The invention relates primarily to the treatment of ocular hypertension, such as that associated with glaucoma, with agents comprising prostaglandin analogs or derivatives wherein treatment with the agent results in minimal or absent ocular hyperpigmentation effects. Additionally, the invention concerns methods to identify and design prostaglandin F analogs which lack the ability to effect hyperpigmentation.
Certain prostaglandins and their analogs and derivatives, such as the PGF2xcex1, derivative latanoprost, sold under the trademark XALATAN(copyright), have been established as compounds useful in treating ocular hypertension and glaucoma. However, latanoprost, the first prostaglandin approved by the United States Food And Drug Administration for this indication, is a prostaglandin derivative possessing the undesirable side effect of producing an increase in brown pigment in the iris of 5-15% of human eyes. The change in color results from an increased number of melanosomes (pigment granules) within iridial melanocytes. See e.g., Watson et al., Ophthalmology 103:126 (1996) (this and all references cited herein are incorporated by reference). While it is still unclear whether this effect has additional and deleterious clinical ramifications, from a cosmetic standpoint alone such side effects are undesirable.
It would be desirable to devise methods of treating a patient having glaucoma with a prostaglandin, prostaglandin analog or prostaglandin derivative that either lacks or has a reduced ability to stimulate an increase in iridial pigmentation as compared to a synthetic ocular hypertensive prostaglandin derivative, such as latanoprost. By prostaglandin derivative is meant a compound having structural similarity to a prostaglandin, preferably a PGF prostaglandin, having carboxylic acid groups, and esters thereof.
It would additionally be desirable to have a method for identifying compounds that are useful for the treatment of ocular hypertension which do not have this undesirable side effect. The present invention provides a method to design and identify PGF2xcex1 analogs and/or derivatives which are useful in lowering ocular pressure, but which substantially lack this undesired side effect, as well as methods of glaucoma treatment with such compounds.
It is known that pigmentation in the iris is due to formation of melanin and that the most essential enzyme in melanin biosynthetic pathway is tyrosinase; tyrosine is absolutely required for melanin production. See, for example, Hearing B. J., et al., FASEB J. (1991) 5:2902-2909. Melanin is typically synthesized in melanocytes and the effects of various factors on melanin production in melanocytes has been studied, for example, by Morelli, J. G., et al., J. Invest Dermatol (1993) 100:191S-195S. In addition, it has been shown that although melanocyte-stimulating hormone (MSH) appears to stimulate the activity of tyrosinase in melanocytes, MSH does not increase the expression or activity of other enzymes involved in the melanin biosynthetic pathway, specifically the enzymes designated TRP2 and TRP1. Aroca, P., et al., J. Biol. Chem. (1993) 268:25650-25655.
Attempts have been made to evaluate the mechanism for pigmentation stimulation by studying the effects of prostaglandins and analogs or derivatives thereof on melanogenesis and cell proliferation in the S91 xe2x80x9cCloudmanxe2x80x9d melanoma cell line. Using this cell line, Krauss and co-workers (Krauss, A. H.-P., et al., ARVO abstracts 39:S804 (1998)) demonstrated that although MSH was able to simulate melanogenesis in a predictable way, PGF2xcex1 and similar compounds were able to stimulate melanin production in this melanoma cell line only at quite high concentrations, indicating the involvement of receptors other than the natural PGF2xcex1 receptor, FP. Treatment of cells with butaprost and ligands for prostaglandin receptors other than FP indicated the involvement of EP2 and IP receptors. These studies indicated that S91 cells contain EP2 and IP receptors, but not FP receptors, which are known to respond to latanoprost. This was further reported by Shi, L., et al., PASPCR abstracts 11:186(1998).
Subsequent studies by Krauss, A. H.-P, et al., Internat. Congress of Eye Res. Abstracts 14:S78(2000) showed that FP receptors were absent in S91 cells, as RT-PCR failed to amplify FP receptor mRNA and stimulation with PGF2xcex1 (the natural ligand for the FP receptor) failed to stimulate a calcium signal. When these cells were stably transfected with an FP receptor and permitted to overexpress the protein, RT-PCR produced FP receptor cDNA and the receptor was displayed on the cell surface. However, PGF2xcex1, did not generate a calcium signal in these transfected S91 cells. A calcium signal is typically associated with FP receptor stimulation in other cells. The effects of PGF2xcex1 on DOPA oxidase (tyrosinase) activity and melanin formation in these FP receptor transfectants were indistinguishable from the responses contained in native, untransfected S91 cells. Krauss, A. H.-P., et al., ARVO abstracts 41:S250 (2000); Krauss, A. H.-P., et al., IPCC abstracts Supp. 7, 68(1999). Thus, these data suggest that although melanocytes lack significant FP receptor expression (which PGF2xcex1 is known to stimulate), modifying these cells to provide this receptor is not sufficient to remedy the inability of melanocytes to synthesize melanin in response to this prostaglandin or its analogs.
It has now been found that in order for PGF2xcex1 to stimulate melanin production in cell culture, the medium in which the melanocytes are thus affected must contain fibroblasts. Based on this knowledge, it is possible to identify and design compounds which will reduce intraocular pressure, while lacking the side effect of hyperpigmentation.
The invention takes advantage of the discovery that the mechanism for melanin production resulting from treatment with prostaglandin analogs involves an intermediate step, specifically thought to be agonism by the analog on the FP receptor of fibroblasts, or cells of fibroblast lineage in the presence of melanocytes (or melanocyte-derived cells), probably followed by the fibroblasts"" elaboration of a soluble signal detectable by the melanocytes which in turn serves to stimulate melanin production.
In one embodiment of the invention, compounds are assayed for their ability to stimulate the FP receptor population present on fibroblasts. Detection of FP receptor activity may be direct or indirect. Compounds which fail to stimulate the FP receptor expressed in fibroblasts but which are nevertheless able to control intraocular pressure are suitable candidates for the design of therapeutic agents exhibiting absent or reduced hyperpigmentation.
In another embodiment of the invention, the present inventors have discovered that prostaglandin analogs that are non-acidicxe2x80x94i.e., lack the carboxyl group characteristic of PGF2xcex1, are unable to stimulate the FP receptor. Thus, compounds can be designed for glaucoma treatment which are neutral or basic at physiological pH and such compounds will exhibit reduced or absent hyperpigmentation.
Thus, in one aspect, the invention is directed to a method of identifying a compound comprising a prostaglandin, prostaglandin derivative or prostaglandin analog capable of lowering intraocular pressure which compound lacks the undesirable property of stimulating iridial hyperpigmentation, said method comprising contacting said compound with fibroblast cells expressing the FP receptor and directly or indirectly assessing the ability of the compound to stimulate said FP receptor, wherein a failure to stimulate the FP receptor is an indication that said compound lacks said property.
As indicated, the determination of whether FP receptor activity is stimulated may be direct, as for example, by detecting stimulation of an intracellular Ca++ signal, or may be indirect. Such indirect means may include detecting the presence or absence of a compound or physical phenomenon whose presence or absence, respectively, is characteristic of FP receptor stimulation.
In a preferred embodiment of the invention fibroblasts and melanocytes (or melanoma cells) are co-cultured in the presence of a test compound, and the synthesis of melanin by the melanocyte is compared with the synthesis of melanin in a control culture, e.g., by the same cell type in a similar cell co-culture in the absence of the test compound, or by the same cell type and agent incubated in the absence of fibroblasts. Melanin production can be detected directly or indirectly. Direct detection methods include, without limitation, extraction and detection of melanin (including either or both eu-malanin or phaeo-melanin) from the incubated melanocytes, HPLC analysis of a melanocyte lysate, correlation of a peak (or absence thereof) with a melanin standard, observation of light absorbency in a cell lysate at a wavelength characteristic of melanin, and radiolabeling of melanin precursors and detection of radiolabeled melanin following stimulation. Indirect detection includes, without limitation, detection of tyrosinase activity in the melanocytes. Currently this latter method is preferred by the Applicants.
The fibroblasts and melanocytes used in the assays of the present invention may be primary cells or may be immortal cell lines derived from these primary cell types. Preferably the cells are immortal cell lines derived from each of the two cell types.
In another embodiment, the invention is directed to a method to select intraocular pressure-lowering PGF2xcex1 analogs or derivatives which display absent or reduced iridial pigmentation when compared to a standard, preferably latanoprost free acid, comprising designing a prostaglandin F2xcex1 analog or derivative which is neutral or basic rather than acidic. A large number of prostaglandin analogs and derivatives are known. Many are set forth, for example, in International Patent Publication No. WO 94/06433 and U.S. Pat. No. 5,688,819, both incorporated herein by reference in their entirety. Many such compounds are commercially available, including latanoprost, fluprostenol (both of which contain carboxylic acid functions and thus according to the present invention would be expected to stimulate iridial hyperpigmentation). Other analogs are available for synthesis, such as prostamide F2xcex1 and other non-acidic analogs such as, without limitation, those set forth below. Finally, arachidonic acid and anandamide are biosynthetic precursors of various prostaglandins and prostamides, respectively. 
By taking advantage of the invention disclosed herein, it is possible to predict which of these analogs, and any newly synthesized analogs or derivatives, will have lessened or absent ability to stimulate hyperpigmentation.