor is a non-steroidal imidazole drag capable of inhibiting aromatase.

28. The composition of claim 27, wherein said imidazole is selected from the group consisting of vorozole, letrozole, itraconazole and anastrozole.

29. The composition of claim 27, wherein said imidazole is present in suitable amounts to block the reductive action of aromatase.

30. The composition of claim 29, wherein said imidazole is present in amounts of about 1 xl0^"6mg to about 1000 mg.

31. The composition of claim 2, wherein said liposome carrier component comprising said attached protein or protein fragment is present in suitable amounts to obtain specific binding to target cells.

32. The composition of claim 1 , wherein said liposome carrier component comprising said attached moiety is present in amounts of about 1 to about 1500 nM.

33. The composition of claim 1, wherein said liposome carrier component comprising said attached moiety is present in amounts of about 10 to about 150 nM.

34. A pharmaceutical composition targeted for endometriotic implants comprising: (a) a liposome carrier component having an external phospholipid layer and an internal phospholipid layer;

(b) a moiety targeted for an endometriotic implant or leaky blood vessels within said implant, said moiety being chemically bound to said external layer; and

(c) an aromatase-inhibiting imidazole drug and/or a radiolabeled complex, said drag and/or complex being encapsulated by said liposome component.

35. The composition of claim 34, wherein said moiety is an antibody Fab fragment specific for said endometriotic implant.

36. The composition of claim 34, wherein said moiety is a polyethylene glycol chain.

37. The composition of claim 36, wherein an antibody Fab fragment specific for said endometriotic implant is attached to the distal ends of said polyethylene glycol chains.

38. The composition of claim 34, further comprising a label chemically bound to said imidazole drag, which label is capable of external detection.

39. The composition of claim 34, wherein said label is a radioactive nuclide.

40. The composition of claim 34, wherein said imidazole drag is selected from the group consisting of vorozole, letrozole, itraconazole and anastrozole.

41. The composition of claim 39, wherein said radioactive nuclide is a positron-emitter or γ-emitter.

42. The composition of claim 41 , wherein said positron-emitter is selected from the group consisting of ⁿC , ^I8F, ⁷⁶Br, ⁷⁷Br and ⁸⁹Zr.

43. The composition of claim 41, wherein said γ-emitter is ⁶⁷Ga or ^{n ι}In.

44. The composition of claim 34, wherein said complex includes a complexing/chelating agent selected from the group consisting of oxine, ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid.

45. The composition of claim 44, wherein said complex further includes a positron- emitter or γ-emitter.

46. The composition of claim 35, wherein said antibody fragment is specific for endometriotic cells in said implant.

47. The composition of claim 35, wherein said antibody fragment is specific for a cytokine, cell or enzyme present in increased amounts in said implant as compared to normal tissues.

48. The composition of claim 35, wherein said antibody fragment is specific for a cytokine, cell or enzyme present in increased amounts in response to inflammation in the implant.

49. A method for treating endometriosis comprising administering to a patient a drug delivery system, said system comprising:

(i) a liposome carrier component;

(ii) a moiety targeted for an endometriotic implant or leaky blood vessels within said implant, said moiety being attached to said liposome carrier component; and (iii) an aromatase-inhibiting imidazole encapsulated by said liposome carrier component.

50. The method of claim 49, wherein said imidazole is administered in amounts of about 1 xl0^"6mg to about 1000 mg.

51. The method of claim 49, wherein said administering comprises parenteral administration.

52. The method of claim 49, wherein said parenteral administration is by intravenous injection or transdermal administration of said drug delivery system.

53. The method of claim 49, wherein said delivery system further includes a label capable of external detection, said label being chemically bound to said aromatase inhibitor.

54. The method of claim 49, wherein said delivery system further includes a radiolabeled complex capable of external detection.

55. A method for detecting an endometrial implant and/or diagnosing the presence of endometriosis in a patient, the method comprising:

(a) administering to a patient a drug delivery system, said system comprising: (i) a liposome carrier component;

(ii) a moiety targeted for an endometriotic implant or leaky blood vessel within said implant, said moiety being attached to said liposome carrier component; and (iii) a radiolabeled, aromatase-inhibiting imidazole drug and/or a radiolabeled complex, said labeled drug and/or labeled complex being encapsulated by said liposome component; and (b) monitoring the presence of said liposome component in said endometrial implant by detecting said labeled drug and/or labeled complex.

56. The method of claim 55, wherein said radiolabeled drag is quantitatively measured.

57. The method of claim 55, wherein said radiolabeled drug is administered in amounts of about 1 xl0^"6mg to about 1 xlO^"3 mg.

58. The method of claim 55, wherein said complex includes a complexing/chelating agent selected from the group consisting of oxine, ethylenediamineteteaacetic acid and diethylenetriaminepentaacetic acid.

59. The method of claim 58, wherein said complex further includes a positron-emitter or γ-emitter.

60. The method of claim 55, wherein said administering comprises intravenous injection or transdermal administration of said drag delivery system.

61. The method of claim 55, wherein said monitoring comprises Positron Emission Tomography (PET) or tomography (γ-camera), and wherein the radiolabel associated with said drag and/or complex is a positron-emitter or γ-emitter, respectively.

62. A method of preparing a drag delivery composition comprising:

(a) providing a liposome component having an external phospholipid layer and an internal phospholipid layer;

(b) attaching a moiety targeted for an endometriotic implant or leaky blood vessels within said implant to said external layer to form a targeted liposome component; and

(c) combining said liposome component with an aromatase inhibitor and/or a radiolabeled complex under suitable conditions for said inhibitor and/or complex to become encapsulated by said liposome component.

63. The method of claim 62, further comprising the step of radiolabeling said aromatase inhibitor prior to said encapsulation.

64. The method of claim 62, wherein said complex includes a complexing/chelating agent selected from the group consisting of oxine, ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid.

65. The method of claim 64, wherein said complex further includes a positron-emitter or γ-emitter.

66. The method of claim 62, wherein said moiety is an antibody fragment which is specific for a cytokine, cell, or enzyme present in increased amounts in said implant as compared to normal tissues.

67. The method of claim 62, wherein said moiety is a polyethylene glycol (PEG) chain, said PEG liposomes being capable of exiting from the blood stream to said implant from said leaking blood vessels in said implant.

68. The method of claim 62, further comprising the step of purifying said liposome component to remove material that is not encapsulated.

69. A drag composition useful in the treatment and/or detection of diseases associated with the upregulation of aromatase comprising:

(a) a liposome carrier component;

(b) a moiety targeted for cells or tissues which are growth-stimulated by the upregulation of aromatase, said moiety being attached to said liposome carrier component; and (c) an aromatase inhibitor and/or radiolabeled complex encapsulated by said liposome carrier component.

70. The composition of claim 69, wherein said moiety is an antibody Fab fragment which binds a marker produced by or associated with an endometriotic cell.

71. The composition of claim 69, wherein said moiety is an antibody Fab fragment which is specific for a cytokine, cell or enzyme present in increased amounts in said growth- stimulated tissues as compared to normal tissues.

72. The composition of claim 69, wherein said moiety is a polyethylene glycol (PEG) chain, said PEG liposomes being capable of exiting from the blood stream to said growth- stimulated tissues from leaky blood vessels in said growth-stimulated tissues.

73. The composition of claim 69, wherein said upregulation comprises increased production or activity of said aromatase.

74. The composition of claim 69, wherein said cells are stromal cells, endothelial cells or endometriotic cells.

75. The composition of claim 69, wherein said diseases are selected from the group consisting of breast cancer and endometriosis

76. The composition of claim 69, further comprising a label chemically bound to said aromatase inhibitor, which label is capable of external detection.

77. The composition of claim 76, wherein said label is a radioactive nuclide.

78. The composition of claim 69, wherein said complex includes a complexing/chelating agent selected from the group consisting of oxine, ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid.

79. The method of claim 78, wherein said complex further includes a positron-emitter or γ-emitter.

80. The composition of claim 69, wherein said liposome caπier component comprises an external phospholipid layer and an internal phospholipid layer.

81. The composition of claim 80, wherein said moiety is attached to said external phospholipid layer.

82. A method of modulating and/or inhibiting the metabolism of steroid hormones by aromatase comprising administering to a mammal a modulating or inhibiting amount of a composition comprising:

(a) a liposome carrier component; (b) a moiety targeted for a tissue or cell in which said metabolism by aromatase occurs, said moiety being attached to said liposome carrier component; and (c) an aromatase inhibitor encapsulated by said liposome carrier component.

83. The method of claim 82, wherein said tissue or cell is characterized by aberrant activity or expression of aromatase.

84. The method of claim 82, wherein said steroid hormone is androstenedione or testosterone.

85. The method of claim 82, wherein said tissue or cell is selected from the group consisting of brain, testes, adipose, breast, skin, bone, heart, prostate, endometriotic implant, ovary, placenta, stromal, Leydig cells, adrenal and combinations thereof.

86. The method of claim 82, wherein said moiety is an antibody fragment.

87. The method of claim 82, wherein said moiety is a peptide, polypeptide, protein, or glycoprotein.

88. The method of claim 82, wherein said aromatase inhibitor is an aromatase-inhibiting imidazole.

89. The method of claim 82, further comprising a label chemically bound to said aromatase inhibitor, which label is capable of external detection.

90. The method of claim 82, wherein said composition further includes a radiolabeled complex capable of external detection.

91. The method of claim 82, wherein said complex includes a complexing/chelating agent selected from the group consisting of oxine, ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid.

92. The method of claim 91, wherein said complex further includes a positron-emitter or γ-emitter.