(W0930-02-09) Development of a Sustained Transdermal Delivery System of Amiloride for Management of Resistant Hypertension

Purpose: Resistant hypertension is a global health challenge, for both developing and developed countries. In America, the National Health and Nutrition Examination Survey (NHANES) estimates the prevalence of apparent treatment-resistant hypertension in the general public to be about 12.8%. Resistant hypertension, is a condition in which blood pressure remains above the ideal value (120/80mmHg), despite concurrent use of three antihypertensive agents of different classes taken at maximally tolerated doses, including a diuretic. Over time, uncontrolled hypertension damages the arteries, leads to stroke, heart attack, heart failure and other heart conditions. Amiloride, a potassium-sparing diuretic agent, when added to the treatment regimen of these drugs has been found suitable for the management of resistant hypertension, especially in diabetic patients and those resistant to a similar diuretic, spironolactone. Currently, it is available as an oral tablet, administered once daily. The oral bioavailability of amiloride is 50%, which gets reduced to 30% when administered with food. In addition, gastrointestinal side effects are also reported. Patient’s adherence to the multi-drug treatment regimen has been found to be low in patients with resistant hypertension and hence, administering amiloride in the oral forms may not solve the problem, in spite of its proven pharmacological efficacy in such situations. Thus, considering the low oral bioavailability, associated side-effects, and prospects of better patient compliance with a skin patch of amiloride, our long term goal is to design a long-acting skin patch for transdermal delivery of amiloride in patients with resistant hypertension. We have investigated the passive and active permeation of amiloride through an In vitro permeation study using microneedles. We aim to evaluate the effects of chemical and other physical enhancement techniques on its permeation through dermatomed porcine ear skin.

Methods: Reverse phase High performance liquid chromatography (HPLC) method for amiloride was developed. Absence of skin interference in the assay was confirmed using blank skin extract. Solubility of amiloride was screened in different solvents, some of which included propylene glycol, phosphate buffered saline, oleic acid in propylene glycol, e.t.c. In vitro permeation of amiloride across intact and microneedle-treated porcine ear skin was evaluated using Franz diffusion cells over 30h. Amiloride dissolved in propylene glycol was added as the drug solution in the donor chamber of the Franz diffusion cell (Figure1). This set up provided an effective diffusion area of 0.64cm². In order to maintain the skin temperature at 32^◦ C, the receptor compartment was maintained at a temperature of 37^oC using a recirculating water bath system. Each cell was washed and filled with 5 mL each of PBS: PG (9:1) to maintain sink conditions. To achieve microneedle treatment of skin samples and characterization of microchannels, Dr. Pen (36 needle array) was used for microporation on skin samples with insertions at a speed of 13,000 insertions/min. The needle length employed was 500 µm and duration of array application was 2 min. The permeation study samples collected over predetermined time intervals were analyzed via RP-HPLC. The results for each group (control and test) were reported as mean ±SE.

Results: The optimized reverse-phase HPLC method involved isocratic elution on Kinetex^®5µm, 100A⁰, 250x4.6mm C18 column using 100% mobile phase (0.2M phosphate buffer 4.5) at a flow rate of 0.8 mL/min, column temperature of 40°C, and UV detection at 360 nm. Drug retention time was found to be around 4 min. Amiloride was found to be most soluble in propylene glycol (57.18 ± 2.41 mg/mL) with least solubility in phosphate buffer saline (0.311 ± 0.004 mg/mL). Microneedles were found to significantly enhance the permeation flux of amiloride by 16 folds as compared to the control intact skin (p < 0.05, Figure 2). The amount of amiloride permeation after 30 h was 557.92 ± 48.77 µg/mL across microporated skin, that was significantly higher than the control group (16.63 ± 4.12 µg/mL, p< 0.05, Figure 3).

Conclusion: Microneedles were found to enhance the permeation of amiloride across skin. The observation of the in vitro study is very encouraging and future studies would focus on developing a weekly microneedle patch for extended drug release.

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2. Pimenta E, Calhoun DA. Resistant Hypertension: Incidence, Prevalence, &Prognosis. Circulation.2012 Apr3 125(13):1594-6. Doi: 10.1161/Circulationaha.112.097345. Epub 2012 Feb 29. Pmid: 22379111;