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Protection of red blood cell acetylcholinesterase by oral huperzine A against ex vivo soman exposure: Next generation prophylaxis and sequestering of acetylcholinesterase over butyrylcholinesterase
Chemico-Biological Interactions 175 (2008) 380–386
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Protection of red blood cell acetylcholinesterase by oral huperzine A against ex vivo soman exposure: Next generation prophylaxis and sequestering of acetylcholinesterase over butyrylcholinesterase Julian R. Haigh a , Scott R. Johnston a , Adam Peppernay a , Patrick J. Mattern a , Gregory E. Garcia a , Bhupendra P. Doctor a , Richard K. Gordon a,∗,1 , Paul S. Aisen b a b
Walter Reed Army Institute of Research, Division of Biochemistry, 503 Robert Grant Road, Silver Spring, MD 20910-7500, USA Department of Neurology, Georgetown University Medical Center, 3800 Reservoir Road NW, Washington, DC 20007, USA
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Article history: Available online 3 May 2008 Keywords: Acetylcholinesterase Alzheimer’s disease Butyrylcholinesterase Huperzine A Pyridostigmine bromide Soman WRAIR whole blood cholinesterase assay
a b s t r a c t As part of a phase Ib clinical trial to determine the tolerability and safety of the highly specific acetylcholinesterase (AChE) inhibitor huperzine A, twelve (12) healthy elderly individuals received an escalating dose regimen of huperzine A (100, 200, 300, and 400 g doses, twice daily for a week at each dose), with three (3) individuals as controls receiving a placebo. Using the WRAIR whole blood cholinesterase assay, red blood cell AChE and plasma butyrylcholinesterase (BChE) were measured in unprocessed whole blood samples from the volunteers following each dose, and then for up to 48 h following the final and highest (400 g) dose to monitor the profile of inhibition and recovery of AChE. Significant inhibition of AChE was observed, ranging from 30–40% after 100 g to >50% at 400 g, and peaking 1.5 h after the last dose. Gradual recovery of AChE activity then occurs, but even 48 h after the last dose red blood cell AChE was about 10% below control (pre-dose) values. Huperzine A levels in plasma peaked 1.5 h after the final 400 g dose (5.47 ± 2.15 ng/mL). Plasma BChE was unaffected by huperzine A treatment (as expected). Aliquots of huperzine A-containing (from three individuals) and placebo blood samples were exposed ex vivo to the irreversible nerve agent soman (GD) for 10 min, followed by removal of unbound huperzine and soman from the blood by passing through a small C18 reverse phase spin column. Eluted blood was diluted in buffer, and aliquots taken at various time intervals for AChE and BChE activity measurement to determine the time taken to achieve full return in activity of the free enzyme (dissociation from the active site of AChE by huperzine A), and thus the proportion of AChE that can be protected from soman exposure. Huperzine A-inhibited red blood cell (RBC) AChE activity was restored almost to the level that was initially inhibited by the drug. The increased doses of huperzine A used were well tolerated by these patients and in this ex vivo study sequestered more red blood cell AChE than has been previously demonstrated for pyridostigmine bromide (PB), indicating the potential improved prophylaxis against organophosphate (OP) poisoning. Published by Elsevier Ireland Ltd
1. Introduction ∗ Corresponding author. Tel.: +1 301 319 9987; fax: +1 301 319 9571. E-mail address: [email protected] (R.K. Gordon). 1 The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the U.S. Department of the Army or the Department of Defense. 0009-2797/$ – see front matter. Published by Elsevier Ireland Ltd doi:10.1016/j.cbi.2008.04.033
Red blood cell acetylcholinesterase and serum butyrylcholinesterase (BChE) can be used as sensitive biomarkers of cholinergic function in the central and peripheral nervous systems. In response to organophosphate (OP) chemical warfare agent (CWA’s) or pesticide exposure, and
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in several neurodegenerative diseases such as Alzheimer’s, there is a selective reduction in AChE or BChE activity which can be monitored by several sensitive assays employed in clinical and research laboratories [1,2]. For these studies, we used the WRAIR whole blood cholinesterase assay. Huperzine A, a reversible and highly specific acetylcholinesterase (AChE) inhibitor in both the peripheral and central nervous systems [3,4], is a natural alkaloid isolated from the Chinese moss Huperzia serrata. In China it is used as a traditional herbal medicine and a number of randomized controlled clinical trials have demonstrated that huperzine A can enhance cognitive function in individuals with memory impairments or dementia [5,6]. In China huperzine A is currently in a phase IV trial as an anti-Alzheimer’s drug. In most cases, the drug was well tolerated (dizziness, nausea, and gastrointestinal side effects were generally mild). Several preclinical studies have demonstrated that huperzine A may have some advantages over the currently used AChE inhibitors (e.g., donepezil or rivastigmine) for the treatment of Alzheimer’s disease (AD) as it is highly selective for AChE, exhibits good CNS penetration, and lacks significant cholinergic toxicity [6–10]. In addition, huperzine A can exert neuroprotective effects against glutamate toxicity by direct antagonism of NMDA receptors in rat neuronal cultures [11]. In addition, clinical trials with huperzine A in China have demonstrated a significant reduction in blood markers of oxidative stress (including plasma and erythrocyte lipoperoxides), suggesting a systemic antioxidant effect [12]. Outside China, studies to assess huperzine A safety and toxicity have been limited, and huperzine A is currently only allowed for sale in the US as an over-the-counter ‘nutraceutical’ supplement for memory enhancement. However, in the US it is being evaluated for safety and efficacy in a phase II trial as a therapy for AD (http://www.clinicaltrials.gov; Identifier: NCT00083590). In humans, huperzine A has a relatively long half-life, with T1/2 values of 5–6 h [13], and functions to reversibly sequester the active site of AChE. When exposed to lethal doses of OP nerve agents such as soman (the treatment options for which are extremely limited as a result of rapid ‘aging’ of the inhibited AChE), huperzine A can cross the blood brain barrier and protects brain AChE from excessive acetylcholine leading to seizures, neuropathological damage, and death. For example, high doses of huperzine A (500 g/kg), which reversibly inhibits blood and brain AChE by 60–70%, can significantly reduce soman (GD) lethality (protective ratios of 2–3) [14]. Lower, subchronic doses of huperzine A (yielding 20–30% inhibition of RBC-AChE) has little or no affinity for muscarinic or NMDA receptors and does not induce neuropathological damage in guinea pig hippocampus. Although there is compelling evidence of huperzine A’s ability to enhance cognitive function in humans, there is currently relatively little quantitative data on the protective effects of huperzine A pretreatments on human AChE in blood, peripheral tissue (e.g., diaphragm) and the CNS. Since 2003, pyridostigmine bromide (PB) has been FDAapproved as the U.S. Army’s pretreatment against potential
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soman exposure. PB (given as a single 30 mg oral dose every 8 h) provides temporary protection (by carbamylation) of peripheral tissue and red blood cell AChE, but is limited as it is fairly short acting, and cannot penetrate the blood brain barrier (BBB) to protect the CNS against seizures and subsequent neuropathological states induced by nerve agent exposure. We have previously shown [15] that oral administration of PB in human volunteers can protect a critical proportion (30–40%) of red blood cell AChE from irreversible inhibition by ex vivo addition of soman. We also showed that huperzine A specifically inhibits red blood cell AChE significantly (50–60%) and without serious side effects when given up to 200 g twice daily in normal elderly individuals [15]. As a pretreatment, huperzine A is a specific and highly selective inhibitor of red blood cell AChE with serum BChE being unaffected (thus preserving the well-documented OP bioscavenging capability of serum BChE) [16,17]. This lack of inhibition of BChE represents an additional advantage of huperzine A being used for prevention of OP toxicity, and helps to explain in part the increase in tolerance of huperzine A-pretreated animals to the lethal effects of soman in comparison to animals pretreated with PB. In this report, we have extended our evaluation of huperzine A, using increased oral doses ranging from 100 g (bid) to 400 g (bid) as part of a clinical study to determine the maximal tolerated dose (MTD) of huperzine A in healthy elderly human volunteers. In view of the fact that huperzine A exhibits good CNS penetration, the questions we have posed are (i) does ex vivo soman exposure in human whole blood containing huperzine A demonstrate adequate protection of red blood cell AChE from soman toxicity? (ii) from a military perspective, would pretreatment by huperzine A (in addition to post-exposure treatment with atropine and an oxime) represent an improved strategy against CWA exposure (and crucially protecting the CNS) that is currently ascribed for pyridostigmine bromide? 2. Methods 2.1. Human subjects and dosing with huperzine A The study design consisted of fifteen randomly assigned healthy elderly volunteers. Twelve (12) individuals were given an escalating dose regimen (orally, twice daily) of 100 g of huperzine A in week 1, followed by increasing doses of 200 g (week 2), 300 g (week 3), and finally 400 g in week 4. Three (3) individuals served as controls and received a placebo. Aliquots of whole blood were collected by a phlebotomist using 3 mL vacutainers containing sodium heparin and labeled with randomly generated identification numbers. Blood samples were collected at various time intervals for RBC-AChE and plasma BChE activity determination before (pre-dose), and after dosing with huperzine A (on day 1 and day 3 of each week). After the highest (400 g) and final dose was given in week 4, blood was withdrawn at various time intervals up to 48 h to monitor the profile of AChE and BChE activity. Samples were flash frozen on dry ice, sent to the Division of Biochemistry, Walter Reed Army Institute of Research (WRAIR) for storage
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at −80 ◦ C prior to the WRAIR whole blood cholinesterase assay. 2.2. Measurement of red blood cell AChE and plasma BChE in huperzine A-containing samples using the WRAIR whole blood (WB) assay Red blood cell AChE and plasma BChE were measured simultaneously in 10 L aliquots of thawed human blood diluted 20-fold in distilled water, as previously described in detail [16]. Briefly, the final assay mixture (300 L in each well of a 96-well microtiter plate) also contained (in separate wells): 1 mM each of acetylthiocholine iodide (ATCh), propionylthiocholine iodide (PTCh), and butyrylthiocholine iodide (BTCh); all wells also contained 0.2 mM of the chromophore 4,4 -dithiodipyridine (DTP), and 50 mM sodium phosphate buffer, pH 8.0. ChE activity was determined colorimetrically at 324 nm (UV) at 25 ◦ C on each plate using a Molecular Devices SpectraMax Plus384 microtiter spectrophotometer (Sunnyvale, CA), and was automated using a bench top robotic platform (Biomek 2000, Beckman-Coulter, Fullerton, CA). After a 4 min kinetic assay endpoint values were determined at 415 and 445 nm for sample normalization. Data (mAbs change/min) were subjected to linear least squares analysis, and AChE and BChE activities (expressed as U/mL and run in duplicate or triplicate) were calculated using SoftMax v5.0 and an Excel spreadsheet. 2.3. Measurement of protection afforded to RBC-AChE by huperzine A after ex vivo soman treatment To evaluate the protection afforded to RBC-AChE by huperzine A, blood samples stored at −80 ◦ C were thawed and then exposed to the nerve agent soman. These experiments were performed at USAMRICD, Aberdeen Proving Ground, MD. To each 70 L blood sample in a PCR tube, 1 M (final concentration) of GD in 0.9% NaCl was added for 10 min at room temperature. Free huperzine A and GD were removed from the blood by using small (6 cm) chromatography spin columns (Bio-Rad, Hercules, CA) containing 300 mg of C18 (Waters, Milford, MA) and a small plug of cotton wool on top. Columns were washed with 3 mL × 1 mL of methanol, and rinsed with 3 mL × 1 mL deionized water under a low vacuum. After adding 2.5 L of saponin (50%, w/v) to the blood and vortexing for 30 s, two 10 L aliquots were removed for measurement of AChE and BCE activity, and 50 L of the remaining lysed whole blood was placed on the cotton wool plug and the column was centrifuged at 1000 × g for 2 min (see Fig. 1). About 90% of the added volume was recovered after centrifugation, and 10 L aliquots were removed for AChE and BChE measurement immediately post-column. A 30 L post-column sample was diluted 100-fold in 50 mM sodium phosphate buffer, pH 8.0, left on the bench at room temperature for 3 h, and aliquots removed for AChE and BChE activity measurement to determine full return in activity (dissociation of huperzine A from the active site of AChE), and thus the proportion of RBC-AChE that can be protected from OP exposure. Post-column (100-fold diluted) blood samples were assayed for ChE activity as described above,
Fig. 1. Spin column chromatography for rapid and efficient removal of huperzine A and soman from hemolysed human blood. Bio-Spin columns contained 300 mg of C18 with a small cotton wool plug on top. AChE and BChE are not retained in the column matrix and recovery of inhibited AChE is measured after dilution of the eluted blood using the WRAIR whole blood ChE assay.
with the appropriate dilution factor correction taken into account. The % recovery of ChE activity is calculated as follows: (i) % inhibited (huperzine A or placebo samples) = 100 × (ChE U/mL of samples at times post-dosing)/(ChE U/mL pre-dosing). After ex vivo exposure to GD and removal of free GD and huperzine A by the spin column, recovery of ChE activity is calculated as follows: (ii) % recovery (huperzine A or placebo samples exposed to GD) = 100 × (ChE U/mL of GD-treated samples at times post-dosing)/(ChE U/mL pre-dosing). 2.4. Liquid chromatography–mass spectrometry (LC–MS) assays of huperzine A concentrations in human plasma Huperzine A was extracted from clarified human plasma samples by solid phase extraction (SPE) using Strata-C 96-well plates (Phenomenex, Inc.). Samples in wells were washed sequentially with 1 mM HCL and 50:50 (v/v) methanol:acetonitrile, and huperzine A eluted with 2 mL × 0.9 mL of 47.5:47.5:5 (v/v/v) methanol:acetonitrile:ammonium hydroxide. Elutes were transferred to 2 mL screw-capped bullet tubes, concentrated by lyophilization, and dried samples stored at −30 ◦ C.
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Huperzine A concentrations were determined by LC–MS (Beckman 128 pumps, Leap PAL temperature-controlled autosampler (5 ± 3 ◦ C), and a ZQ single-quadrupole MS (Waters, Inc.). The eluting solutions were buffer A (BA): 50 mM ammonium acetate pH 4.5 and buffer B (BB): 150 mM ammonium acetate pH 4.5 in 50% (v/v) acetonitrile and 40% water. Huperzine A was separated using a HS F5 HPLC column, 2.1 mm × 50 mm, (Supelco Inc.) at a flow rate of 0.2 mL/min, consisting of a gradient of 98% BA/2% BB for 1 min followed by a linear increase of BB to 60% for 10 min. Lyophilized samples were reconstituted in 120 L of BA, mixed, and clarified by centrifugation at 14,500 rpm for 1 min at 4 ◦ C. Sample (100 L) was injected on to the column and MS run in positive electrospray ionization (ESI) mode. The source capillary temperature was 250 ◦ C with 500 L h−1 N2 (g) desolvation gas. The cone parameters were 120 ◦ C, 35 V, and 50 L h−1 N2 . Huperzine A was detected by selected ion monitoring of the (M+H) ion (m/z 243). Huperzine A plasma concentrations were determined by comparison of peak-area-under-the-curve values for 243 m/z for matrix matched control huperzine A-spiked (to 1.33 g/mL) and then serially diluting with human pooled control plasma with final concentrations of 6.66, 13.3, 133, 1,333 ng/mL (Interstate Blood Bank, Inc., TN). Plasma huperzine A concentrations are given as ng/mL, (mean ± S.E. for n individual determinations). Standard (−)huperzine A stock solutions (Calbiochem Inc., San Diego, CA) were prepared as 1 mg/mL in 5 mM HCl and 50 mM sodium phosphate buffer, pH 8.0, clarified by centrifugation and stored at −80 ◦ C. Huperzine A concentrations were determined by absorbance using a molar extinction coefficient of 1.047 × 104 M/cm at 307 nm. 3. Results 3.1. Inhibition and recovery of RBC-AChE activity after oral huperzine A and ex vivo soman exposure: removal of huperzine A and soman from blood using a small spin column In this study, we have analyzed blood AChE (and BChE) activities in human blood from volunteers receiving up to 400 g huperzine A twice daily, as part of a larger clinical study to evaluate the safety and tolerability of increasing doses of huperzine A in healthy elderly individuals. Huperzine A reversibly binds to AChE, and thereby protects the enzyme from reaction with OPs. The activity of the huperzine A-protected but inhibited AChE will be restored once the drug–AChE complex spontaneously dissociates, which occurs after soman is removed from the blood. We have demonstrated this using rapid centrifugation of the soman and huperzine A-containing blood samples through a small C18 column to remove any free soman and drug, which bind to the column matrix while allowing 97–100% of the AChE and BChE activity to pass through. Under these circumstances, any RBC-AChE not protected by huperzine A would be irreversibly inhibited by soman. In contrast, the RBC-AChE protected by huperzine A would spontaneously dissociate over time, and this enzyme’s activity would be restored. In the example shown in Fig. 2, the solid bars represent RBC-AChE without any huperzine A (con-
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Fig. 2. Effects of soman (GD) on RBC-AChE activity in whole blood after 400 g huperzine A taken orally. Control (pre-dose) RBC-AChE (solid bar) is taken as the 100% value (3.34 ± 0.6 U/mL AChE activity, mean ± S.D., n = 3). AChE is maximally inhibited by 60%, 1.5 h post-huperzine A dose (speckled bar, arrow). After GD exposure, huperzine A and GD were removed using a small C18 spin column, as described in Section 2 (postcolumn treatment). The huperzine A-protected AChE recovery is complete within 3 h post-column. Hatched bars show the subsequent return of AChE activity of GD exposed blood due to dissociation of the protected enzyme after huperzine A and GD removal (3 h post-column arrow). Thus, the % difference between the pre-dose control (arrow, pre-dose control) and the huperzine A-inhibited control (arrow, speckled, 400 g huperzine A; 60% inhibition) is close to the % recovery of GD-inhibited RBC-AChE protected by huperzine A (arrow, hatched bar; 55% AChE activity recovered).
trol, pre-dose), while the stippled bar represents AChE from an individual that received a 400 g dose of huperzine A (60 ± 1.1% inhibition). In the first part, after soman treatment, no AChE activity is observed by the WRAIR assay in either the control or huperzine A treated volunteers (solid bars close to 0 U/mL). However, after the spin column removal of uncomplexed soman and huperzine A, dilution of the eluted blood sample and a 3 h period to allow for complete dissociation, the AChE is almost restored to the level that was initially inhibited by huperzine A (60% inhibition by huperzine A before the column vs. 55% returned AChE activity post-column in this individual; Fig. 2). Even after 3 h post-column recovery of AChE, the pre-dose control (soman exposed but not huperzine A treated) sample remains irreversibly inhibited (Fig. 2). These results definitively demonstrate that huperzine A is highly effective in protecting a significant proportion of RBC-AChE from ex vivo soman exposure. 3.2. Huperzine A protection and recovery of AChE activity in blood following ex vivo soman exposure Using the spin column technique described above, we have analyzed the complete profile of inhibition and recovery (protection by huperzine A) of RBC-AChE in three out of the twelve subjects receiving huperzine A, and the three placebo’s, over the full duration of the clinical trial, from
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pre-dose (control), through the incremental huperzine A dose increases over four weeks, and finally the 48 h lag phase following the final (and highest) 400 g dose. These results are shown in Fig. 3. Most of the decrease in RBCAChE (30–40%) occurs during week 1 (100 g twice daily), with further declines to a maximum of 48 ± 5% of control during week 4 (400 g twice daily) (solid line in Fig. 3). Following the last dose, recovery of RBC-AChE is followed for the next 48 h, and as Fig. 3 shows, significant inhibition persists for at least a further 12 h (53 ± 4% after 1 h, 54 ± 3% after 2 h, and even 44 ± 4% after 12 h post-dose). The gradual recovery of RBC-AChE activity continues but even 48 h after the last dose red blood cell AChE was still ∼10% below control (pre-dose) values (see Section 3.4, below). Fig. 3 also shows the time required to achieve full return in RBC-AChE activity (dissociation from the active site of AChE by huperzine A), and thus the proportion of red blood cell AChE that can be protected from OP exposure, after these samples were passed through the spin column to remove free huperzine A and soman (dashed line in Fig. 3). Huperzine A-inhibited AChE was restored almost to the level that was initially inhibited by the drug, and this is demonstrated by the line (solid triangles) at the bottom of Fig. 3 which represents the overall difference in the % AChE inhibition pre-column vs. % returned AChE activity post-column across all the time points. RBC-AChE returns to within 2.3% of the control (pre-column) activity level (labeled , delta, in Fig. 3). Finally, Fig. 3 also demonstrates the RBC-AChE activities in three placebo samples (top line, solid diamonds) remain constant at 95–100% of control values, as expected for
individuals that did not receive any oral doses of huperzine A for the duration of this study. Placebo samples exposed to soman (i.e., with no huperzine A present to protect AChE) and passed through the spin column to remove soman remained irreversibly inhibited at all time points (>95%, not shown). 3.3. Plasma BChE levels after huperzine A dosing In the same blood samples and doses of huperzine A (three individuals) or placebo (three individuals), we have also followed the effects on plasma BChE activities. As shown in Fig. 4 (top lines) plasma BChE is completely unaffected by huperzine A and remains at 98 ± 3% of control activity at all dosing and sampling time points. After treatment with soman (which completely inhibits BChE) and passage through the spin column to remove free soman and huperzine A, plasma BChE activities were inhibited by 86 ± 4% of control (pre-column) activity values, and were similar to the BChE in the placebo samples exposed to soman (which were inhibited by >90%). 3.4. Huperzine A concentrations in human plasma Huperzine A concentrations in plasma were analyzed by HPLC/MS and measured immediately after the first daily 200 g dose during week 2, and then after the final 400 g dose at the end of the study period. After the 200 g dose, the huperzine A concentration in plasma was 2.54 ± 1.92 ng/mL (n = 11). Immediately after the last (400 g) dose, the huperzine A concentration in plasma was 4.64 ± 1.28 ng/mL (n = 8), peaking at 5.47 ± 2.15 ng/mL
Fig. 3. Complete profile of inhibition and recovery of RBC-AChE after four weeks of an increasing dose regimen of huperzine A in three human volunteers and three placebos. Control (pre-dose) RBC-AChE is taken as the 100% value (3.43 ± 0.6 U/mL AChE activity, mean ± S.D., n = 6). Arrows (top) show huperzine A dose, and although given twice daily, only AChE activities on day 1 and day 3 of each week are shown (vertical dashed lines). Note that this figure is not to scale with left side representing weekly treatments, and right side time elapsed (in hours) after last dose. Solid triangles (bottom line) represent the overall difference in the % AChE inhibition pre-column vs. % returned AChE activity post-column across all the time points. RBC-AChE activity returns to within 2.3% of the huperzine A-treated (but not GD exposed) activity level.
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Fig. 4. Profile of plasma BChE after four weeks of an increasing dose regimen of huperzine A in three human volunteers and three placebos. Control (predose) plasma BChE is taken as the 100% value (1.74 ± 0.35 U/mL BChE activity, mean ± S.D., n = 6). Same legend as Fig. 3 for huperzine A dosing. Huperzine A has no effect on plasma BChE levels and there was no difference between pre- and post-column activity (BChE recovery huperzine A-treated or BChE recovery, placebo); not shown.
(n = 10) 1 h post-dose. A residual amount of huperzine A was barely detectable within the limits of the method (1.1 ± 0.9 ng/mL, n = 10) at 48 h post-dose (corresponding to about 10% inhibition of RBC-AChE, see Fig. 3), but the AChE activity is within a few percent of the placebo activity after 48 h. It is clear that huperzine A is a potent RBCAChE inhibitor since smaller concentrations of the drug were required to achieve greater RBC-AChE inhibition than shown previously with PB [15]. 4. Discussion and conclusions It is now well established that huperzine A, a reversible and highly selective AChE inhibitor isolated from the Chinese moss Huperzia serrata and used in traditional Chinese herbal medicine, is well tolerated in humans and animals. Animal and human clinical studies with huperzine A have demonstrated rapid absorption, high bioavailability, long biological half-life, and efficient penetration of the blood brain barrier to protect the central nervous system. In China, clinical trials of healthy individuals and patients with various neurological disorders have indicated that huperzine A is safe and well tolerated. Outside China, there have been very few controlled studies to assess its safety and toxicity. In the US, huperzine A is presently undergoing clinical trials to determine safety and efficacy as a potential treatment for Alzheimer’s disease, and is presently only approved as an over-the-counter nutritional supplement for memory enhancement. In a previous study [15], we have shown that huperzine A is a more potent inhibitor of RBC-AChE than PB, another reversible AChE inhibitor that has been specifically FDAapproved for military personnel as a pretreatment against soman, a potent and irreversible OP nerve agent. We showed that a 200 g dose of huperzine A in human
volunteers can protect a significant proportion (30–40%) of red blood cell AChE from inhibition from soman measured in an ex vivo assay using the WRAIR whole blood cholinesterase method, and that the AChE inhibition is fairly long lasting. The WRAIR ChE assay can quickly determine the profile of AChE (and BChE) in whole blood samples using high throughput screening and can be used to monitor exposure to nerve agents, pesticides, and medically useful drugs (e.g., anti-cholinergics) in the treatment of neurological diseases [15]. In this work, the effects of even higher oral doses of huperzine A on red blood cell AChE in humans have been described using increased oral doses ranging from 100 g (bid) to 400 g (bid) over a four week period, as part of a clinical study to determine the tolerability and safety of huperzine A in healthy elderly human volunteers. This step-wise increase in huperzine A at weekly intervals, from 100 g (bid) to 400 g (bid) was employed to mitigate the occurrence of gastrointestinal side effects. This high oral dose produced 50–60% inhibition of RBC-AChE (Fig. 3), with peak inhibition of AChE and plasma huperzine A concentrations occurring 1 h after the last huperzine A dose. This effect persists for several hours followed by a gradual dissociation of the huperzine A–AChE complex and restoration of AChE activity, although, as Fig. 3 shows, AChE activity is still inhibited by 50% after 12 h, and more than 20% inhibition remains even after 48 h post-dose. This long duration of action is a result of the tightly bound complex which forms between huperzine A and AChE [4] and the pharmacokinetics of huperzine A in vivo; the reported half-life of huperzine A given orally in humans was 4.8 h, with peak blood levels of huperzine A being reached after 80 min [13]. Similarly, there is a long duration (6 h) of AChE inhibition in rat brain [10]. In a recent study of 18 healthy male Chinese volunteers given a single 0.2 mg (200 g) oral dose of
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huperzine A [18], the maximal concentration (Cmax ) measured in plasma was 2.47 ± 0.47 ng/mL and reached a peak level after 80 min. Using a sensitive HPLC/MS method, very similar results for huperzine A plasma concentrations were observed in the elderly volunteers in this study (see Section 3). Recently, real time measurement of AChE activity using immobilized human red blood cells as an in vitro model after pretreatment inhibition (50%) with pyridostigmine bromide and huperzine A, demonstrated lower levels AChE recovery following subsequent exposure to soman for 30 min (28.5% and 7% with PB and huperzine A, respectively) [19]. In this assay recovery of AChE activity following disassociation of huperzine A from the active site of AChE is very rapid (t1/2 = 4.6 min) compared with the decarbamylation rate of PB (t1/2 = 27 min), and thus the amount of sequestered AChE after removal of huperzine A is rapidly diminished because of the significantly longer soman exposure time (30 min) and ‘aging’ [20]. However, a longer dissociation rate (∼60 min) for huperzine A has been reported [3]. Likewise, in Fig. 2 we showed very rapid recovery of AChE activity following complete removal of huperzine A and soman from red blood cells using the spin column, but are able to demonstrate much higher sequestering (protection) of the enzyme (after 3 h) because of the lack of soman in the post-column eluate for irreversible inhibition and ‘aging’. The inhibition of RBC-AChE persists for significantly longer (∼45% inhibition after 6 h; ∼25% inhibition after 24 h post-dose, Fig. 3) than huperzine A levels in the plasma. Based on huperzine A’s half-life in plasma and its tight binding to the enzyme [4], (Fig. 2 demonstrated that full recovery of full AChE activity only occurs after 3 h (following removal of any free huperzine A bound to the C18 spin column. As Fig. 3 also shows, a residual amount of inhibition (∼10%) is a value almost indistinguishable from the placebo. Therefore, it is likely that complete recovery of RBC-AChE is apparent at 48 h post-dose. The observed lack of inhibition on BChE (Fig. 3) represents an additional advantage of huperzine A being used for prevention of OP toxicity, and helps to explain the increase in tolerance of huperzine A-pretreated animals to the lethal effects of soman in comparison to animals pretreated with PB. 5. Conclusions Huperzine A is a potential second-generation drug for prophylaxis against organophosphate poisoning. Unlike PB, huperzine A can protect the CNS from the anti-cholinergic effects of OP’s and may have fewer side effects. Given the potential rise in urban terrorism that may include the use of chemical warfare organophosphate agents, Federal, State, and local authorities need a fast acting, inexpensive, and potent pretreatment measure that can protect both CNS and peripheral AChE. Acknowledgements We would like to thank Dr. Benedict Capacio, USAMRICD, Aberdeen Proving Ground, MD, for permission to perform exposure of human blood samples to soman (GD).
The studies were in compliance with WRAIR Human Use Protocol (No. 1242), and the Georgetown University Human Use Review Committee. References [1] P. Taylor, Anticholinesterase agents, in: L.L. Brunton, J.S. Lazo, K.L. Parker (Eds.), Goodman and Gilman’s The Pharmacological Basis of Therapeutics, eleventh edition, McGraw-Hill, New York, 2006, pp. 201–216. [2] H.N. Nigg, J.B. Knaak, Blood cholinesterases as human biomarkers of organophosphorus pesticide exposure, Rev. Environ. Contam. Toxicol. 163 (2000) 29–111. [3] Y. Ashani, J.O. Peggins, B.P. Doctor, Mechanism of inhibition of cholinesterases by huperzine A, Biochem. Biophys. Res. Commun. 30 (184) (1992) 719–726. [4] M.L. Raves, M. Harel, Y.P. Pang, I. Silman, A.P. Kozikowski, J.L. Sussman, Structure of acetylcholinesterase complexed with the nootropic alkaloid, (−)-huperzine A, Nat. Struct. Biol. 4 (1997) 57–63. [5] R.W. Zhang, X.C. Tang, Y.Y. Han, G.W. Sang, Y.D. Zhang, Y.X. Ma, C.L. Zhang, R.M. Yang, Drug evaluation of huperzine A in the treatment of senile memory disorders, Chung Kuo Yao Li Hsueh Pao 12 (1991) 250–252. [6] J.T. Little, S. Walsh, P.S. Aisen, An update on huperzine A as a treatment for Alzheimer’s disease, Expert. Opin. Investig. Drugs 17 (2) (2008) 209–215. [7] H. Jiang, X. Luo, D. Bai, Progress in clinical, pharmacological, chemical and structural biological studies of huperzine A: a drug of traditional medicine origin for the treatment of Alzheimer’s disease, Curr. Med. Chem. 10 (21) (2003) 2231–2252. [8] D.H. Cheng, X.C. Tang, Comparative studies of huperzine A, E2020, and tacrine on behavior and cholinesterase activities, Pharmacol. Biochem. Behav. 60 (1998) 377–386. [9] T. Wang, X.C. Tang, Reversal of scopolamine-induced deficits in radial maze performance by (−)-huperzine A: comparison with E2020 and tacrine, Eur. J. Pharm. 349 (2–3) (1998) 137–142. [10] D. Bai, Development of huperzine A and B for treatment of Alzheimer’s disease, Pure Appl. Chem. 79 (4) (2007) 469–479. [11] R.K. Gordon, S.V. Nigam, J.A. Weitz, J.R. Dave, B.P. Doctor, H.S. Ved, The NMDA receptor ion channel: a site for binding of huperzine A, J. Appl. Toxicol. 21 (Suppl. 1) (2001) S47–S51. [12] S.S. Xu, Z.Y. Cai, Z.W. Qu, Huperzine-A in capsules and tablets for treating patients with Alzheimer’s disease, Acta Pharmacol. Sin. 20 (1999) 486–490. [13] B.C. Qian, M. Wang, Z.F. Zhou, K. Chen, R.R. Zhou, G.S. Chen, Pharmacokinetics of tablet huperzine A in six volunteers, Chung Kuo Yao Li Hsueh Pao 16 (1995) 396–398. [14] G. Lallement, V. Baille, D. Baubichon, P. Carpentier, J.M. Collombet, P. Filliat, A. Foquin, E. Four, C. Masqueliez, G. Testylier, L. Tonduli, F. Dorandeu, Review of the value of huperzine A as pretreatment of organophosphate poisoning, Neurotoxicology 23 (2002) 1–5. [15] R.K. Gordon, J.R. Haigh, G.E. Garcia, S.R. Feaster, M.A. Riel, D.E. Lenz, P.S. Aisen, B.P. Doctor, Oral administration of pyridostigmine bromide and huperzine A protects human whole blood cholinesterases from ex vivo exposure to soman, Chem. Biol. Interact. 157–158 (2005) 239–246. [16] B.P. Doctor, L. Raveh, A.D. Wolfe, D.M. Maxwell, Y. Ashani, Enzymes as pretreatment drugs for organophosphate toxicity, Neurosci. Biobehav. Rev. 15 (1) (1991) 123–128. [17] O. Cohen, C. Kronman, L. Raveh, O. Mazor, A. Ordentlich, A. Shafferman, Comparison of polyethylene glycol-conjugated recombinant human acetylcholinesterase and serum human butyrylcholinesterase as bioscavengers of organophosphate compounds, Mol. Pharmacol. 70 (3) (2006) 1121–1131. [18] W. Li, J. Li, Q. Hu, Determination of huperzine A in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study on Chinese volunteers, Biomed. Chromatogr. 22 (4) (2008) 354–360. [19] S. Eckert, P. Eyer, F. Worek, Reversible inhibition of acetylcholinesterase by carbamates or huperzine A increases residual activity of the enzyme upon soman challenge, Toxicology 233 (2007) 180–186. ¨ [20] S. Eckert, P. Eyer, H. Muckter, F. Worek, Kinetic analysis of the protection afforded by reversible inhibitors against irreversible inhibition of acetylcholinesterase by highly toxic organophosphorus compounds, Biochem. Pharmacol. 72 (2006) 344–357.
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Protection of red blood cell acetylcholinesterase by oral huperzine A against ex vivo soman exposure: Next generation prophylaxis and sequestering of acetylcholinesterase over butyrylcholinesterase Julian R. Haigh a , Scott R. Johnston a , Adam Peppernay a , Patrick J. Mattern a , Gregory E. Garcia a , Bhupendra P. Doctor a , Richard K. Gordon a,∗,1 , Paul S. Aisen b a b
Walter Reed Army Institute of Research, Division of Biochemistry, 503 Robert Grant Road, Silver Spring, MD 20910-7500, USA Department of Neurology, Georgetown University Medical Center, 3800 Reservoir Road NW, Washington, DC 20007, USA
a r t i c l e
i n f o
Article history: Available online 3 May 2008 Keywords: Acetylcholinesterase Alzheimer’s disease Butyrylcholinesterase Huperzine A Pyridostigmine bromide Soman WRAIR whole blood cholinesterase assay
a b s t r a c t As part of a phase Ib clinical trial to determine the tolerability and safety of the highly specific acetylcholinesterase (AChE) inhibitor huperzine A, twelve (12) healthy elderly individuals received an escalating dose regimen of huperzine A (100, 200, 300, and 400 g doses, twice daily for a week at each dose), with three (3) individuals as controls receiving a placebo. Using the WRAIR whole blood cholinesterase assay, red blood cell AChE and plasma butyrylcholinesterase (BChE) were measured in unprocessed whole blood samples from the volunteers following each dose, and then for up to 48 h following the final and highest (400 g) dose to monitor the profile of inhibition and recovery of AChE. Significant inhibition of AChE was observed, ranging from 30–40% after 100 g to >50% at 400 g, and peaking 1.5 h after the last dose. Gradual recovery of AChE activity then occurs, but even 48 h after the last dose red blood cell AChE was about 10% below control (pre-dose) values. Huperzine A levels in plasma peaked 1.5 h after the final 400 g dose (5.47 ± 2.15 ng/mL). Plasma BChE was unaffected by huperzine A treatment (as expected). Aliquots of huperzine A-containing (from three individuals) and placebo blood samples were exposed ex vivo to the irreversible nerve agent soman (GD) for 10 min, followed by removal of unbound huperzine and soman from the blood by passing through a small C18 reverse phase spin column. Eluted blood was diluted in buffer, and aliquots taken at various time intervals for AChE and BChE activity measurement to determine the time taken to achieve full return in activity of the free enzyme (dissociation from the active site of AChE by huperzine A), and thus the proportion of AChE that can be protected from soman exposure. Huperzine A-inhibited red blood cell (RBC) AChE activity was restored almost to the level that was initially inhibited by the drug. The increased doses of huperzine A used were well tolerated by these patients and in this ex vivo study sequestered more red blood cell AChE than has been previously demonstrated for pyridostigmine bromide (PB), indicating the potential improved prophylaxis against organophosphate (OP) poisoning. Published by Elsevier Ireland Ltd
1. Introduction ∗ Corresponding author. Tel.: +1 301 319 9987; fax: +1 301 319 9571. E-mail address: [email protected] (R.K. Gordon). 1 The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the U.S. Department of the Army or the Department of Defense. 0009-2797/$ – see front matter. Published by Elsevier Ireland Ltd doi:10.1016/j.cbi.2008.04.033
Red blood cell acetylcholinesterase and serum butyrylcholinesterase (BChE) can be used as sensitive biomarkers of cholinergic function in the central and peripheral nervous systems. In response to organophosphate (OP) chemical warfare agent (CWA’s) or pesticide exposure, and
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in several neurodegenerative diseases such as Alzheimer’s, there is a selective reduction in AChE or BChE activity which can be monitored by several sensitive assays employed in clinical and research laboratories [1,2]. For these studies, we used the WRAIR whole blood cholinesterase assay. Huperzine A, a reversible and highly specific acetylcholinesterase (AChE) inhibitor in both the peripheral and central nervous systems [3,4], is a natural alkaloid isolated from the Chinese moss Huperzia serrata. In China it is used as a traditional herbal medicine and a number of randomized controlled clinical trials have demonstrated that huperzine A can enhance cognitive function in individuals with memory impairments or dementia [5,6]. In China huperzine A is currently in a phase IV trial as an anti-Alzheimer’s drug. In most cases, the drug was well tolerated (dizziness, nausea, and gastrointestinal side effects were generally mild). Several preclinical studies have demonstrated that huperzine A may have some advantages over the currently used AChE inhibitors (e.g., donepezil or rivastigmine) for the treatment of Alzheimer’s disease (AD) as it is highly selective for AChE, exhibits good CNS penetration, and lacks significant cholinergic toxicity [6–10]. In addition, huperzine A can exert neuroprotective effects against glutamate toxicity by direct antagonism of NMDA receptors in rat neuronal cultures [11]. In addition, clinical trials with huperzine A in China have demonstrated a significant reduction in blood markers of oxidative stress (including plasma and erythrocyte lipoperoxides), suggesting a systemic antioxidant effect [12]. Outside China, studies to assess huperzine A safety and toxicity have been limited, and huperzine A is currently only allowed for sale in the US as an over-the-counter ‘nutraceutical’ supplement for memory enhancement. However, in the US it is being evaluated for safety and efficacy in a phase II trial as a therapy for AD (http://www.clinicaltrials.gov; Identifier: NCT00083590). In humans, huperzine A has a relatively long half-life, with T1/2 values of 5–6 h [13], and functions to reversibly sequester the active site of AChE. When exposed to lethal doses of OP nerve agents such as soman (the treatment options for which are extremely limited as a result of rapid ‘aging’ of the inhibited AChE), huperzine A can cross the blood brain barrier and protects brain AChE from excessive acetylcholine leading to seizures, neuropathological damage, and death. For example, high doses of huperzine A (500 g/kg), which reversibly inhibits blood and brain AChE by 60–70%, can significantly reduce soman (GD) lethality (protective ratios of 2–3) [14]. Lower, subchronic doses of huperzine A (yielding 20–30% inhibition of RBC-AChE) has little or no affinity for muscarinic or NMDA receptors and does not induce neuropathological damage in guinea pig hippocampus. Although there is compelling evidence of huperzine A’s ability to enhance cognitive function in humans, there is currently relatively little quantitative data on the protective effects of huperzine A pretreatments on human AChE in blood, peripheral tissue (e.g., diaphragm) and the CNS. Since 2003, pyridostigmine bromide (PB) has been FDAapproved as the U.S. Army’s pretreatment against potential
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soman exposure. PB (given as a single 30 mg oral dose every 8 h) provides temporary protection (by carbamylation) of peripheral tissue and red blood cell AChE, but is limited as it is fairly short acting, and cannot penetrate the blood brain barrier (BBB) to protect the CNS against seizures and subsequent neuropathological states induced by nerve agent exposure. We have previously shown [15] that oral administration of PB in human volunteers can protect a critical proportion (30–40%) of red blood cell AChE from irreversible inhibition by ex vivo addition of soman. We also showed that huperzine A specifically inhibits red blood cell AChE significantly (50–60%) and without serious side effects when given up to 200 g twice daily in normal elderly individuals [15]. As a pretreatment, huperzine A is a specific and highly selective inhibitor of red blood cell AChE with serum BChE being unaffected (thus preserving the well-documented OP bioscavenging capability of serum BChE) [16,17]. This lack of inhibition of BChE represents an additional advantage of huperzine A being used for prevention of OP toxicity, and helps to explain in part the increase in tolerance of huperzine A-pretreated animals to the lethal effects of soman in comparison to animals pretreated with PB. In this report, we have extended our evaluation of huperzine A, using increased oral doses ranging from 100 g (bid) to 400 g (bid) as part of a clinical study to determine the maximal tolerated dose (MTD) of huperzine A in healthy elderly human volunteers. In view of the fact that huperzine A exhibits good CNS penetration, the questions we have posed are (i) does ex vivo soman exposure in human whole blood containing huperzine A demonstrate adequate protection of red blood cell AChE from soman toxicity? (ii) from a military perspective, would pretreatment by huperzine A (in addition to post-exposure treatment with atropine and an oxime) represent an improved strategy against CWA exposure (and crucially protecting the CNS) that is currently ascribed for pyridostigmine bromide? 2. Methods 2.1. Human subjects and dosing with huperzine A The study design consisted of fifteen randomly assigned healthy elderly volunteers. Twelve (12) individuals were given an escalating dose regimen (orally, twice daily) of 100 g of huperzine A in week 1, followed by increasing doses of 200 g (week 2), 300 g (week 3), and finally 400 g in week 4. Three (3) individuals served as controls and received a placebo. Aliquots of whole blood were collected by a phlebotomist using 3 mL vacutainers containing sodium heparin and labeled with randomly generated identification numbers. Blood samples were collected at various time intervals for RBC-AChE and plasma BChE activity determination before (pre-dose), and after dosing with huperzine A (on day 1 and day 3 of each week). After the highest (400 g) and final dose was given in week 4, blood was withdrawn at various time intervals up to 48 h to monitor the profile of AChE and BChE activity. Samples were flash frozen on dry ice, sent to the Division of Biochemistry, Walter Reed Army Institute of Research (WRAIR) for storage
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at −80 ◦ C prior to the WRAIR whole blood cholinesterase assay. 2.2. Measurement of red blood cell AChE and plasma BChE in huperzine A-containing samples using the WRAIR whole blood (WB) assay Red blood cell AChE and plasma BChE were measured simultaneously in 10 L aliquots of thawed human blood diluted 20-fold in distilled water, as previously described in detail [16]. Briefly, the final assay mixture (300 L in each well of a 96-well microtiter plate) also contained (in separate wells): 1 mM each of acetylthiocholine iodide (ATCh), propionylthiocholine iodide (PTCh), and butyrylthiocholine iodide (BTCh); all wells also contained 0.2 mM of the chromophore 4,4 -dithiodipyridine (DTP), and 50 mM sodium phosphate buffer, pH 8.0. ChE activity was determined colorimetrically at 324 nm (UV) at 25 ◦ C on each plate using a Molecular Devices SpectraMax Plus384 microtiter spectrophotometer (Sunnyvale, CA), and was automated using a bench top robotic platform (Biomek 2000, Beckman-Coulter, Fullerton, CA). After a 4 min kinetic assay endpoint values were determined at 415 and 445 nm for sample normalization. Data (mAbs change/min) were subjected to linear least squares analysis, and AChE and BChE activities (expressed as U/mL and run in duplicate or triplicate) were calculated using SoftMax v5.0 and an Excel spreadsheet. 2.3. Measurement of protection afforded to RBC-AChE by huperzine A after ex vivo soman treatment To evaluate the protection afforded to RBC-AChE by huperzine A, blood samples stored at −80 ◦ C were thawed and then exposed to the nerve agent soman. These experiments were performed at USAMRICD, Aberdeen Proving Ground, MD. To each 70 L blood sample in a PCR tube, 1 M (final concentration) of GD in 0.9% NaCl was added for 10 min at room temperature. Free huperzine A and GD were removed from the blood by using small (6 cm) chromatography spin columns (Bio-Rad, Hercules, CA) containing 300 mg of C18 (Waters, Milford, MA) and a small plug of cotton wool on top. Columns were washed with 3 mL × 1 mL of methanol, and rinsed with 3 mL × 1 mL deionized water under a low vacuum. After adding 2.5 L of saponin (50%, w/v) to the blood and vortexing for 30 s, two 10 L aliquots were removed for measurement of AChE and BCE activity, and 50 L of the remaining lysed whole blood was placed on the cotton wool plug and the column was centrifuged at 1000 × g for 2 min (see Fig. 1). About 90% of the added volume was recovered after centrifugation, and 10 L aliquots were removed for AChE and BChE measurement immediately post-column. A 30 L post-column sample was diluted 100-fold in 50 mM sodium phosphate buffer, pH 8.0, left on the bench at room temperature for 3 h, and aliquots removed for AChE and BChE activity measurement to determine full return in activity (dissociation of huperzine A from the active site of AChE), and thus the proportion of RBC-AChE that can be protected from OP exposure. Post-column (100-fold diluted) blood samples were assayed for ChE activity as described above,
Fig. 1. Spin column chromatography for rapid and efficient removal of huperzine A and soman from hemolysed human blood. Bio-Spin columns contained 300 mg of C18 with a small cotton wool plug on top. AChE and BChE are not retained in the column matrix and recovery of inhibited AChE is measured after dilution of the eluted blood using the WRAIR whole blood ChE assay.
with the appropriate dilution factor correction taken into account. The % recovery of ChE activity is calculated as follows: (i) % inhibited (huperzine A or placebo samples) = 100 × (ChE U/mL of samples at times post-dosing)/(ChE U/mL pre-dosing). After ex vivo exposure to GD and removal of free GD and huperzine A by the spin column, recovery of ChE activity is calculated as follows: (ii) % recovery (huperzine A or placebo samples exposed to GD) = 100 × (ChE U/mL of GD-treated samples at times post-dosing)/(ChE U/mL pre-dosing). 2.4. Liquid chromatography–mass spectrometry (LC–MS) assays of huperzine A concentrations in human plasma Huperzine A was extracted from clarified human plasma samples by solid phase extraction (SPE) using Strata-C 96-well plates (Phenomenex, Inc.). Samples in wells were washed sequentially with 1 mM HCL and 50:50 (v/v) methanol:acetonitrile, and huperzine A eluted with 2 mL × 0.9 mL of 47.5:47.5:5 (v/v/v) methanol:acetonitrile:ammonium hydroxide. Elutes were transferred to 2 mL screw-capped bullet tubes, concentrated by lyophilization, and dried samples stored at −30 ◦ C.
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Huperzine A concentrations were determined by LC–MS (Beckman 128 pumps, Leap PAL temperature-controlled autosampler (5 ± 3 ◦ C), and a ZQ single-quadrupole MS (Waters, Inc.). The eluting solutions were buffer A (BA): 50 mM ammonium acetate pH 4.5 and buffer B (BB): 150 mM ammonium acetate pH 4.5 in 50% (v/v) acetonitrile and 40% water. Huperzine A was separated using a HS F5 HPLC column, 2.1 mm × 50 mm, (Supelco Inc.) at a flow rate of 0.2 mL/min, consisting of a gradient of 98% BA/2% BB for 1 min followed by a linear increase of BB to 60% for 10 min. Lyophilized samples were reconstituted in 120 L of BA, mixed, and clarified by centrifugation at 14,500 rpm for 1 min at 4 ◦ C. Sample (100 L) was injected on to the column and MS run in positive electrospray ionization (ESI) mode. The source capillary temperature was 250 ◦ C with 500 L h−1 N2 (g) desolvation gas. The cone parameters were 120 ◦ C, 35 V, and 50 L h−1 N2 . Huperzine A was detected by selected ion monitoring of the (M+H) ion (m/z 243). Huperzine A plasma concentrations were determined by comparison of peak-area-under-the-curve values for 243 m/z for matrix matched control huperzine A-spiked (to 1.33 g/mL) and then serially diluting with human pooled control plasma with final concentrations of 6.66, 13.3, 133, 1,333 ng/mL (Interstate Blood Bank, Inc., TN). Plasma huperzine A concentrations are given as ng/mL, (mean ± S.E. for n individual determinations). Standard (−)huperzine A stock solutions (Calbiochem Inc., San Diego, CA) were prepared as 1 mg/mL in 5 mM HCl and 50 mM sodium phosphate buffer, pH 8.0, clarified by centrifugation and stored at −80 ◦ C. Huperzine A concentrations were determined by absorbance using a molar extinction coefficient of 1.047 × 104 M/cm at 307 nm. 3. Results 3.1. Inhibition and recovery of RBC-AChE activity after oral huperzine A and ex vivo soman exposure: removal of huperzine A and soman from blood using a small spin column In this study, we have analyzed blood AChE (and BChE) activities in human blood from volunteers receiving up to 400 g huperzine A twice daily, as part of a larger clinical study to evaluate the safety and tolerability of increasing doses of huperzine A in healthy elderly individuals. Huperzine A reversibly binds to AChE, and thereby protects the enzyme from reaction with OPs. The activity of the huperzine A-protected but inhibited AChE will be restored once the drug–AChE complex spontaneously dissociates, which occurs after soman is removed from the blood. We have demonstrated this using rapid centrifugation of the soman and huperzine A-containing blood samples through a small C18 column to remove any free soman and drug, which bind to the column matrix while allowing 97–100% of the AChE and BChE activity to pass through. Under these circumstances, any RBC-AChE not protected by huperzine A would be irreversibly inhibited by soman. In contrast, the RBC-AChE protected by huperzine A would spontaneously dissociate over time, and this enzyme’s activity would be restored. In the example shown in Fig. 2, the solid bars represent RBC-AChE without any huperzine A (con-
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Fig. 2. Effects of soman (GD) on RBC-AChE activity in whole blood after 400 g huperzine A taken orally. Control (pre-dose) RBC-AChE (solid bar) is taken as the 100% value (3.34 ± 0.6 U/mL AChE activity, mean ± S.D., n = 3). AChE is maximally inhibited by 60%, 1.5 h post-huperzine A dose (speckled bar, arrow). After GD exposure, huperzine A and GD were removed using a small C18 spin column, as described in Section 2 (postcolumn treatment). The huperzine A-protected AChE recovery is complete within 3 h post-column. Hatched bars show the subsequent return of AChE activity of GD exposed blood due to dissociation of the protected enzyme after huperzine A and GD removal (3 h post-column arrow). Thus, the % difference between the pre-dose control (arrow, pre-dose control) and the huperzine A-inhibited control (arrow, speckled, 400 g huperzine A; 60% inhibition) is close to the % recovery of GD-inhibited RBC-AChE protected by huperzine A (arrow, hatched bar; 55% AChE activity recovered).
trol, pre-dose), while the stippled bar represents AChE from an individual that received a 400 g dose of huperzine A (60 ± 1.1% inhibition). In the first part, after soman treatment, no AChE activity is observed by the WRAIR assay in either the control or huperzine A treated volunteers (solid bars close to 0 U/mL). However, after the spin column removal of uncomplexed soman and huperzine A, dilution of the eluted blood sample and a 3 h period to allow for complete dissociation, the AChE is almost restored to the level that was initially inhibited by huperzine A (60% inhibition by huperzine A before the column vs. 55% returned AChE activity post-column in this individual; Fig. 2). Even after 3 h post-column recovery of AChE, the pre-dose control (soman exposed but not huperzine A treated) sample remains irreversibly inhibited (Fig. 2). These results definitively demonstrate that huperzine A is highly effective in protecting a significant proportion of RBC-AChE from ex vivo soman exposure. 3.2. Huperzine A protection and recovery of AChE activity in blood following ex vivo soman exposure Using the spin column technique described above, we have analyzed the complete profile of inhibition and recovery (protection by huperzine A) of RBC-AChE in three out of the twelve subjects receiving huperzine A, and the three placebo’s, over the full duration of the clinical trial, from
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pre-dose (control), through the incremental huperzine A dose increases over four weeks, and finally the 48 h lag phase following the final (and highest) 400 g dose. These results are shown in Fig. 3. Most of the decrease in RBCAChE (30–40%) occurs during week 1 (100 g twice daily), with further declines to a maximum of 48 ± 5% of control during week 4 (400 g twice daily) (solid line in Fig. 3). Following the last dose, recovery of RBC-AChE is followed for the next 48 h, and as Fig. 3 shows, significant inhibition persists for at least a further 12 h (53 ± 4% after 1 h, 54 ± 3% after 2 h, and even 44 ± 4% after 12 h post-dose). The gradual recovery of RBC-AChE activity continues but even 48 h after the last dose red blood cell AChE was still ∼10% below control (pre-dose) values (see Section 3.4, below). Fig. 3 also shows the time required to achieve full return in RBC-AChE activity (dissociation from the active site of AChE by huperzine A), and thus the proportion of red blood cell AChE that can be protected from OP exposure, after these samples were passed through the spin column to remove free huperzine A and soman (dashed line in Fig. 3). Huperzine A-inhibited AChE was restored almost to the level that was initially inhibited by the drug, and this is demonstrated by the line (solid triangles) at the bottom of Fig. 3 which represents the overall difference in the % AChE inhibition pre-column vs. % returned AChE activity post-column across all the time points. RBC-AChE returns to within 2.3% of the control (pre-column) activity level (labeled , delta, in Fig. 3). Finally, Fig. 3 also demonstrates the RBC-AChE activities in three placebo samples (top line, solid diamonds) remain constant at 95–100% of control values, as expected for
individuals that did not receive any oral doses of huperzine A for the duration of this study. Placebo samples exposed to soman (i.e., with no huperzine A present to protect AChE) and passed through the spin column to remove soman remained irreversibly inhibited at all time points (>95%, not shown). 3.3. Plasma BChE levels after huperzine A dosing In the same blood samples and doses of huperzine A (three individuals) or placebo (three individuals), we have also followed the effects on plasma BChE activities. As shown in Fig. 4 (top lines) plasma BChE is completely unaffected by huperzine A and remains at 98 ± 3% of control activity at all dosing and sampling time points. After treatment with soman (which completely inhibits BChE) and passage through the spin column to remove free soman and huperzine A, plasma BChE activities were inhibited by 86 ± 4% of control (pre-column) activity values, and were similar to the BChE in the placebo samples exposed to soman (which were inhibited by >90%). 3.4. Huperzine A concentrations in human plasma Huperzine A concentrations in plasma were analyzed by HPLC/MS and measured immediately after the first daily 200 g dose during week 2, and then after the final 400 g dose at the end of the study period. After the 200 g dose, the huperzine A concentration in plasma was 2.54 ± 1.92 ng/mL (n = 11). Immediately after the last (400 g) dose, the huperzine A concentration in plasma was 4.64 ± 1.28 ng/mL (n = 8), peaking at 5.47 ± 2.15 ng/mL
Fig. 3. Complete profile of inhibition and recovery of RBC-AChE after four weeks of an increasing dose regimen of huperzine A in three human volunteers and three placebos. Control (pre-dose) RBC-AChE is taken as the 100% value (3.43 ± 0.6 U/mL AChE activity, mean ± S.D., n = 6). Arrows (top) show huperzine A dose, and although given twice daily, only AChE activities on day 1 and day 3 of each week are shown (vertical dashed lines). Note that this figure is not to scale with left side representing weekly treatments, and right side time elapsed (in hours) after last dose. Solid triangles (bottom line) represent the overall difference in the % AChE inhibition pre-column vs. % returned AChE activity post-column across all the time points. RBC-AChE activity returns to within 2.3% of the huperzine A-treated (but not GD exposed) activity level.
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Fig. 4. Profile of plasma BChE after four weeks of an increasing dose regimen of huperzine A in three human volunteers and three placebos. Control (predose) plasma BChE is taken as the 100% value (1.74 ± 0.35 U/mL BChE activity, mean ± S.D., n = 6). Same legend as Fig. 3 for huperzine A dosing. Huperzine A has no effect on plasma BChE levels and there was no difference between pre- and post-column activity (BChE recovery huperzine A-treated or BChE recovery, placebo); not shown.
(n = 10) 1 h post-dose. A residual amount of huperzine A was barely detectable within the limits of the method (1.1 ± 0.9 ng/mL, n = 10) at 48 h post-dose (corresponding to about 10% inhibition of RBC-AChE, see Fig. 3), but the AChE activity is within a few percent of the placebo activity after 48 h. It is clear that huperzine A is a potent RBCAChE inhibitor since smaller concentrations of the drug were required to achieve greater RBC-AChE inhibition than shown previously with PB [15]. 4. Discussion and conclusions It is now well established that huperzine A, a reversible and highly selective AChE inhibitor isolated from the Chinese moss Huperzia serrata and used in traditional Chinese herbal medicine, is well tolerated in humans and animals. Animal and human clinical studies with huperzine A have demonstrated rapid absorption, high bioavailability, long biological half-life, and efficient penetration of the blood brain barrier to protect the central nervous system. In China, clinical trials of healthy individuals and patients with various neurological disorders have indicated that huperzine A is safe and well tolerated. Outside China, there have been very few controlled studies to assess its safety and toxicity. In the US, huperzine A is presently undergoing clinical trials to determine safety and efficacy as a potential treatment for Alzheimer’s disease, and is presently only approved as an over-the-counter nutritional supplement for memory enhancement. In a previous study [15], we have shown that huperzine A is a more potent inhibitor of RBC-AChE than PB, another reversible AChE inhibitor that has been specifically FDAapproved for military personnel as a pretreatment against soman, a potent and irreversible OP nerve agent. We showed that a 200 g dose of huperzine A in human
volunteers can protect a significant proportion (30–40%) of red blood cell AChE from inhibition from soman measured in an ex vivo assay using the WRAIR whole blood cholinesterase method, and that the AChE inhibition is fairly long lasting. The WRAIR ChE assay can quickly determine the profile of AChE (and BChE) in whole blood samples using high throughput screening and can be used to monitor exposure to nerve agents, pesticides, and medically useful drugs (e.g., anti-cholinergics) in the treatment of neurological diseases [15]. In this work, the effects of even higher oral doses of huperzine A on red blood cell AChE in humans have been described using increased oral doses ranging from 100 g (bid) to 400 g (bid) over a four week period, as part of a clinical study to determine the tolerability and safety of huperzine A in healthy elderly human volunteers. This step-wise increase in huperzine A at weekly intervals, from 100 g (bid) to 400 g (bid) was employed to mitigate the occurrence of gastrointestinal side effects. This high oral dose produced 50–60% inhibition of RBC-AChE (Fig. 3), with peak inhibition of AChE and plasma huperzine A concentrations occurring 1 h after the last huperzine A dose. This effect persists for several hours followed by a gradual dissociation of the huperzine A–AChE complex and restoration of AChE activity, although, as Fig. 3 shows, AChE activity is still inhibited by 50% after 12 h, and more than 20% inhibition remains even after 48 h post-dose. This long duration of action is a result of the tightly bound complex which forms between huperzine A and AChE [4] and the pharmacokinetics of huperzine A in vivo; the reported half-life of huperzine A given orally in humans was 4.8 h, with peak blood levels of huperzine A being reached after 80 min [13]. Similarly, there is a long duration (6 h) of AChE inhibition in rat brain [10]. In a recent study of 18 healthy male Chinese volunteers given a single 0.2 mg (200 g) oral dose of
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huperzine A [18], the maximal concentration (Cmax ) measured in plasma was 2.47 ± 0.47 ng/mL and reached a peak level after 80 min. Using a sensitive HPLC/MS method, very similar results for huperzine A plasma concentrations were observed in the elderly volunteers in this study (see Section 3). Recently, real time measurement of AChE activity using immobilized human red blood cells as an in vitro model after pretreatment inhibition (50%) with pyridostigmine bromide and huperzine A, demonstrated lower levels AChE recovery following subsequent exposure to soman for 30 min (28.5% and 7% with PB and huperzine A, respectively) [19]. In this assay recovery of AChE activity following disassociation of huperzine A from the active site of AChE is very rapid (t1/2 = 4.6 min) compared with the decarbamylation rate of PB (t1/2 = 27 min), and thus the amount of sequestered AChE after removal of huperzine A is rapidly diminished because of the significantly longer soman exposure time (30 min) and ‘aging’ [20]. However, a longer dissociation rate (∼60 min) for huperzine A has been reported [3]. Likewise, in Fig. 2 we showed very rapid recovery of AChE activity following complete removal of huperzine A and soman from red blood cells using the spin column, but are able to demonstrate much higher sequestering (protection) of the enzyme (after 3 h) because of the lack of soman in the post-column eluate for irreversible inhibition and ‘aging’. The inhibition of RBC-AChE persists for significantly longer (∼45% inhibition after 6 h; ∼25% inhibition after 24 h post-dose, Fig. 3) than huperzine A levels in the plasma. Based on huperzine A’s half-life in plasma and its tight binding to the enzyme [4], (Fig. 2 demonstrated that full recovery of full AChE activity only occurs after 3 h (following removal of any free huperzine A bound to the C18 spin column. As Fig. 3 also shows, a residual amount of inhibition (∼10%) is a value almost indistinguishable from the placebo. Therefore, it is likely that complete recovery of RBC-AChE is apparent at 48 h post-dose. The observed lack of inhibition on BChE (Fig. 3) represents an additional advantage of huperzine A being used for prevention of OP toxicity, and helps to explain the increase in tolerance of huperzine A-pretreated animals to the lethal effects of soman in comparison to animals pretreated with PB. 5. Conclusions Huperzine A is a potential second-generation drug for prophylaxis against organophosphate poisoning. Unlike PB, huperzine A can protect the CNS from the anti-cholinergic effects of OP’s and may have fewer side effects. Given the potential rise in urban terrorism that may include the use of chemical warfare organophosphate agents, Federal, State, and local authorities need a fast acting, inexpensive, and potent pretreatment measure that can protect both CNS and peripheral AChE. Acknowledgements We would like to thank Dr. Benedict Capacio, USAMRICD, Aberdeen Proving Ground, MD, for permission to perform exposure of human blood samples to soman (GD).
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