Limitations of the Anticholinergic Activity Assay and Assay-Based Anticholinergic Drug Scales.

OBJECTIVE: The anticholinergic activity (AA) assay is a common method to determine a patient's anticholinergic load. Several limitations, however, are expected when applying the AA assay to patients or using drug scales to estimate anticholinergic burden based on AA levels. This study aims to demonstrate common pitfalls in an experimental setting and outline their clinical consequences. METHODS: The AA was analyzed for five drugs with reported interaction with muscarinic receptors. Concentration-response curves were constructed for furosemide (weak anticholinergic), diphenhydramine (moderate anticholinergic), the strong anticholinergic amitriptyline and its metabolite nortriptyline, and the cholinergic pilocarpine. The Combination Index (CI) was used to assess the interaction of three drug combinations with amitriptyline. RESULTS: All compounds displaced the radioactive tracer from its receptor binding site in a concentration-dependent manner, and full displacement was reached for all compounds except furosemide (Emax 16%). The CI indicated that amitriptyline and thioridazine have antagonistic effects (CI = 1.46) at low and synergistic effects (CI = 0.88) at higher concentrations (p < 0.0001), whereas synergistic effects (CI = 0.47-0.48) were observed for amitriptyline in any concentration combined with pilocarpine (p < 0.001). CONCLUSION: When the patient's anticholinergic load is estimated using AA levels, the actual exposure, combination of anticholinergic drugs, their active metabolites, and also drugs with an opposite pharmacologic action will contribute to AA levels, whereas weak anticholinergic drugs in therapeutic concentrations are rather negligible.

Human paraoxonase 1 is the enzyme responsible for pilocarpine hydrolysis.

Pilocarpine has been widely used in ophthalmic preparations for the treatment of glaucoma and in oral preparations for the treatment of radiation-induced xerostomia and Sjögren syndrome. The major metabolic pathways of pilocarpine in human are hydrolysis and hydroxylation. It was found that CYP2A6 is responsible for the 3-hydroxylation, but the enzymes responsible for the hydrolysis have not been characterized. In this study, we attempted to identify esterases responsible for pilocarpine hydrolysis. Pilocarpine hydrolase activities in human liver microsomes and plasma were stimulated by the addition of CaCl(2), suggesting that the calcium-dependent esterase, paraoxonase (PON), was responsible for pilocarpine hydrolysis. To confirm this hypothesis, the pilocarpine hydrolase activity was measured using the recombinant human PONs (PON1, PON2, and PON3) established in this study, and the result was that only PON1 showed pilocarpine hydrolase activity. The effect of PON1 polymorphism (Q192R) on pilocarpine hydrolase activity was analyzed using recombinant human PON1 192Q and 192R and human plasma from 50 volunteers. The results showed that recombinant PON1 192R revealed significantly higher catalytic efficiency than PON1 192Q. In human plasma, the activity of the R/R genotype (117.0 ± 25.2 pmol · min(-1) · µl(-1), n = 23) was significantly higher than those of the Q/R and Q/Q genotypes (97.3 ± 21.0 pmol · min(-1) · µl(-1), n = 20 and 90.4 ± 26.2 pmol · min(-1) · µl(-1), n = 7, respectively). It is suggested that this polymorphism affects pilocarpine hydrolase activity. In this study, we found that human PON1 is the major enzyme for the catalytic efficiency of pilocarpine hydrolysis.

Pharmacokinetics of pilocarpine in subjects with varying degrees of renal function.

Data from three separate single-center studies were combined to assess the pharmacokinetics of orally administered pilocarpine. Pilocarpine concentration-time data were used to generate a data set including 42 subjects (34 males, 8 females) with varying degrees of renal function (average of two estimated creatinine clearance rates of 10 to 112 mL/min). Age ranged from 19 to 88 years. Subjects received single oral doses (range: 2.5-20 mg) of pilocarpine. Plasma samples were collected at time 0; at 20 and 40 minutes; and at 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 24 hours following dose administration. Cmax and AUC were normalized to a 5 mg exposure in those subjects who received doses other than 5 mg. Plasma pilocarpine concentrations were determined by gas chromatography/mass spectrometry. The pharmacokinetic parameters (elimination rate constant, Cmax, tmax, AUC, Vd/F, and Cl/F) in subjects with impaired renal function were similar to results found in other pharmacokinetic studies involving normal healthy volunteers with only Cmax being significantly higher (p < 0.05). No significant regression relationships were noted between creatinine clearance and pilocarpine elimination rate constant, tmax, Vd/F, Cl/F, or AUC. Pilocarpine clearance does not appear to be impaired in patients with varying degrees of renal insufficiency.

A pilot study of the disposition of pilocarpine in plasma, saliva and urine after a single oral dose.

Concentrations of pilocarpine in plasma, saliva and urine from three healthy male volunteers were measured using a fluorescence derivatisation method, following administration of a single 10 mg oral dose. Pharmacokinetic parameter values were estimated from concentration-time profiles. Linear correlations between plasma and saliva pilocarpine concentrations (r2=0.945, n=10, p<0.001; r2=0.954, n=12, p<0.001) and plasma concentrations and salivation rate (r2=0. 863, n=12, p<0.001; r2=0.862, n=15, p<0.001) were established. Pilocarpine and an unidentified metabolite, respectively 20.3% and 34.7% of the oral dose, were excreted into urine.

Determination of pilocarpic acid in human plasma by capillary gas chromatography with mass-selective detection.

A novel, highly sensitive method for the determination of pilocarpic acid (PA) in human plasma is described. In addition, the method provides for the conversion of the lactone, pilocarpine (P), to PA so that a total drug presence can be determined. Using novel high-performance liquid chromatographic conditions capable of separating P, isopilocarpine (I-P), PA and isopilocarpic acid (I-PA) from each other and from endogenous plasma impurities, it was confirmed that P exclusively and quantitatively converts to PA in heparinized human plasma during storage. For the determination of PA, the selective extraction of PA from protein-free plasma was accomplished using two different solid-phase extraction (SPE) cartridges in two consecutive SPE steps. After extraction, PA was lactonized with trifluoroacetic acid back to P, and both P and an internal standard were acylated using heptafluorobutyric anhydride (HFBA). The trifluoroacetylated derivatives were monitored using gas chromatography (GC) with mass spectrometric (MS) detection. This procedure allowed the sensitive and reliable determination of PA with a limit of quantification (LOQ) of 1 ng/ml, which could not be achieved using previously described methods. The assay was validated in the concentration range of 1 to 10 ng/ml with an intra-day precision (expressed as the coefficient of variation, C.V.) ranging from 9.9 to 0.5%. Inter-day precision for the quality control standard at 2.5 ng/ml showed a C.V. of 10.2%. Accuracy ranged from 94 to 102%. The assay was used to monitor the maximum systemic exposure to P, administered by the ocular route, in terms of total plasma PA (P and PA).

Determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine by high-performance liquid chromatography with tandem mass spectrometric detection.

A method is described for the determination of pilocarpine and its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine. The method is based on a simple sample preparation step -- ultrafiltration for plasma and dilution for urine samples -- followed by a reversed-phase liquid chromatographic separation of the analytes and detection by means of tandem mass spectrometry. Parameters affecting the performance of these steps are discussed. The high sensitivity and selectivity of the method allow low ng/ml concentrations to be determined for all compounds in plasma and undiluted urine, which enables the investigation of the metabolic fate and elimination of pilocarpine after oral administration to humans.

Pilot study of controlled-release pilocarpine in normal subjects.

OBJECTIVES: Current systemic treatments with sialogogues for patients with xerostomia are limited because of minimal efficacy, short duration of activity, or problems with side effects. The purpose of this pilot study was an initial assessment of safety, efficacy, duration of action, multiple dose tolerance, and side effects of a controlled-release formulation of pilocarpine hydrochloride. STUDY DESIGN: Eight healthy hospitalized subjects were given 15 mg of a controlled-release pilocarpine formulation every 12 hours for three doses. Saliva and blood samples were collected at assigned intervals. Repeated measures analysis and paired t tests were used for statistical analyses. RESULTS: A significant (p < 0.05) increase in both parotid and whole saliva output followed all three doses beginning within 1 hour of dosing and lasting over 10 hours. Mean plasma pilocarpine concentration reached a maximum of 8.2 ng/ml at approximately 1 hour after the first dose, 11.5 ng/ml after the third dose, and declined to near baseline (0.06 ng/ml) 24 hours after the final dose. None of the participants showed evidence of adverse effects including complaints of sweating or gastrointestinal discomfort. CONCLUSIONS: A controlled-release formulation of pilocarpine may overcome the therapeutic weaknesses of current pilocarpine preparations by prolonging salivary secretion and reducing undesirable side effects.

A pharmacokinetic and pharmacodynamic study of intravenous pilocarpine in humans.

Pilocarpine (P) is of potential utility in the treatment of xerostomia. Because optimal development of P dosage forms for humans requires that its pharmacokinetics and pharmacodynamics be defined, this intravenous study of its disposition and associated salivary responses was performed. In a hospital setting, two healthy female subjects were given a series of graded doses of intravenous P or placebo to stimulate salivary secretion. Plasma levels of P, heart rate, blood pressure, and respiratory rate were simultaneously monitored. Other objective and subjective physiological parameters were assessed. Plasma concentrations of P declined either mono- or bi-exponentially with time, and brisk initial salivation was followed by prolonged salivation at doses > or = 1 mg. At doses between 0.5 and 3.5 mg, dose-independent pharmacokinetic parameters included a small steady-state volume of distribution (2.4 to 3.0 L/kg), a high plasma clearance (0.026 to 0.03 L/kg/min), and a mean residence time of approximately 100 min. The cumulative volume of whole saliva secreted during the first 3 h post-dose was linearly related to the area under the plasma concentration-time curve. Plasma concentrations from 1 to 42 ng/mL were associated with significant levels of salivation. The pharmacokinetic linearity of the system and proportionality between the area under plasma concentration-time curves and overall salivary response have important implications for the design and utilization of pilocarpine dosage forms.

Comparison of enzymatic hydrolysis of pilocarpine prodrugs in human plasma, rabbit cornea, and butyrylcholinesterase solutions.

Various bispilocarpic acid diesters (double prodrugs of pilocarpine) were synthesized, and their in vitro esterase catalyzed hydrolysis was evaluated in diluted human plasma, rabbit cornea homogenate, and specific butyrylcholinesterase solution. The structural changes greatly affected the rate of enzymatic hydrolysis of the prodrugs. Bispilocarpic acid with 2 cyclopropane substituents was the most stable derivative, whereas bispilocarpic acid with 2 cyclobutane substituents was the most labile derivative. The charged bispilocarpic acid diester hydrolyzed more slowly than the unchanged form. Comparison of the results obtained from different plasma and cornea homogenate batches is difficult because of the variety of the enzyme systems involved. This variety also makes comparing the results between different laboratories difficult.

Salivary flow induction by buccal permucosal pilocarpine in anesthetized beagle dogs.

We tested whether permucosal delivery of pilocarpine nitrate could be used to elicit significant salivary secretion. Pilocarpine (pKa 6.6 at 37 degrees C) was applied as solutions (pHs 5.6, 6.6, 7.6; 15 mg/mL) to the buccal mucosa (2.8 cm2) of 6 anesthetized dogs. Saliva was collected continuously from cannulated submandibular and parotid ducts and blood sampled during and after drug administration. Plasma pilocarpine levels were determined by reversed-phase HPLC. Absorption rates were determined by use of data from separate zero-order intravenous infusions to the same dogs. Pilocarpine was buccally absorbed at a constant rate of 72.9 +/- 38.5 micrograms/kg/h following its application at pH 7.6. At this pH of the drug solution, the time to appearance of pilocarpine in blood plasma was 0.31 +/- 0.08 h, and the time to appearance of salivary flow was 0.86 +/- 0.32 h. A threshold dose of 32.9 +/- 7.5 micrograms/kg was required to induce secretion with the pH 7.6 drug, the steady-state submandibular flow rate was 0.14 +/- 0.11 mL/min/gland pair. Salivary flow induction was symmetrical and reached levels as high as 0.35 mL/min/submandibular gland pair without apparent tachyphylaxis. Results at pHs 5.6, 6.6, and 7.6 were consistent with the hypothesis that pilocarpine is primarily absorbed as un-ionized drug. The data indicate that transmucosal delivery of pilocarpine, avoiding "first pass" hepatic loss, may hold promise for the treatment of xerostomia.

High-performance liquid chromatographic determination of pilocarpine in plasma.

A high-performance liquid chromatographic procedure requiring neither derivatization nor complex sample work-up is reported for reproducibly and sensitively determining pilocarpine in plasma. Following stabilization of pilocarpine against in vitro hydrolysis using sodium fluoride, plasma samples were extracted and the extracts chromatographed on a 5-microns, low-carbon-load (6%) C18 reversed-phase column. The assay was linear between 10 and 300 ng/ml (r = 0.998). It had sufficient sensitivity to quantitate pilocarpine at concentrations as low as 10 ng/ml (signal-to-noise ratio > or = 4) using a 500-microliters sample. The assay appears to be the first published specifically for plasma determinations and has proven capable of supporting pharmacokinetics studies of pilocarpine disposition in the anesthetized dog.

Pharmacokinetic basis for nonadditivity of intraocular pressure lowering in timolol combinations.

The authors determined whether the ocular absorption of topically applied timolol in the pigmented rabbit was affected significantly by coadministration with either pilocarpine or epinephrine in the same drop to explain the nonadditivity in intraocular pressure lowering (IOP) seen clinically. They instilled 25 microliters of 0.65% timolol maleate solution (equivalent to 0.5% timolol), both in the presence and absence of 2.6% pilocarpine nitrate or 1% epinephrine bitartrate, into pigmented rabbit eyes. The time course of timolol concentration in the conjunctiva, anterior sclera, corneal epithelium, corneal stroma, aqueous humor, iris-ciliary body, and lens was monitored for 360 min by using reversed-phase high-performance liquid chromatography. The area under the timolol concentration-time curve in all but one of the anterior segment tissues was reduced by 20-50% (mean, 40%) when timolol was coadministered with pilocarpine and by 20-70% (mean, 42%) when timolol was coadministered with epinephrine. Such an effect was not a result of alterations in corneal permeability or aqueous humor turnover rate, nor was it related to the extent of systemic absorption caused by pilocarpine and epinephrine. Rather, the reduction in ocular timolol absorption may have been caused by the accelerated washout of timolol by tears stimulated by the coadministered drugs and, to a lesser extent, by the loss of timolol through binding to the increased amount of tear proteins induced by the coadministered drugs. Thus, the nonadditivity in IOP lowering from timolol-pilocarpine and timolol-epinephrine combinations is probably caused by changes in precorneal timolol clearance.

Determination of physicochemical properties, stability in aqueous solutions and serum hydrolysis of pilocarpic acid diesters.

New alkyl and aralkyl pilocarpic acid diesters, prodrugs of pilocarpine, were synthesized with the aim of improving the bioavailability of pilocarpine by increasing its corneal permeability. These esters were several orders of magnitude more lipophilic than pilocarpine as determined by their apparent partition coefficients between 1-octanol and phosphate buffer (pH 7.40) (log P). Good correlation between log P and HPLC capacity factors of the compounds was observed. All the compounds are stable in acidic aqueous solution; in serum, however, pilocarpic acid diesters are hydrolysed enzymatically to pilocarpic acid monoester, which undergoes spontaneous cyclization to active pilocarpine and inactive isopilocarpine. The half-lives of the diesters in serum varied from 6-232 min. In addition to the direct effects of the R2, R1 moiety had a remarkable effect on the rate of enzyme-catalysed hydrolysis taking place in moiety R2. The formed pilocarpine was analysed with a new HPLC method which allowed good resolution of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid. Rates for pilocarpine formation were both determined by experiment and calculated using the STELLA simulation programme with known degradation rate constants of pilocarpic acid diesters and monoesters. Since the simulations were in good agreement with the experimental results, it is concluded that STELLA simulation programme is useful in predicting pilocarpine formation.

Monitoring compliance through analysis of drug and metabolite levels.

Cold analytical methodology is usually available for drug and metabolite monitoring during clinical trials, since a procedure is required for the bioavailability, pharmacokinetic, and dose proportionality studies that must be conducted by the sponsor. Such methods can and have been applied to monitoring patient compliance. Examples from several classes of drugs with different pharmacokinetic profiles illustrate the type of data that can be obtained, along with their applicability and inherent limitation in assessing compliance. The effects of concomitant medication, drug half-life, volume of distribution, and sampling time on observed levels are also discussed. Several other approaches involving trace metals, microtaggants, and an electronic monitor are also presented.