Technical note: the comparison of hypointense lesions from 'pseudo-T1' and T1-weighted images in secondary progressive multiple sclerosis.

There is a large historical database of dual-echo conventional spin-echo (CSE) magnetic resonance images in multiple sderosis (MS). If new analysis techniques can be developed then this database could provide valuable information. We have investigated a technique in which the late echo of a dual-echo data set is subtracted from the corresponding early echoyielding images, which appear qualitatively similar to T1-weighted images. This study investigated whether the hypointense lesions on the 'pseudo-T1' images (created as described above) were related to hypointense lesions on conventional T1-weighted images. The hypointense lesion areas were measured by a blinded observer using a computer-assisted contouring technique applied to pseudo-T1 and T1-weighted CSE images obtained from 17 patients with secondary progressive MS (SPMS). The mean hypointense lesion area from T1-weighted images was 2218 +/- 2072 mm2, compared to 1426 +/- 1353 mm2 from pseudo-T1 images (p = 0.008). There was, however, a strong correlation between the values obtained from the two sets of images (r = 0.93, p < 0.001). The strong correlation between the values obtained from the two sets of images suggests that pseudo-T1 images may be useful to investigate a subgroup of more destructive lesions in MS from historical databases and in future prospective studies when imaging time is limited.

The effect of IFNbeta-1b on the evolution of enhancing lesions in secondary progressive MS.

BACKGROUND: After the resolution of contrast enhancement, the majority of new MS lesions become isointense with surrounding white matter on T1-weighted MRI. Less commonly, a hypointense T1 lesion develops, representing the development of more severe focal tissue damage. Interferon beta (IFNbeta) reduces both the number of new enhancing lesions and the duration of contrast enhancement. OBJECTIVE: To determine if IFNbeta affects the degree of tissue damage within new lesions and if its effects are related to lesion size. METHODS: One hundred twenty-five patients with secondary progressive MS from seven European sites were randomized to receive either IFNbeta-1b or placebo. Monthly, contrast-enhanced T1-weighted MR images were acquired at baseline, at months 1 to 6, and at months 19 to 24. The size of all new enhancing lesions developing between months 1 and 6 was recorded and their appearance at follow-up documented. RESULTS: In the first 6 months, fewer new enhancing lesions occurred in the IFNbeta-1b arm. This difference was greater for small (70% decrease) than for large (46% decrease) lesions. Hypointense T1 lesions were more likely to form from large (25%) than from small (9%) enhancing lesions in both treatment arms. Patients taking IFNbeta-1b developed fewer hypointense T1 lesions; however, the proportion of enhancing lesions developing into hypointense T1 lesions was similar in both arms. CONCLUSION: IFNbeta-1b reduced the number of new enhancing lesions, with a greater effect on small lesions. However, when a new enhancing lesion did become established, treatment with IFNbeta-1b did not alter its subsequent course.

Clinical-MRI correlations in a European trial of interferon beta-1b in secondary progressive MS.

BACKGROUND: The recently completed placebo-controlled multicenter randomized trial of interferon beta-1b (Betaferon) in 718 patients with secondary progressive MS shows significant delay of disease progression and reduction of relapse rate. This study provides an opportunity to assess the level of relationship between clinical and MRI outcomes in this cohort of patients with secondary progressive MS. METHODS: Brain T2-weighted lesion volume was measured annually in all available patients, with visual analysis to identify any new or enlarging (active) T2 lesions at each annual time point. A subgroup of 125 patients had monthly gadolinium-enhanced, T1-weighted imaging at months 0 to 6 and 18 to 24. Relapses were documented and expanded disability status scale (EDSS) was measured every 3 months. RESULTS: For the annual MRI outcomes, a significant but modest correlation was identified between the change in T2 lesion volume from baseline to the final scan and the corresponding change from baseline in EDSS (r = 0.17, p < 0.0001). There were significant correlations between the cumulative number of active T2 lesions and 1) change in EDSS (r = 0.18, p < 0.0001) and 2) relapse rate (r = 0.24, p < 0.0001). In the subgroup of 125 patients undergoing monthly imaging, MRI lesion activity was correlated with relapse rate over months 0 to 24 (r = 0.24, p = 0.006) but not with change in EDSS. CONCLUSIONS: These results confirm that the clinical-MRI relationships previously identified in relapsing-remitting MS still are apparent in the secondary progressive phase of the disease and support the use of MRI as a relevant outcome measure. In view of the relatively modest nature of the correlations, it seems unwise to rely on such MRI measures alone as primary efficacy variables in secondary progressive MS trials.

The effect of interferon beta-1b treatment on MRI measures of cerebral atrophy in secondary progressive multiple sclerosis. European Study Group on Interferon beta-1b in secondary progressive multiple sclerosis.

The recently completed European trial of interferon beta-1b (IFNbeta-1b) in patients with secondary progressive multiple sclerosis (SP multiple sclerosis) has given an opportunity to assess the impact of treatment on cerebral atrophy using serial MRI. Unenhanced T(1)-weighted brain imaging was acquired in a subgroup of 95 patients from five of the European centres; imaging was performed at 6-month intervals from month 0 to month 36. A blinded observer measured cerebral volume on four contiguous 5 mm cerebral hemisphere slices at each time point, using an algorithm with a high level of reproducibility and automation. There was a significant and progressive reduction in cerebral volume in both placebo and treated groups, with a mean reduction of 3.9 and 2.9%, respectively, by month 36 (P = 0.34 between groups). Exploratory subgroup analyses indicated that patients without gadolinium (Gd) enhancement at the baseline had a greater reduction of cerebral volume in the placebo group (mean reduction at month 36: placebo 5.1%, IFNbeta-1b 1.8%, P < 0.05) whereas those with Gd-enhancing lesions showed a trend to greater reduction of cerebral volume if the patient was on IFNbeta-1b (placebo 2.6%, IFNbeta-1b 3.7%; P > 0.05). These results are consistent with ongoing tissue loss in both arms of this study of secondary progressive multiple sclerosis. This finding is concordant with previous observations that disease progression, although delayed, is not halted by IFNbeta. The different pattern seen in patients with and without baseline gadolinium enhancement suggests that part of the cerebral volume reduction observed in IFNbeta-treated patients may be due to the anti-inflammatory/antioedematous effect of the drug. Longer periods of observation and larger groups of patients may be needed to detect the effects of treatment on cerebral atrophy in this population of patients with advanced disease.

The precision of T1 hypointense lesion volume quantification in multiple sclerosis treatment trials: a multicenter study.

The volume of hypointense lesions on T1 weighted brain MRI represents an increasingly used MR endpoint in phase III MS treatment trials. In this study we evaluated the reproducibility of hypointense T1 lesion volume quantification in a cohort of Multiple Sclerosis (MS) patients. The gadolinium enhanced T1 weighted brain MR images of 33 MS patients from three European centers were used in this study. These images were acquired as part of a phase III trial of interferon beta-1b in secondary progressive MS. The MRI machine manufacturers and imaging parameters varied according to the MRI acquisition center. Three experienced observers used a semi-automated local thresholding technique to quantify the hypointense T1 lesion volume on two occasions, separated by a delay. The intra and inter observer coefficients of variation were 3.7% and 4.9% respectively, with similar values derived for images obtained at all three sites. There was a generally high level of agreement between the lesion volumes obtained by the three raters. However, a modest but significant measurement drift was identified between the first and second sessions for one of the three raters, highlighting the very real possibility of measurement drift even for experienced observers. Our results support the increasing role for T1 hypointense lesion volume as an outcome measure in multicenter phase III MS treatment trials. Multiple Sclerosis (2000) 6 237 - 240

Lesion heterogeneity in multiple sclerosis: a study of the relations between appearances on T1 weighted images, T1 relaxation times, and metabolite concentrations.

OBJECTIVES: Multiple sclerosis lesions appear as areas of high signal on T2 weighted MRI. A proportion of these lesions, when viewed on T1 weighted MRI, appear hypointense compared with surrounding white matter. These hypointense T1 lesions are thought to represent areas of greater tissue damage compared with the more non-specific, total T2 lesion load. This study aimed to better characterise the properties of high signal T2 lesions with differing appearances on T1 weighted MRI using quantitative MR techniques. METHODS: Eleven patients with secondary progressive multiple sclerosis were studied. Two high signal T2 lesions were selected from each patient-one of which appeared hypointense and one isointense on a T1 weighted image. A voxel was positioned around each lesion and for this volume of brain the metabolite concentrations were estimated using proton MR spectroscopy ((1)H-MRS) and the T1 relaxation time within each voxel calculated from a T1 map generated using a multislice technique. RESULTS: Compared with isointense T1 lesions, hypointense T1 lesions exhibited a significantly lower absolute concentration of N-acetyl derived metabolites (tNAA) and a significantly higher absolute concentration of myo-inositol (Ins). T1 relaxation time correlated significantly with both tNAA (r=-0.8, p < 0.001) and Ins (r=0.5, p=0. 012). There was no correlation between T1 relaxation times and creatine/phosphocreatine or choline containing compounds. CONCLUSIONS: Prolonged T1 relaxation times seem to reflect the severity of axonal damage or dysfunction (inferred by a low tNAA) and possibly also gliosis (inferred by a high Ins) in chronic multiple sclerosis lesions.

The use of magnetic resonance imaging in multiple sclerosis treatment trials: power calculations for annual lesion load measurement.

Phase III definitive treatment trials of new multiple sclerosis (MS) therapies now routinely incorporate an annual magnetic resonance imaging protocol, with change in T2-weighted brain lesion load providing an important outcome measure. To date the accepted strategy has been to perform a core imaging protocol on all patients in such studies. The aim of this study was to provide power calculations based on this MRI endpoint. Serial MRI data from 128 patients with either relapsing remitting (RR) or secondary progressive (SP) MS were used to calculate sample size requirements using a repeated measures analysis of variance design. We provide sample size calculations based on various follow-up intervals and effect sizes. Sample sizes for the SPMS cohort were substantially larger than for the RRMS group, reflecting the greater variance in lesion load changes between patients in the SPMS group. With a follow-up of 3 years, we estimate that only 12 and 33 patients per arm are needed to show stabilisation of MRI lesion load in the RRMS and SPMS groups, respectively. Our results suggest that ongoing phase III treatment trials are more than adequately powered to detect even subtle treatment effects, and indicate that incorporating measurements from longer follow-up durations increases power substantially. We conclude that an annual imaging protocol provides a robust and powerful tool for assessing effects on the radiological appearance of the disease process.

Effect of interferon-beta1b on magnetic resonance imaging outcomes in secondary progressive multiple sclerosis: results of a European multicenter, randomized, double-blind, placebo-controlled trial. European Study Group on Interferon-beta1b in secondary progressive multiple sclerosis.

A randomized placebo-controlled trial of interferon-beta1b was performed on 718 patients with secondary progressive multiple sclerosis with follow-up of up to 3 years. In addition to clinical variables, serial magnetic resonance imaging (MRI) studies were performed to determine the effect of treatment on the pathological evolution of the disease. All patients eligible for MRI had annual proton density/T2-weighted brain scans from which total lesion volume was measured and the number of new and enlarging lesions noted. A subgroup of 125 patients also underwent monthly gadolinium-enhanced and proton density/T2-weighted brain MRI from months 0 to 6 and 18 to 24 to determine the effect of treatment on the frequency of new lesion activity, defined as new enhancing lesions and new/enlarging T2 lesions not enhancing with gadolinium. The difference in total lesion volume between treatment groups was highly significant. In the placebo group, there was an increase of 15% from baseline to last scan, whereas in the interferon-beta1b group, a reduction of 2% was seen. Within the placebo group, there was a significant year-on-year increase in total lesion volume, with a mean increase of 16% at year 3 compared with baseline. In the treated group, there was a significant reduction at year 1 (4%) and year 2 (5%) compared with baseline; the 2% decrease at year 3 was not significant. The number of new or enlarging proton density/T2 lesions was also significantly reduced by treatment. In the frequent MRI subgroup, treatment was associated with a significant 65% reduction in new lesion activity between months 1 and 6, and 78% reduction from months 19 to 24. Interferon-beta1b has a substantial and sustained effect on reducing the accumulation of new inflammatory disease foci in secondary progressive MS. This therapeutic mechanism may contribute to the positive clinical benefits of treatment on the progression of sustained neurological disability and relapse activity that were also identified in this trial.

Quantitative MRI in patients with secondary progressive MS treated with monoclonal antibody Campath 1H.

BACKGROUND: To assess the long-term effect of the lymphocyte-depleting humanized monoclonal antibody Campath 1H on MR markers of disease activity and progression in secondary progressive MS patients. METHODS: Twenty-five patients participated in a crossover treatment trial with monthly run-in MR scans for 3 months, followed (after a single pulse of Campath 1H) by monthly MR scans from months 1 to 6 and again from months 12 to 18. MR analysis was performed to provide measurements of the number and volume of gadolinium (Gd)-enhancing lesions as well as the hypointense lesion volume on a T1-weighted sequence. In addition, serial measurements of T2 brain lesion volume, brain volume, and spinal cord cross-sectional area were made over the duration of the study. The relationship between clinical and MR measures of disease evolution was also assessed. RESULTS: Treatment was associated with a reduction in the number and volume of Gd-enhancing lesions (p < 0.01). Despite this, a decrease in brain volume was seen in 13 patients during the 18 months post-treatment. The mean pretreatment Gd-enhancing lesion volume was predictive of subsequent reduction in brain volume (r = 0.77, p = 0.002). Reduction in brain volume also correlated with the change in T1 hypointense lesion volume after treatment (r = 0.53, p < 0.01). A reduction in spinal cord area was also seen throughout the study duration, and this correlated with an increase in disability (r = 0.65, p = 0.01). CONCLUSION: Campath 1H treatment was associated with a sustained and marked reduction in the volume of Gd enhancement, indicating suppression of active inflammation. Nevertheless, many patients developed increasing brain and spinal cord atrophy, T1 hypointensity, and disability. This study highlights the potential role for novel MR techniques in monitoring the effect of treatment on the pathologic process in MS.

Proton MR spectroscopy in clinically isolated syndromes suggestive of multiple sclerosis.

The concentration of the metabolite N-acetyl aspartate (NAA), thought to be a marker of axonal loss or damage, has been shown to be reduced in lesions, as demonstrated by high signal areas on T2-weighted MRI, and in normal-appearing white matter (NAWM) in established multiple sclerosis (MS). The stage of the disease when these changes first appear is not known. To try to determine this we studied 20 patients with clinically isolated syndromes, many of whom will be at the earliest clinical stages of MS, and 20 age- and sex-matched controls with single-voxel proton magnetic spectroscopy (MRS). MRS was performed using a General Electric 1.5T Signa EchoSpeed scanner (TR 3000 ms, TE 30 ms, PRESS). Absolute metabolite concentrations were determined using the LCModel fitting software. No significant reduction of NAA concentration was evident in the NAWM of the patients (patients: median 7.3 mM; controls: median 7.7 mM; P=0.19). There was, however, a significantly lower concentration of NAA in lesions (median 6.6 mM, P=0.015). Absolute values of choline-containing compounds, creatine and myo-inositol were significantly raised in the lesions (P=0.007, P=0.011 and P=0.002 respectively). The low NAA in lesions is consistent with axonal loss, damage or dysfunction occurring focally at the earliest clinical phase of the disease. The lack of any significant reduction in NAA in patient NAWM demonstrates that more widespread axonal changes are not yet detectable at this early clinical stage. A larger cohort and follow-up will be necessary to determine whether or not MRS findings have any prognostic significance for individual patients or sub-groups. This will also enable the clarification of the time course, pathogenesis and pathophysiological significance of the development of the low NAA, which is found in the NAWM of many patients with established MS.

Statistical power of MRI monitored trials in multiple sclerosis: new data and comparison with previous results.

OBJECTIVES: To evaluate the durations of the follow up and the reference population sizes needed to achieve optimal and stable statistical powers for two period cross over and parallel group design clinical trials in multiple sclerosis, when using the numbers of new enhancing lesions and the numbers of active scans as end point variables. METHODS: The statistical power was calculated by means of computer simulations performed using MRI data obtained from 65 untreated relapsing-remitting or secondary progressive patients who were scanned monthly for 9 months. The statistical power was calculated for follow up durations of 2, 3, 6, and 9 months and for sample sizes of 40-100 patients for parallel group and of 20-80 patients for two period cross over design studies. The stability of the estimated powers was evaluated by applying the same procedure on random subsets of the original data. RESULTS: When using the number of new enhancing lesions as the end point, the statistical power increased for all the simulated treatment effects with the duration of the follow up until 3 months for the parallel group design and until 6 months for the two period cross over design. Using the number of active scans as the end point, the statistical power steadily increased until 6 months for the parallel group design and until 9 months for the two period cross over design. The power estimates in the present sample and the comparisons of these results with those obtained by previous studies with smaller patient cohorts suggest that statistical power is significantly overestimated when the size of the reference data set decreases for parallel group design studies or the duration of the follow up decreases for two period cross over studies. CONCLUSIONS: These results should be used to determine the duration of the follow up and the sample size needed when planning MRI monitored clinical trials in multiple sclerosis.

The sensitivity of thin-slice fast spin echo, fast FLAIR and gadolinium-enhanced T1-weighted MRI sequences in detecting new lesion activity in multiple sclerosis.

Fast fluid-attenuated inversion-recovery (FLAIR) and proton density/T2-weighted fast spin echo (FSE) brain images with 3-mm slices were acquired monthly for 7 months in 37 multiple sclerosis patients. New and enlarging lesions were counted and compared according to the site of lesions seen with each sequence. In addition, the number of new enhancing lesions seen on gadolinium-enhanced T1-weighted brain magnetic resonance imaging at the same time points was counted. All sequences used 3-mm contiguous axial slices. Overall, 126 new or enlarging lesions were seen on FSE and 135 on fast FLAIR (P = 0.25, Wilcoxon signed ranks test). Regional comparisons revealed significantly more fast FLAIR lesions only in the cortical/subcortical areas. There was a total of 295 new enhancing lesions over the same period -- a gain in the number of 'active lesions' of 234% seen with FSE and 218% with FLAIR. It is concluded that serial thin slice fast FLAIR is only slightly superior to FSE in detecting new and enlarging multiple sclerosis lesions but the difference is not sufficient to recommend that FLAIR should replace FSE in short-term, exploratory trials in MS using monthly scanning. Gadolinium-enhanced imaging is more then twice as sensitive as either FSE or fast FLAIR to new multiple sclerosis lesion activity, and enhancing lesions should provide the primary outcome measure in such studies.

Visual analysis of serial T2-weighted MRI in multiple sclerosis: intra- and interobserver reproducibility.

We evaluated the effect of consensus formation and training on the agreement between observers in scoring the number of new and enlarging multiple sclerosis (MS) lesions on serial T2-weighted MRI studies. The baseline and month 9 MRI studies of 16 patients with a range of MRI activity were used (dual-echo conventional spin-echo sequence, TR 2000, TE 34 and 90 ms, 5 mm contiguous slices, inplane resolution 1 mm). First, the serial studies were visually analysed for the presence of new and enlarging lesions, on two occasions, by five experienced observers, without adopting any consensus strategy and in isolation. Next, the observers met to identify the common sources of inconsistencies in reporting between observers and formulate consensus rules. Finally, a further independent reading session was performed on the same MRI dataset, this time applying the consensus rules. Agreement between observers was assessed using kappa scores. Without the consensus rules, interobserver kappa scores for the first and second reading sessions for new lesions were only 0.51 and 0.39 respectively; agreement for enlarging lesions was even worse. The mean intraobserver kappa score for new lesions was higher at 0.72, reflecting the fact that the observers were consistently applying their individual assessment strategies. Application of the consensus rules did not lead to a significant improvement in inter observer kappas; the kappa scores adopting the guidelines were 0.46 and 0.21 for new and enlarging lesions respectively. Consensus guidelines thus did not improve the reproducibility of visual analysis of serial T2-weighted MRI, and the level of agreement between observers remained only moderate. Suboptimal repositioning is likely to be a major source of residual variability and this suggests a future role for image registration strategies; until then, a single observer, or pair of observers working in consensus, should be used in MS studies.

The effect of section thickness on MR lesion detection and quantification in multiple sclerosis.

BACKGROUND AND PURPOSE: The purpose of our study was to investigate the effect of section thickness on MR detection of brain lesion volume and measurement precision in patients with multiple sclerosis (MS). METHODS: Eight subjects with known MS were studied on a 1.5-T MR system. We used a 3D fast fluid-attenuated inversion-recovery sequence to obtain contiguous axial brain images at section thicknesses of 5 mm, 3 mm, and 1 mm. Two sets of images were acquired at each section thickness during two sessions, between which the patient was removed from the scanner. Lesion volumes were measured at each section thickness using a semiautomated local thresholding technique. RESULTS: We found that progressive reduction in section thickness led to detection of smaller lesions, resulting in a significant (8%) increase in lesion volume on MR images as section thickness was reduced from 5 mm to 3 mm. However, despite a further increase in lesion detection at a section thickness of 1 mm, this did not result in an increase in total lesion volume. This finding indicates that the relationship between section thickness and lesion volume on MR images is not linear. Scan-rescan reproducibility was improved by reducing section thickness, at the cost of increased analysis time. CONCLUSION: This study shows that acquisition of very thin sections increases the sensitivity and precision of MS lesion measurement. Serial studies assessing lesion changes over time are needed to define the impact of this increase on sample size requirements for MS treatment trials.

Precision and reliability for measurement of change in MRI lesion volume in multiple sclerosis: a comparison of two computer assisted techniques.

OBJECTIVE: The serial quantification of MRI lesion load in multiple sclerosis provides an effective tool for monitoring disease progression and this has led to its increasing use as an outcome measure in treatment trials. Segmentation techniques must display a high degree of precision and reliability if they are to be responsive to small changes over time. This study has evaluated the performance of two such techniques, the manual outlining and contour methods, in serial lesion load quantification. METHODS: Sixteen patients with clinically definite multiple sclerosis were scanned at baseline and after two years. Scan analysis was performed twice, independently by three observers using each technique. RESULTS: For the absolute lesion volumes the median intrarater coefficient of variation (CV) was 3.2% for the contour technique and 7.6% for the manual outlining method (p < 0.005), the interrater CVs were 3.8% and 6.1% respectively (p < 0.01) and the reliability of both techniques was very high. For the change in lesion volume the intrarater and interrater repeatability coefficients were respectively 2.6 cm3 and 2.8 cm3 for the contour technique, and 3.3 cm3 and 3.7 cm3 for the manual outlining method (lower values reflect higher precision). The values for intrarater and interrater reliability for measuring change in lesion volume were respectively, 0.945 and 0.944 for the contour technique, and 0.939 and 0.921 for the manual outline method (perfect reliability = 1.0). CONCLUSIONS: With such high values for reliability, the impact of measurement error in lesion segmentation on sample size requirements in multiple sclerosis treatment trials is minor. This study shows that a change in lesion volume can be measured with a higher level of precision and reliability with the contour technique and this supports its further application in serial studies.

Correlations between monthly enhanced MRI lesion rate and changes in T2 lesion volume in multiple sclerosis.

Magnetic resonance imaging (MRI) provides a powerful tool for assessing disease activity in multiple sclerosis (MS), and its role as a surrogate marker for monitoring treatment efficacy is now becoming established. The most commonly used MRI parameters in treatment trials are (1) monthly gadolinium-enhanced MRI, with the number of active lesions serving as the outcome measure, and (2) annual lesion load quantification, in which change in MS lesion volume provides the MRI endpoint. We evaluated clinical/MRI correlations and the relationship between these two markers of disease activity in 73 patients with clinically definite MS. Quantification of T2 lesion load was performed at study entry and exit, with a median study duration of 11 months (range, 9 to 14 months). Monthly postgadolinium T1-weighted images were acquired between these time points. Lesion load at study entry was significantly correlated with the baseline Expanded Disability Status Scale (EDSS) score, but no significant longitudinal correlation was demonstrated. The number of enhancing lesions on the entry scan was predictive of subsequent relapse rate over the study duration and also correlated with the subsequent enhancing lesion activity over the study period. A significant correlation was found between change in lesion load and disease activity on the monthly scans. Our results suggest that annual lesion load quantification provides an efficient measure of ongoing disease activity, and this supports its application as a surrogate marker of disease evolution in phase III treatment trials.

Relative bioavailability of pilocarpine from a novel ophthalmic delivery system and conventional eyedrop formulations.

Pupillary responses to pilocarpine following topical application in a novel ophthalmic delivery system (NODS) and a conventional eyedrop formulation have been compared in eight healthy subjects in a single dose crossover study. The magnitude of the miotic and light reflex responses to NODS 40, 80, and 170 micrograms and to a single 2% Minims eyedrop (delivering 518 micrograms) were recorded by infrared television pupillography over periods of 24 hours. Dosage comparisons of drug responses were obtained by interpolation and yielded the equivalence of one eyedrop to 67 +/- 11 micrograms pilocarpine from the NODS formulation. These findings indicate that in a comparison of total doses delivered pilocarpine has an approximately eight-fold greater bioavailability from NODS than from a conventional eyedrop formulation.