D Garteiz, TexMS Analytical Services, Houston, TX
A Varian 1200L LC/MS/MS system was used for the quantitation of the plasma levels oxybutynin obtained by topical delivery of therapeutic agents to the urinary tract using a urethra suppository mode of administration.
Background Urinary tract disease is a major health problem in the U.S. and will continue to grow with an increasingly aging population. Urinary incontinence accounts for over ten million patients suffering from urinary tract disease in the United States. Although oral delivery of drugs is an effective method of treating incontinence, many patients are unable to continue oral drug therapy because of severe side effects such as dry mouth, nausea, and tachycardia. One method of satisfying the clinical need of treating patients refractory to oral medication is through topical delivery of therapeutic agents to the urinary tract. The urethra provides an accessible and potentially effective method for achieving topical drug delivery to the urinary tract and surrounding organs and tissues.
One of the most commonly used drugs for urinary tract disease is oxybutynin chloride (Figure 1). Because of the severity of the drugs side effects, low doses are administered, leading to oxybutynin plasma levels that can range from the low pg/mL to low ng/mL. The present study investigates the feasibility of providing therapeutic agents to the urinary tract by absorption through the urethra.
Using a rabbit model, urethral suppositories, formulated with oxybutynin chloride were placed in the urethra of female rabbits. The suppositories liquefy in the urethra and transfer the oxybutynin through the urethral mucosal into the systemic circulation. Safety of urethral absorption of oxybutynin was established by comparing the plasma levels of urethral absorption of oxybutynin versus levels achieved by intravesical administration (injection in the bladder) of the drug.
Varian 1200L LC/MS/MS equipped with Electrospray Ionization (ESI) source
Materials and Reagents
Oxybutynin chloride from Sigma-Aldrich, Catalog Number O2881.
Diphenhydramine hydrochloride from Sigma-Aldrich, Catalog Number D3630.
All other chemicals are reagent grade or HPLC grade.
A 0.5 mL aliquot of rabbit EDTA plasma containing oxybutynin chloride is spiked with 10 μL of diphenhydramine (0.5 ng/mL) as the internal standard (ISTD). Plasma is made basic and extracted with methyl t-butyl ether.
After separation of the phases by centrifugation, the organic layer is transferred by pouring into a conical tube and is then evaporated to dryness. The dry residue is reconstituted in 200 μL of methyl t-butyl ether and back extracted using 1% formic acid.
Results and Discussion
The ratio of the intensities of the product ions for oxybutynin to that of its internal standard [(m/z 142.3)/(m/z 167.5)] is used to calculate the oxybutynin concentration in unknown plasma samples using a calibration curve. The calibration curve is generated from the analysis of a drug free rabbit plasma matrix fortified with various amounts of oxybutynin and a fixed amount of the internal standard. The calibration curve range for the analyte is from 0.1 ng to 5.0 ng per mL of rabbit plasma
Visual examination of the calibration curve in Figure 2 shows that good linearity was obtained with the log/log curve-fitting model used. Additional sensitivity can easily be obtained by increasing the sample volume used for the assay. Figures 3 and 4 show excellent response and peak shape for both the LLOQ and ULOQ.
The pharmacokinetic plot in Figure 5 shows that oxybutynin levels are present in plasma with urethral delivery at lower doses versus intravesical delivery at higher doses. Comparing the same dose of oxybutynin for urethral and intravesical delivery methods, the urethral delivery had a lower peak level but appears to maintain a higher drug level over a greater period of time.
This study shows that the urethral delivery of oxybutynin results in a lower plasma level of drug at the same dose as intravesical delivery. The lower plasma levels resulting from urethral delivery may reduce the frequency and or severity of the undesirable side effects that have been described following intravesical administration of this drug. At the same time, the topical nature of the delivery method may improve the efficacy of the drugs pharmacological response because it is being delivered to the site of action.
1. H. Budzikiewicz and C. Djerassi, Mass Spectrometry of Organic Compounds. Holden-Day, Inc., Publishers. 1967.