Update on the mechanism of action of bimatoprost: a review and discussion of new evidence

SURVEY OF OPHTHALMOLOGY

VOLUME 49 • SUPPLEMENT 1 • MARCH 2004

Update on the Mechanism of Action of Bimatoprost: A Review and Discussion of New Evidence Achim H.-P. Krauss, PhD, and David F. Woodward, PhD Allergan, Inc, Irvine, California, USA

Abstract. Bimatoprost is a pharmacologically unique and highly efficacious anti-glaucoma agent. It appears to mimic the activity of the prostamides, which are biosynthesized from the natural endocannabinoid anandamide by the enzyme cyclo-oxygenase 2 (COX-2). Bimatoprost has also been suggested to lower intraocular pressure by behaving as a prodrug or, alternatively, by stimulating FP receptors directly. These three distinctly different hypotheses for the mechanism of bimatoprost activity are discussed in the light of current evidence. (Surv Ophthalmol 49(Suppl 1):S5–S11, 2004. 쑖 2004 Elsevier Inc. All rights reserved.) Key words. bimatoprost prostamide

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glaucoma

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intraocular pressure

Bimatoprost is a novel and highly efficacious ocular hypotensive agent indicated for the treatment of primary open-angle glaucoma and ocular hypertension.5,30 Bimatoprost is a synthetic molecule structurally and pharmacologically similar to prostaglandin F2α 1-ethanolamide, more conveniently referred to in the contracted form prostamide F2α. Studies on the ocular distribution and metabolism of bimatoprost in living monkeys support the view that bimatoprost lowers intraocular pressure as the intact, prostamide-like molecule.39 Long-term treatment of primates with bimatoprost leads to a remodeling of the conventional and uveoscleral outflow pathways suggestive of an enhancement of both conventional and uveoscleral outflow.25 This is consistent with the drug’s reported improvement of pressure-sensitive (presumed trabecular) and pressure-insensitive (presumed uveoscleral) outflow in humans.4 The extremely efficacious ocular hypotensive activity of bimatoprost is undisputed. The pharmacology underlying the profound effect of bimatoprost on

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metabolism

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prostaglandin

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intraocular pressure is presently contentious and three distinctly different explanations have been advanced. Bimatoprost has been variously described as a prostanoid FP receptor agonist, an ester prodrug equivalent, and a prostamide mimetic. Prostaglandin ester prodrugs for glaucoma therapy and the prostanoid FP receptor were technologies that emerged in the 1980s and matured in the 1990s. It is, perhaps, inevitable that these established concepts would be contemplated in initial attempts to explain the activity of bimatoprost. In contrast, the prostamides and other COX-2 products of the endocannabinoids are very much an emerging technology of the 21st century. At present, there is much to be learned about the biological significance of the prostamides and related substances. More importantly, the prostamide hypothesis deserves consideration as an explanation for the effects of bimatoprost as it fits all the facts. In this review, the relative merits of these hypotheses will be examined and discussed. S5

쑖 2004 by Elsevier Inc. All rights reserved.

0039-6257/04/$–see front matter doi:10.1016/j.survophthal.2003.12.014

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Pharmacology of Bimatoprost To date studies on the pharmacology of bimatoprost, involving the cat iris sphincter, cat lung parenchyma, and human ciliary smooth muscle cells, have demonstrated potent activity.19,38,39 It appears that bimatoprost possesses a unique pharmacological profile, mimicking that of prostamide F2α. The pharmacological activities of these compounds may be mediated by a novel receptor. Bimatoprost, like prostamide F2α, has no meaningful activity at prostaglandin receptors.38,39 Bimatoprost also fails to stimulate a diverse variety of other known receptors that may be involved in mediating effects on intraocular pressure. Hence, its target receptor appears to be a novel prostamide-sensitive receptor.38,39 The conclusion that this novel receptor is different from prostaglandin receptors, most notably prostaglandin FP receptors, has been demonstrated in tissues and cells naturally expressing PG-sensitive receptors and in cells overexpressing recombinant prostanoid receptors. These results are exemplified by a study comparing the Ca2⫹ signal to PGF2α and bimatoprost in human fibroblasts constitutively expressing FP receptors and in genetically engineered cells overexpressing human FP receptors. The data are shown in Fig. 1. It may be noted that PGF2α is at least 1,000 times more potent than bimatoprost, which has no more than residual activity at the FP receptor. In cells overexpressing the recombinant FP receptor, a measurable response is apparent at concentrations of 10⫺6 and 10⫺5 M bimatoprost (Fig. 1 right). Results obtained in systems where receptors are overexpressed do not, however, adequately represent reality. In cells expressing the FP receptor at natural constitutive levels bimatoprost exerts no measurable

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effect until a very high 10⫺5 M concentration is achieved (Fig. 1 left).28 Important structural features account for the differences in the pharmacological profiles of prostamides and prostaglandins. Prostamides, such as prostamide F2α and bimatoprost, are fatty acid amides whereas prostaglandins are fatty acids. Fatty acid amides are neutral lipids and do not carry the negative charge associated with the carboxylic acid group of fatty acids. This is an important structural difference and is responsible for the fact that these neutral lipids are pharmacologically distinct from the corresponding charged lipids. In addition to being pharmacologically unique like bimatoprost, prostamide F2α is biosynthesized from anandamide by a pathway involving COX-2.16 In a similar manner PG-glyceryl esters are also formed from the endocannabinoid 2-arachidonyl glycerol. The biological significance of these COX-2 products of endocannabinoids remains to be elucidated. At present, studies on prostamide E226 and prostamide F2α38 suggest that they are both pharmacologically novel. Further, the effects of bimatoprost on intraocular pressure are consistent with its prostamide-mimetic activity according to the following evidence: 1) bimatoprost has potent inherent pharmacological activity in prostamide-sensitive tissues and cells, which is sufficient to explain its potent and efficacious ocular hypotensive activity, and 2) bimatoprost essentially remains intact in the living primate eye. An alternative explanation for the activities of bimatoprost is that it behaves as an FP receptor agonist.15,28 This assertion has been based on the very weak interaction of high concentrations of bimatoprost with human FP receptors overexpressed in

Fig. 1. Activity of bimatoprost (circles) and PGF2α (squares) at human prostaglandin FP receptors in normal human dermal fibroblasts (left) and HEK-293(EBNA) cells stably overexpressing FP receptors (right). Receptor activation was determined functionally as an increase in cytosolic calcium levels. Data represent mean and SEM; n ⫽ 4 (left) or n ⫽ 3 (right).

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HEK-293 cells (EC50 of ∼3,000 nM, Ki of ∼6,000 to 10,000 nM) and FP receptors in rat and mouse cell lines. In another report on studies in cells overexpressing human FP receptors,27 bimatoprost was claimed to be approximately 10 times more potent than in the other publications. The inconsistency between various publications, with respect to the potency of bimatoprost is most likely due to the use of genetically manipulated cells overexpressing human FP receptors. Overexpression of receptors provides more receptor targets on the cell surface, hence renders these genetically modified cells more sensitive to drugs. As a result, agonists appear more potent than in normal cells, which constitutively express the receptor. This was evident when we compared the activities of bimatoprost and PGF2α in normal human FP receptor expressing cells (human dermal fibroblasts, Fig. 1 left) and HEK-293 cells overexpressing human FP receptors (Fig. 1 right). Whereas a very high bimatoprost concentration of 10,000 nM produced only a marginal effect in human fibroblasts (EC50 [10,000 nM), the same concentration provided a more pronounced stimulation of HEK-293 cells overexpressing human FP receptors (EC50 ⫽ 10,000 nM). For pharmacological studies, genetically manipulated, transfected cells are useful tools for comparative drug testing to determine the rank order of potency for different drugs at a receptor of interest. If the absolute potency of drugs in a more physiological setting is of interest, normal unaltered human cells, such as dermal fibroblasts (Fig. 1 left) or trabecular meshwork cells28 naturally expressing FP receptors on their cell surface, are more relevant models. It should be kept in mind that the extraordinarily high concentrations (in the range of 10,000 nM and higher) required for bimatoprost to at least minimally activate FP receptors are not achieved in intraocular tissues—this will be discussed in more detail below. Despite the very weak interaction of bimatoprost with FP receptors, one group has classified bimatoprost as an FP receptor agonist.15,29 It is interesting to note in this context that travoprost acid loses its selectivity for FP receptors and stimulates other types of prostaglandin receptors at similar micromolar concentrations.11 It appears that different criteria for the pharmacological classification of travoprost acid and bimatoprost have been applied. While discounting micromolar affinities of travoprost acid at a variety of prostaglandin receptors, travoprost acid was described as a “highly selective” FP receptor agonist.11 A similar very low affinity of bimatoprost at FP receptors was regarded as representing its primary pharmacology.15,29 The substantially higher potency of bimatoprost at prostamide-sensitive receptors38,39

was not taken into consideration. Thus, the bimatoprost concentration required to stimulate FP receptors in normal, unaltered human cells is on the order of approximately 1,000 times higher than the concentration required to stimulate prostamide-sensitive preparations such as the cat iris sphincter,38 cat lung parenchyma,39 or human ciliary smooth muscle cells.19 Taking all available information into consideration, both compounds, travoprost acid and bimatoprost, should indeed be considered selective agonists at their respective target receptors: travoprost acid a selective FP agonist, bimatoprost a selective prostamide agonist. As will be discussed later, the bimatoprost concentrations achieved in intraocular tissues of primates after topical treatment are sufficient to activate prostamide-sensitive receptors but too low to stimulate FP receptors. Thus, in the eye, bimatoprost clearly works as a prostamide.

Ocular Hypotensive Activity and Mechanism of Action Bimatoprost highly effectively lowers IOP in cynomolgus monkeys.38,39 A single dose administered topically to the eyes of ocular hypertensive primates produced a 32% (0.01% bimatoprost) or 40% (0.1% bimatoprost) decrease in IOP. In these non-human ocular hypertensive primates the efficacy of bimatoprost is similar to its efficacy in human glaucoma and ocular hypertensive patients.13,18,22 It has been demonstrated in humans that bimatoprost lowers IOP by a dual outflow mechanism. Bimatoprost significantly enhances both pressure-sensitive and pressure-insensitive aqueous humor outflow, which presumably reflect trabecular and uveoscleral outflow, respectively.4 To assess the mechanism of ocular hypotensive action in cynomolgus monkeys, the effects of bimatoprost on aqueous humor dynamics were studied directly with established invasive and non-invasive techniques: fluorophotometry (aqueous flow); two-level constant pressure perfusion technique (total outflow facility1); and uveoscleral outflow using a modified36 tracer technique originally developed by Bill.3 These studies revealed that, in cynomolgus monkeys, bimatoprost significantly stimulated uveoscleral outflow by approximately 42% without affecting aqueous humor flow or total outflow facility. Increased uveoscleral outflow appears to result from a remodeling of the extracellular matrix in these primates leading to the formation of organized fluid pathways in the ciliary muscle.25 Direct determination of uveoscleral outflow in these primates, which cannot be accomplished in humans, provides supporting evidence that the calculated increase in pressure-insensitive outflow in humans4 most likely reflects uveoscleral outflow.

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Humans and non-human primates appear to differ with respect to bimatoprost effects on outflow facility, which is significantly enhanced in the former but unaltered in the latter species—at least after shortterm treatment. It has been suggested that bimatoprost treatment may render the uveoscleral outflow pathway pressure-sensitive. However, no data to support this contention have been presented. If that were the case one would expect an increase in outflow facility in the primate study similar to that seen in humans. This was not found. A more plausible explanation is that during short-term treatment for up to 5 days, bimatoprost has a direct effect on trabecular, conventional outflow in humans but not in cynomolgus monkeys. This scenario is more in line with the long-held belief that at least a large component of pressure-sensitive aqueous humor outflow is composed of trabecular outflow. Morphological examination of primate eyes after chronic treatment with bimatoprost for 1 year revealed changes in conventional outflow tissues which are perhaps suggestive of an effect of the drug on conventional, pressure-dependent outflow in primates as well.25 In-summary, accumulating evidence suggests that long-term therapy with bimatoprost increases both pressure-dependent trabecular outflow and pressure-independent uveoscleral outflow4 via remodeling of extracellular matrix in the trabecular meshwork and ciliary muscle, respectively.25 These effects are likely to involve multifunctional, extracellular matrix–associated signaling proteins, such as Cyr61.19

Ocular Penetration and Distribution Permeability studies carried out in vitro revealed that bimatoprost penetrates substantially better through the human sclera than the human cornea.38 Apparent permeability coefficients were 14.5 × 10⫺6 cm/sec and 3.24 × 10⫺6 cm/sec for sclera and cornea, respectively (Fig. 2). The conjunctiva had been removed from the scleral specimens for these permeability studies; an intervention which could have affected the scleral permeability to bimatoprost. To answer this question and to assess the ocular distribution of bimatoprost and potential ocular metabolites in a “real world” scenario, cynomolgus monkeys were bilaterally treated with 0.1% [3H]-bimatoprost and the eyes collected at various post-treatment time points (one animal was sacrificed at each time point). Radio-labeled drug was used to ensure accurate quantification in ocular tissues and fluids. Two independent investigations were carried out, one using a single topical treatment, and another one using BID treatment for 9.5 days. These studies are described in detail.39 In both studies the levels of bimatoprost

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Fig. 2. Corneal and scleral penetration of bimatoprost in humans. The scleral penetration rate (14.5 × 10⫺6 cm/sec) is approximately four times faster than the corneal penetration rate (3.24 × 10⫺6 cm/sec).

were on the order of 10 to 100 times higher in the ciliary body and iris compared to the aqueous humor. Additionally, bimatoprost was highly resistant to metabolism in the living eye and was the predominant molecular species detected. The implications of these results are discussed in the Ocular Metabolism section. Taken together, these findings provide strong additional support for a preferred scleral penetration route for bimatoprost in the intact eyes of living animals. If scleral permeability had been limited and access to the eye were to occur primarily via the cornea, the aqueous humor would be the vehicle carrying the drug to and into the intraocular tissues. Under those circumstances, one would expect equal or higher drug concentrations in the aqueous humor compared to the iris and the ciliary body. This was not the case. Tissue levels were found to be substantially higher than aqueous humor levels. Thus, these findings argue very strongly in favor of the sclera, and against the cornea, as the preferred route of entry for bimatoprost into the living eye. The conjunctiva does not appear to present a significant permeability barrier for the drug. The data indicate that the sclera provides direct access for bimatoprost to intraocular

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target tissues, such as the ciliary body and trabecular meshwork (Fig. 2). The exclusive stimulation of uveoscleral outflow by bimatoprost in cynomolgus monkeys indicates that the ciliary body represents the target tissue in this species for the ocular hypotensive activity of this drug, at least under the short-term treatment regimen employed in these studies. Peak levels (Cmax) in the ciliary body, equivalent to 848 nM and 2.6 µM, were rapidly achieved within 30 minutes in single and multiple dose studies, respectively.39 Considering the potent activity of bimatoprost at prostamide-sensitive receptors (EC50 of 34 nM), it is apparent that the ciliary body concentrations, achieved in both single and multiple-dose studies, are more than adequate to account for its ocular hypotensive activity. This contention remains valid even in light of the fact that a somewhat higher concentration (0.1%) was used in the primate studies than is present in a Lumigan bottle (0.03%).

Ocular Metabolism It was of particular importance to investigate the metabolic fate of bimatoprost in the eye since the potential hydrolysis product (17-phenyl PGF2α, which has sometimes been referred to as bimatoprost acid) is a potent prostaglandin FP receptor agonist. More pertinent, 17-phenyl PGF2α has an EC50 value of 112 nM, according to a significant increase in phosphatidylinositol (PI) turnover in human trabecular meshwork cells.28 The primate ocular distribution studies discussed above provide essential information on the ocular distribution of bimatoprost and its metabolic fate. The single-dose study most clearly and unequivocally demonstrates how bimatoprost works in the eye. As described above, topical administration of a single dose yielded pharmacologically relevant bimatoprost levels in the target tissue, the ciliary body. Secondly, 17-phenyl PGF2α as an acidic hydrolysis product of bimatoprost was not detectable in that same tissue. Third, as mentioned above, that same topical dose produces a 40% reduction in IOP in ocular hypertensive animals of the same species.39 A straight-forward conclusion is that bimatoprost works directly to lower IOP. No metabolic conversion is required for its substantial ocular hypotensive activity. Considering the target tissue levels achieved after topical treatment and the pharmacological activity profile of bimatoprost, it is suggested that the unique prostamide pharmacology is responsible for its ocular hypotensive activity. This contention appears also valid for the human eye based on the ability of the intact bimatoprost molecule to independently upregulate a gene (Cyr61) in human ciliary smooth muscle cells, which

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has been implicated in processes leading to a remodeling of the extracellular matrix.19 Such a mechanism can explain the reported morphological alterations of the ciliary muscle,25 leading to an increase in uveoscleral outflow.38 Contrary to the prostamide receptor and prostanoid FP receptor hypotheses, it has also been claimed that bimatoprost behaves in the eye as a prostaglandin prodrug.6,8,12,21 Maxey and colleagues investigated the hydrolysis of bimatoprost in bovine and human corneas;21 however, their experimental design incorporated such high bimatoprost concentrations and such long incubation times that the results are unlikely to be clinically relevant. The bimatoprost concentrations used were 400 µg/ml for bovine corneas and either 50 or 250 µg/ml for human corneas—concentrations that exceed or are close to that of the commercially available product. These concentrations are also 100 to 1,000 times higher than those achieved in the corneas of living primate eyes after topical treatment.39 Moreover, their incubation time was 23 hours—hundreds of times longer than the few minutes an ophthalmic eye drop can be expected to remain on the corneal surface following topical dosing. Despite the exaggerated experimental design, reported hydrolysis rates translate into approximately 0.3% per hour for bovine corneas and approximately 0.4% for human corneas. A more comprehensive in vitro study also demonstrated the excellent metabolic stability of bimatoprost in human ocular tissues—cornea, sclera, ciliary body and iris.6 The authors of this investigation reported that bimatoprost hydrolysis occurred at a very slow rate of 1% or less over a 3-hour incubation period. Thus, the reported hydrolysis rate is in a similar and negligible range to the rate found by Maxey and colleagues.21 A third in vitro study also reported a slow but slightly higher rate of enzymatic hydrolysis, albeit using a higher concentration of bimatoprost.12 The minimal hydrolysis rates in these human ocular tissue studies support the conclusions of Woodward and colleagues38,39 that bimatoprost is metabolically highly stable. If bimatoprost were a prodrug, rapid and complete hydrolysis to an active metabolite would be expected. Such rapid and complete ocular and systemic hydrolysis has consistently been reported for the ester prodrug latanoprost.2,31–35 In fact, latanoprost hydrolysis was so rapid that the isopropylester parent molecule was usually not detectable in the intraocular tissues and fluids in these studies. Eisenberg et al8 based their conclusion that bimatoprost is a prostaglandin prodrug not on any studies of their own but on several other references. It is specifically claimed that amide hydrolysis by ocular tissues has been shown, naming anandamide,20,24

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nepafenac,14 and prostanoid DP receptor agonists10 as specific examples. However, published evidence for the hydrolytic activity of a specific amidase, such as fatty acid amide hydrolase, on bimatoprost does not exist. It is also extremely unlikely that fatty acid amide hydrolase could recognize an oxygenated fatty acid amide with a “hairpin”-shaped conformation. In contrast to the conclusion drawn by Eisenberg et al,8 the ocular hydrolysis of nepafenac was shown to be minimal at best in the referenced study by Ke et al.14 As part of their investigations, the authors examined corneal penetration and metabolism of nepafenac using an in vitro paradigm designed to mimic the topical administration of a drug to the living eye. Briefly, in a device where the cornea separated two fluid filled chambers, the “extraocular” epithelial side of the cornea was incubated with drug solution for 5 minutes, then washed and further incubated for an additional 6 hours with buffer without drug. This study was designed to take into account the limited corneal residence time of an ophthalmic eye drop. After this 5-minute incubation period of the corneal epithelial side with nepafenac, unmetabolized nepafenac but very little acid hydrolysis product exited the cornea on the endothelial as well as epithelial sides during the 6-hour monitoring period. These reported findings demonstrate that the amide nepafenac is largely resistant to corneal hydrolysis under conditions simulating topical ocular instillation. Eisenberg et al8 have also suggested that ocular hydrolysis of amides of prostanoid DP receptor agonists had been demonstrated in a third reference.10 However, no metabolism studies were actually reported by Hellberg and colleagues, nor was there a reference to any such studies. A more plausible explanation is that the referenced amide compounds lowered IOP in a prostamide-like fashion. Considering a number of different studies dealing with the metabolic fate of amides in the eye, it becomes clear that amidases specific for the hydrolysis of amides like bimatoprost do not exist in the eye. Otherwise, efficient hydrolytic activity would have been discovered. Corneal metabolism studies carried out in vitro provide only a snapshot of events taking place in that part of the eye. At the least, the virtual absence of specific amidases in the cornea means that relevant quantities of an acid hydrolysis product of bimatoprost are not produced and entering the eye via this tissue. As previously mentioned, one needs to examine the target tissues themselves to determine the mechanism of action of a drug. A drug can only be active if it reaches its target in sufficient quantities, regardless of whether the active principle is represented by the parent molecule or a metabolite. Thus,

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the primate studies involving topical treatment with bimatoprost, closely resembling treatment of glaucoma patients, provide a much more clear, accurate, and complete picture of the active principle of bimatoprost. These studies clearly demonstrate that bimatoprost works directly to lower IOP. The reported minimal rate of hydrolysis for bimatoprost, even the studies conducted by Maxey et al21 and Davies et al,6 are not inconsistent with that contention. The implications of all these investigations taken together are the following: 1) hydrolysis of bimatoprost is not a prerequisite for its excellent ocular hypotensive activity; and 2) bimatoprost works directly to lower IOP in the human and non-human primate eye by virtue of its prostamide pharmacology.

Clinical Studies Confirm the Unique Pharmacology and Mechanism of Action of Bimatoprost Investigations on the systemic clinical pharmacology of bimatoprost also demonstrated the stability of the bimatoprost molecule to enzymatic hydrolysis. After topical ocular administration to human volunteers, unmetabolized bimatoprost, but again no acid hydrolysis product, was detected in the systemic circulation.38 These findings are consistent with a recent publication demonstrating the metabolic stability of prostamides, as exemplified by prostamide E2, in a variety of biological settings.17 As in the eye, the systemic metabolic stability of prostamides contrasts sharply with the exquisite systemic hydrolysis of latanoprost in humans and primates, where latanoprost acid but no isopropylester parent drug was detectable in the plasma.32–35 Gandolfi and Cimino9 reported recently that bimatoprost substantially lowers IOP in patients who respond poorly to latanoprost. Their investigations provide compelling clinical evidence that bimatoprost and latanoprost work differently in the eye. The fact that bimatoprost is the active molecule in the eye and its unique pharmacology help to explain the high efficacy of bimatoprost,7,13,22,23 and why it is effective in patients non-responsive to latanoprost.9,37

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Drs. Krauss and Woodward are employees of Allergan, Inc. Reprint address: David F. Woodward, PhD, Biological Sciences RO-2C, Allergan Inc., 2525 Dupont Drive, Irvine, CA 92612-1531.