Purpose
Stimulation of nicotinic acetylcholine (nACh) receptors on vascular endothelial cells promotes angiogenesis and vascular permeability in animal models. The safety and bioactivity of topical mecamylamine, an antagonist of nACh receptors, was tested in patients with diabetic macular edema.
Design
A multicenter phase I/II clinical trial.
Methods
Twenty-three patients with chronic diabetic macular edema received 1% mecamylamine topically twice daily for 12 weeks, the primary end point. Patients underwent safety assessments, measurement of best-corrected visual acuity (BCVA), and measurement of foveal thickness using optical coherence tomography at baseline, 1, 4, 8, 12, and 16 weeks.
Results
Mecamylamine drops were well tolerated and there were no drug-related safety problems. Mean improvement in BCVA at 1, 4, 8, 12, and 16 weeks was 2.8, 1.9, 2.4, 0.8, and 3.1 letters, respectively. There was little change in mean excess foveal thickness. There was substantial heterogeneity in response, because 8 patients showed convincing improvement in BCVA, foveal thickness, or both, 9 patients showed equivocal or no substantial changes, and 4 patients showed worsening. Five patients showed a substantial improvement in BCVA, foveal thickness, or both between their last visit while receiving mecamylamine and 1 month after stopping mecamylamine.
Conclusions
This study suggested that administration of topical mecamylamine, a nonspecific nACh receptor blocker, may have heterogeneous effects in patients with diabetic macular edema. Variable expression of nACh receptor subtypes on endothelial cells that have different effects on permeability would provide an explanation for these results and should be investigated, because more specific nACh receptor blockers may dissociate antipermeability and propermeability effects.
Nicotinic acetylcholine (nACh) receptors play an important role in neurotransmission. They are ligand-gated ion channels made up of 5 subunits that form a central pore (for review, see ). Seventeen subunits have been identified, α1 through α10, β1 through β4, γ, δ, and ε. Homomeric and heteromeric pentamers can form so that there are numerous nACh receptor subtypes. Binding of acetylcholine, the endogenous ligand, induces a conformational change that opens the ion pore, causing influx of cations. The heteromeric nAChRs are differentially permeable to sodium, whereas the homomeric nAChRs are differentially permeable to calcium. Sustained exposure to acetylcholine may result in desensitization, a progressive loss of response. Receptor subtypes differ with respect to activation and desensitization depending on their subunit makeup and extent of phosphorylation, resulting in different magnitudes of effect or even opposing effects mediated by different subtypes on the same cell.
In addition to binding acetylcholine, nACh receptors bind nicotine; the activation of heteromeric nAChRs by nicotine is responsible for the addictive effects of cigarette smoke. Binding of nicotine distinguishes nACh receptors from muscarinic acetylcholine receptors, which are activated by acetylcholine, but not nicotine. The muscarinic receptors are G protein-coupled 7-transmembrane spanning receptors that are very different in structure. Nicotine provides a tool for identification of nACh receptors and has been used to determine that they are present on some nonneuronal as well as neuronal cells. Vascular endothelial cells contain nACh receptors that may be activated by exogenous nicotine or endogenous acetylcholine. Endothelial nAChR activation increases proliferation, migration, and tube formation of endothelial cells in vitro and neovascularization in atherosclerotic plaques and tumors in vivo. One source of endogenous acetylcholine is the endothelium itself, as endothelial cells synthesize, store and release acetylcholine resulting in autocrine loops. In the eye, nicotine stimulates choroidal neovascularization, which may contribute to the increased progression to neovascular age-related macular degeneration seen in smokers with nonneovascular age-related macular degeneration. Nicotine also stimulates increased vascular permeability in brain. Endothelial α7-nAChRs have been implicated as the major subtype mediating nicotine-induced vascular permeability and angiogenesis.
There are several nACh receptor antagonists that bind to nACh receptors and reduce activation by agonists. Mecamylamine is a nonspecific nACh receptor antagonist that was approved as an oral antihypertensive agent in the 1950s. It reduces blood pressure by blocking neuronal nAChR-mediated neurotransmission at sympathetic ganglia, permitting blood vessels to dilate. Mecamylamine has a good safety profile at antihypertensive doses of 30 to 90 mg/day, but may cause orthostatic hypotension, as well as anticholinergic side effects including constipation, urinary retention, and dry mouth. Mecamylamine is able to cross the blood-brain barrier and has been used as an antidepressant and to facilitate cessation of smoking.
The previous use of mecamylamine in humans made it a good choice to test whether blocking nAChRs can reduce abnormal retinal vascular permeability in patients with diabetic macular edema (DME). To avoid orthostatic hypotension, constipation, urinary retention and dry mouth, a formulation for local topical ocular delivery was developed. Application of this formulation containing 0.1% or 1% mecamylamine 3 times daily in rabbits resulted in detectable levels of mecamylamine in the retina and suppressed choroidal neovascularization in mice. During 6 hours of sampling after a single application of 1% mecamylamine to the cornea of rabbits, high levels of mecamylamine were detected by high performance liquid chromatography in retina and choroid with mean peak levels of 5000 to 11 300 ng/mL (Kengatharan KM, et al. IOVS 2008; 49: ARVO E-Abstract 3424). The area under the curve was approximately 7500 ng/mL per hour using a trapezoidal rule on a population pharmacokinetic basis, and comparison of levels in different parts of the eye suggested that topically delivered mecamylamine gains access to the choroid and retina by penetration through the conjunctiva and sclera. Topical application of 1% mecamylamine twice daily in normal volunteers was well tolerated with no adverse effects. In this study, we tested the effect of topical mecamylamine on foveal thickness and visual acuity in patients with DME.
Methods
This was a phase I/II, open-label clinical trial conducted at 3 sites in the United States through an Investigational New Drug application granted by the Food and Drug Administration.
Patient Eligibility and Exclusion Criteria
Patients (18 years of age or older) with type 1 or type 2 diabetes and DME were eligible if they had reduction in visual acuity between 20/40 and 20/400 and met the following criteria: (1) center subfield thickness measured by optical coherence tomography (OCT) of 250 μm or more, (2) no potential contributing causes to reduced visual acuity other than DME, (3) no indication of permanent vision loss such as atrophy and pigmentary change in the fovea, (4) no intraocular surgery, laser photocoagulation, or treatment with an antagonist of vascular endothelial growth factor (VEGF) within 3 months, (5) no media problems that would preclude retinal evaluation, (6) no treatment with any investigational agent within 3 months, and (7) hemoglobin A1C levels of 12% or less.
Study Drug
Mecamylamine HCl ophthalmic solution (CoMentis, Inc, South San Francisco, California, USA) is a clear, colorless, buffered aqueous solution containing 0.01% benzalkonium chloride and ethylenediaminetetraacetic acid as a chelating agent. It was supplied as a sterile 1% solution in clear low-density polyethylene bottles with controlled dropper tips for multiple uses.
Study Protocol
Consenting patients were screened for the study by undergoing a medical history investigation, physical examination, measurement of best-corrected visual acuity (BCVA) by an experienced examiner using the Early Treatment Diabetic Retinopathy protocol, a complete eye examination, optical coherence tomography (OCT), fluorescein angiography, and laboratory tests of blood and urine. If both eyes met eligibility criteria, one was selected by the enrolling investigator to be the study eye. Eligible patients returned for a baseline visit and had measurement of BCVA, complete eye examination, and OCT. Patients were given 1% mecamylamine eye drops and were instructed in their use. It was requested that the drops be administered twice daily. Follow-up study visits were at 1, 4, 8, 12, and 16 weeks. At each visit, patients underwent safety assessments, measurement of BCVA, complete eye examination, and OCT. At 12 weeks, the primary end point, patients also underwent a repeat physical examination and hematology and blood chemistry laboratory tests. At one site, patients underwent an anterior chamber tap at baseline, 1 week, and 12 weeks for measurement of aqueous VEGF levels.
Optical Coherence Tomography
OCT scans were performed by an experienced investigator with a StratusOCT3 (Carl Zeiss Meditec, Dublin, California, USA) using the fast macular scan protocol. This protocol consists of 6-line scans that are 6.0 mm long centered on fixation and spaced 30 degrees apart around the circumference of a circle. Each line consists of 128 A-scan measurements. With each A-scan, the OCT software measures the distance between the inner surface of the retina and the anterior border of the retinal pigmented epithelium–choriocapillaris complex based on changes in reflectivity. The center point thickness, also known as the foveolar thickness, is a mean value generated by the StratusOCT software from the 6 central A-scan thickness values of each of the radial lines comprising the fast macular thickness map. We did not use this value generated from only 6 data points for our primary measure of central retinal thickness, but instead used the foveal or central 1-mm thickness, which is an average interpolated value based on central 21 scans of each of the 6 lines passing through the patient’s fixation point. The number of data points used to compute this value is 21 × 6 = 126, which provides a better representation of the thickness of the central retina than a value generated from only 6 points around fixation. Readers at the Wilmer Retinal Imaging Research and Reading Center examined the images for each OCT file to be sure that there were no artifacts such as misidentification of inner or outer surface of the retina. When artifacts were present, corrected measurements were obtained using RetinaTOMOGRAPHER software (version 1.1; Retinal Imaging Research and Reading Center, Baltimore, Maryland, USA). Excess foveal thickness (EFT) was calculated by subtracting the measured foveal thickness value from 212 μm, the upper limit of the normal range of center subfield thickness determined from measurements on a large population of subjects.
Measurement of Vascular Endothelial Growth Factor Levels in Aqueous
Anterior chamber taps were performed under topical anesthesia by biomicroscopic observation of the eye with the patient seated at a slit lamp. A 30-gauge needle was inserted into the anterior chamber and 0.1 mL aqueous was removed and stored frozen at −80 C until assayed. Aqueous VEGF levels were measured with the Human VEGF Quantikine ELISA kit (R&D Systems, Minneapolis, Minnesota, USA) using the manufacturer’s instructions. Three separate assays were performed to ensure that results were reproducible.
Results
The primary objective of this study was to evaluate the safety of topical mecamylamine in patients with DME. A few patients reported transient stinging after application, but in general the drops were well tolerated. Twenty-three patients were enrolled in the study, and 19 completed all study visits. Two patients withdrew consent before the week 1 visit and no data are available for those patients; 1 patient decided that the time commitment was too great and the other decided that an eye drop would be unlikely to be strong enough to help the condition. One patient was withdrawn from the study for alternative treatment because of worsening of DME after the month 2 visit, and this patient’s observations at month 2 were carried forward to months 3 and 4. One patient made all study visits through the primary end point and missed the month 4 visit, for which the data at month 3 were carried forward. Thus there are 21 patients for whom data were evaluated. There were no serious adverse events and no drug-related adverse events.
The secondary objective of the study was to assess for potential bioactivity of topical mecamylamine. In early phase studies, it is useful to examine the course of each patient in great detail, and therefore BCVA and OCT scans are presented for each patient at each study visit.
Subgroup of 8 Patients Showing Improvement
Figure 1 shows the course for 8 patients who showed signs of improvement with topical mecamylamine. Patients 01-008, 03-007, 02-003, and 01-003 showed improvements in BCVA of 9, 6, 14, and 15 letters at the week 12 primary end point along with a substantial reduction in EFT at some point during the course. Red-free photographs from Patient 01-008 showed a reduction in exudates between baseline and 3 months, and fluorescein angiography illustrated a corresponding reduction in leakage and pooling of dye in the macula during the late phase of the angiogram ( Figure 2 ). The concordance of anatomic and functional improvement increases confidence that these patients truly had benefit. Patient 03-005 showed improvement in BCVA of 22 letters despite a modest change in EFT. Patient 01-011 did not show a substantial change in EFT at any time, but showed an improvement in BCVA of more than 2 lines at 1 week (generally a 2-line change is our threshold for taking a BCVA change as clinically significant), and although it fluctuated somewhat thereafter, an improvement was substantiated. In both of these patients, the improvement in vision was substantial, occurred soon after starting the drug, and was reproduced in multiple measurements for the remainder of the study. Both of these patients had modest EFT at baseline and therefore even modest changes may be expected to have a substantial functional impact. Patients 01-002 and 03-009 had mild macular edema at baseline that essentially was completely eliminated by week 12 with normal-appearing scans, but this was not accompanied by substantial improvement in BCVA. Patients may have permanent visual loss from chronic macular edema so that BCVA cannot improve despite elimination of the edema. In retrospect, this situation might have been predicted for these 2 patients, because despite the mild edema at baseline, the BCVA was quite poor.
Subgroup of 9 Patients Showing No Change
Patients 01-010, 02-005, and 03-013 showed a similar pattern in that they showed some evidence of improvement through week 8, but during the last 4 weeks of mecamylamine treatment, there was a substantial drop in BCVA that recovered 1 month after mecamylamine was stopped ( Figure 3 ).
Patients 01-009 and 03-012 showed improvements in BCVA of 8 and 7 letters, respectively, but this was not accompanied by a substantial change in EFT ( Figure 4 ). Patient 03-012 had a reduction in EFT of 41 and 38 μm at 4 and 8 weeks, respectively, and because the reproducibility limit of time-domain OCT is in the range of 30 μm, this may represent a real change that may explain the improvement of 7 letters. However, this patient never had an improvement of 2 lines or more in BCVA at any time point, and therefore is listed as equivocal. Similarly, Patients 01-009 and 03-012 had severe edema at baseline, but did not have a substantial change in EFT. Patients 01-006 and 01-007 showed minimal change in BCVA and no change (Patient 01-006) or modest worsening (Patient 01-007) in EFT. Patients 03-004 and 02-004 showed essentially no change in BCVA (+3 and +1) and a modest improvement (Patient 03-004) or worsening (Patient 02-004) in EFT.
Subgroup of 4 Patients Showing Mecamylamine-Induced Worsening
Patient 03-002 showed a 15-letter reduction in BCVA and a 248-μm increase in excess central subfield thickening over the 3 months while receiving mecamylamine. Notably, 1 month after stopping mecamylamine, there was resolution of the macular edema with a reduction of 516 μm in EFT accompanied by a gain of 14 letters in BCVA ( Figure 5 ). The magnitude of the worsening as well as the rapidity and magnitude of the reversal after mecamylamine was discontinued implicate mecamylamine, rather than a spontaneous change in DME status. Patient 01-005 had a 20-letter reduction in BCVA and a 238-μm increase in EFT over the 3 months of receiving mecamylamine; within 1 month of cessation of mecamylamine, the BCVA improved by 10 letters and the EFT decreased by 20 μm. Patient 03-010 had severe edema with EFT at baseline of 492 μm that was reduced by 128 μm at 8 weeks. However, at 12 weeks, EFT increased by 78 μm, and BCVA fell by 9 letters compared with baseline. After stopping mecamylamine, the edema continued to worsen and the BCVA remained the same. Patient 03-011 was withdrawn from the study for alternative treatment at month 2 because of a reduction in BCVA of 14 letters and an increase in EFT thickness of 288 μm.
Mean and Median Changes in Best-Corrected Visual Acuity and Excess Foveal Thickness
There was no change in mean EFT throughout the study ( Figure 6 , Top). Median EFT was slightly higher than baseline at the primary end point and less than baseline at month 4 ( Figure 6 , Bottom). There was an increase in mean BCVA of 2.8 letters at 1 week after starting mecamylamine, but there was a substantial dip to 0.8 letters at week 12, the primary end point, with rebound to 3.1 letters 4 weeks after stopping mecamylamine. Median improvement in BCVA peaked at 2 letters at month 2, and was 1 letter at the primary end point.
Aqueous Vascular Endothelial Growth Factor Levels
To determine if mecamylamine influenced VEGF levels in patients with DME, aqueous VEGF levels were measured in 9 patients enrolled at 1 site at baseline, day 7, and day 84, the primary end point. There were small reductions in mean and median VEGF levels between baseline and day 84 that were not statistically significant ( Figure 7 , Top). Two of 4 patients in category 1 who showed improvement with mecamylamine had reductions in aqueous VEGF of 105 and 168 pmol/mL during the 12 weeks of treatment, but the other 2 showed increases of 52 and 62 ( Figure 7 , Bottom). Patient 1-005, who worsened while receiving mecamylamine, had a reduction in aqueous VEGF of 93 pmol/mL. Thus, although the numbers are very small that would preclude any firm conclusions in any case, there were no consistent correlations of outcome with alterations of VEGF.
Correlation of Other Patient Characteristics With Outcome
There are a number of things that could have an effect on outcome, and it is useful to determine if they played any role in the variable response to topical mecamylamine. Patients with shorter duration of DME may respond better in terms of visual improvement to pharmacologic agents. There were 4 patients in the study who had DME for only a few months when they entered the study; 3 of these patients showed improvement after 12 weeks of mecamylamine treatment (14, 9, and 9 letters), but 1 patient showed an early improvement of 9 letters after 4 weeks, but a loss of 10 letters at 12 weeks ( Table ). However, 2 patients who improved by 15 letters or more at 12 weeks had a fairly long duration of DME (7 and 27 years). A sudden change in glycemic control, either improvement or worsening, can cause worsening of diabetic retinopathy, but very few patients had any substantial change in hemoglobin A1C during the 12 weeks of treatment with mecamylamine ( Table ). Likewise, patients showed little change in blood pressure, serum creatinine (an indicator of renal function), serum cholesterol, or triglycerides. There was only one patient who smoked cigarettes during the time of the study, and that patient had a good response to topical mecamylamine ( Table ).
