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Topical Pilocarpine Formulation for Diagnosis of Cystic Fibrosis
Journal of Pharmaceutical Sciences xxx (2020) 1-5
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Research Article
Topical Pilocarpine Formulation for Diagnosis of Cystic Fibrosis Tasnim Fatima 1, Srinivas Ajjarapu 1, Vijay Kumar Shankar 1, Srinath Rangappa 1, H. Nanjappa Shivakumar 2, 3, Subrata Kumar Biswas 4, Mozammel Hoque 4, S. Narasimha Murthy 1, * 1
Department of Pharmaceutics of Drug Delivery, University of Mississippi, University, Mississippi 38677 KLE College of Pharmacy, Bengaluru, Karnataka, India 3 Institute for Drug Delivery and Biomedical Research (IDBR), Pharmaceutics, Bengaluru, Karnataka, India 4 Department of Biochemistry, Bangabandhu Sheikh Mujib Medical University, Dhaka, Dhaka District, Bangladesh 2
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 January 2020 Revised 27 January 2020 Accepted 29 January 2020
Cystic fibrosis is diagnosed in infants by estimating the levels of chloride ions present in the sweat induced by iontophoresis of pilocarpine solution. Elevated levels of chloride (60 mMol/L) in sweat are indicative of cystic fibrosis. However, the iontophoretic method of delivering pilocarpine is cumbersome and usually is associated with several side effects such as skin burn, skin rashes, erythema, and so forth. The objective of this study was therefore to develop a topical formulation that delivers adequate amount of pilocarpine. The drug delivery of formulation was compared with iontophoresis of aqueous solution of pilocarpine nitrate in vitro using porcine skin model. The pilocarpine levels in the skin exposed to topical pilocarpine solution under mild hyperthermia was 152.04 ± 52.23 mg/cm2 after 10 min of application, whereas it was 97.05 ± 27.93 mg/cm2 in the skin after 10 min of iontophoresis. The topical formulation was subjected to clinical evaluation to assess the efficacy of the product to induce sweat production. The average amount of the sweat secreted on application of topical formulation was found to be 77.28 ± 18.97 mg. Based on these results, it was found that the topical formulation was successful in delivering pilocarpine and to stimulate sweat secretion. © 2020 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
Keywords: skin permeation enhancer(s) clinical trial(s) formulation iontophoresis
Introduction Cystic fibrosis (CF) is a progressive, genetic disease inherited in an autosomal recessive manner.1 According to the Cystic Fibrosis Foundation Patient Registry, there are around 30,000 people with CF in the United States alone and more than 70,000 people worldwide. Every year, approximately 1000 new cases of CF are diagnosed in the United States.2 CF manifests as a persistent lung infection, aquagenic wrinkling of the palms, exocrine pancreatic insufficiency, and presence of excessive amount of salt in the sweat.3,4
Abbreviations used: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; HPLC, high-performance liquid chromatography; PEG, polyethylene glycol; PDA, photodiode array; rpm, revolutions per minute; SD, standard deviation. Conflict of interest: None. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. * Correspondence to: S. Narasimha Murthy (Telephone: þ1-662-915-5164). E-mail address: [email protected] (S.N. Murthy).
CF is caused because of mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene located on chromosome 7, resulting in malfunctioning of CFTR proteins. CFTR is a membrane protein and chloride channel present in the lungs, pancreas, intestine, sweat glands, and other exocrine glands. CFTR regulates the transport of sodium, chloride, and bicarbonate ions maintaining transepithelial homeostasis.5,6 Defective CFTR proteins affect gland duct reabsorption potential of electrolytes, leading to excess of salt in the sweat.7,8 There are several complications associated with CF such as bronchiectasis, chronic infections, pneumothorax, respiratory failure, and others.9 Management of CF involves maintenance of lung function by clearing airway, administering bronchodilators, antibiotics, and pancreatic enzyme replacement therapy.10,11 Ivacaftor alone or in combination with lumacaftor is used for improvement of defective CFTR’s function.12,13 Although incurable, early diagnosis and close monitoring could help in management of CF. Methods for diagnosis of CF includes sweat testing or genetic test. The levels of chloride ions in sweat are estimated for diagnosis of CF. Elevated levels of chloride (60 mMol/L) in sweat is indicative of CF. The sweat in patients with CF is
https://doi.org/10.1016/j.xphs.2020.01.030 0022-3549/© 2020 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
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T. Fatima et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
Table 1 Formulation Composition of Pilocarpine Nitrate Topical Solutions Formulation
Penetration Enhancer
Penetration Enhancer Concentration (%w/w)
Pilocarpine Nitratea (%w/w)
Ethanol (%w/w)
Water (%w/w)
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11
Transcutol® HP
5% 5% 5% 5% 5% 5% 9% 9% 5% 5% 6%
27% 27% 27% 27% 27% 27% 45% 45% 59.5% 59.5% 45%
100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%
S12
PEG 200 þ menthol þ salicylic acid
5% 10% 5% 10% 5% 10% 2.5% 5% 5% 10% 10% 4% 10% 4% 10%
6%
45%
100% (q.s)
a
PEG 400 PEG 200 Menthol Salicylic acid PEG 200 þ menthol
(q.s) (q.s) (q.s) (q.s) (q.s) (q.s) (q.s) (q.s) (q.s) (q.s) (q.s)
Calculated on basis of pilocarpine base.
induced by stimulation of cholinergic muscarinic receptors. The iontophoretic delivery of pilocarpine (muscarinic receptor agonist) by applying a current of 2.5-4.0 mA over a gauge (5.1 cm 5.1 cm) placed on the forearm soaked in 3 mL of pilocarpine solution (0.2%0.5% w/w) followed by collection of sweat for 30 min with the use of gauze/filter paper or macroduct coils and chloride concentration in sweat is determined using a chloride analyzer.14,15 Sweat test is generally performed in neonates. Application of current for iontophoresis causes discomfort (tingling sensation), with reported incidences of skin burn, rashes, erythema, and so forth. To avoid burns occurring during application of current, an automatic current limiter incorporated will shut down the iontophoresis when current exceeds 1.75 mA. In spite of this safety feature, there still exist burn incidences.16 Hence, developing a compliant topical formulation to deliver sufficient quantity of pilocarpine, which could generate significant quantity of sweat to quantify the chloride concentration, could be beneficial over iontophoresis.17,18
glycol monoethyl ether) was obtained as a gift sample from Gattefosse, USA. Combined acid buffer and chloride standard (100 mmol/L of chloride) were purchased from Nelson Jameson Inc. (Marshfield, WI). Adult Electrodes (3M Red Dot™) and adhesive tape (Transpore™) were purchased from 3M Science, USA. Heat bandages (ThermaCare Heatwraps) were purchased from Walmart, USA. All solvents used in the analysis were of HPLC grade. Methods Preparation of Pilocarpine Topical Formulations Pilocarpine nitrate topical solutions were prepared by using 5 different penetration enhancers (Transcutol® HP, PEG 400, PEG 200, menthol, and salicylic acid) either alone or in combination. The composition of pilocarpine nitrate topical solutions is presented in Table 1. The solutions were prepared by dissolving all the ingredients in a hydroalcoholic solution of water and ethanol with the help of a vortex mixer. The pH of all the solutions was adjusted to ~4 using sodium hydroxide.
Experimental Materials Pilocarpine nitrate (98.0%), salicylic acid (99%), and polyethylene glycol 200 and 400 (PEG 200 and PEG 400) were purchased from TCI America (Portland, OR). Menthol was purchased from Ward’s science (Rochester, NY). Sodium hydroxide was procured from VWR Amresco lifescience. Transcutol® HP (diethylene
In Vitro Penetration Studies In vitro penetration studies were carried out using Franz diffusion apparatus of 1.77 cm2 active diffusion area. Fullthickness porcine skin obtained from a local slaughterhouse was refrigerated at 80 C till the time of study. Porcine skin was thawed and cleaned just before the study and mounted in between the donor and the receiver compartments of the Franz diffusion apparatus. The receiver compartment was filled with
Table 2 Results of the In Vitro Penetration Studies After Application of Pilocarpine Formulations and Control Solution Formulation
Amount Penetrated After 10 Min (mg/cm2)
Amount Penetrated After 40 Min (mg/cm2)
Control (Aq. pilocarpine solution 9% w/w) S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12
43.48 ± 6.94 108.23 ± 46.70 115.54 ± 54.08 79.81 ± 31.35 68.99 ± 0.57 69.81 ± 6.25 57.38 ± 47.65 68.76 ± 8.97 71.26 ± 19.82 106.29 ± 4.46 120.29 ± 27.54 105.45 ± 41.58 145.91 ± 43.07
58.07 ± 15.91 89.27 ± 35.63 82.58 ± 13.23 74.99 ± 13.04 80.20 ± 33.93 106.14 ± 22.42 69.54 ± 8.06 73.43 ± 15.94 78.92 ± 10.18 139.65 ± 34.39 158.97 ± 20.15 140.60 ± 38.46 138.02 ± 23.33
The values represented are mean ± SD (n ¼ 3).
T. Fatima et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
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Table 3 Amount of Pilocarpine Delivered by Lead Topical Formulation and Iontophoresis Mode of Delivery
Formulation
Skin Temperature ( C)
Amount of Pilocarpine Penetrated After 10 Min (mg/cm2)
Amount of Pilocarpine Penetrated After 40 Min (mg/cm2)
Topical Formulation Topical Formulation Iontophoresis
S12 S12 Aqueous Pilocarpine Solution
32 40 32
145.91 ± 43.07 152.04 ± 52.23 97.05 ± 27.93
138.02 ± 23.33 210.27 ± 53.72 140.56 ± 88.66
The values represented are mean ± SD (n ¼ 6).
Comparison of Extent of Pilocarpine Penetration into the Skin by Topical Pilocarpine Formulation Under Normal Condition, Topical Pilocarpine Formulation Under Mild Hyperthermia and Iontophoresis of Aqueous Pilocarpine Solution In vitro penetration studies of the lead topical formulation (S12) were performed under normal condition to determine whether the pilocarpine formulation was as effective as iontophoresis in terms of drug penetration into the skin. In addition, lead topical pilocarpine formulation under mild hyperthermic condition (40 ± 1 C) was also tested, as hyperthermic condition is known to increase the drug penetration into the skin.19 The experimental setup for in vitro penetration studies remained the same as detailed in the section In Vitro Penetration Studies except that the cells were divided into 3 groups such that the skin temperature of first and third groups were maintained at 32 C, whereas the second group was maintained at 40 C. On the first and second groups of the skin, topical pilocarpine solution (S12) was applied, and the third group was subjected to iontophoresis, applying 0.5% w/w aqueous pilocarpine solution. Iontophoresis was a control study in which aqueous pilocarpine solution was applied on epidermal area of the fullthickness porcine skin. A dose of 115 mL/cm2 was selected based on the total amount of pilocarpine solution applied for sweat test (3 mL applied over an area of 26 cm2). Two 3M Red Dot™ adult electrodes were connected to either side of the skin surface, the electrode fixed to the epidermal surface was connected to the
Analytical Method and Sample Preparation for Determination of Pilocarpine Content Pilocarpine in the samples was quantified using reversedphase HPLC technique. Shimadzu UFLC equipped with a LC-20AB
a Amount penetrated (μg/cm2)
Screening of Topical Pilocarpine Formulations by In Vitro Penetration Studies The objective of the study was to develop a formulation that is as efficient as the iontophoretic delivery of pilocarpine. Different topical pilocarpine formulations were prepared with varying concentrations of drug and penetration enhancers. These formulations were screened along with control (9% w/w aq. pilocarpine solution) by in vitro skin penetration studies to evaluate their efficiency to penetrate pilocarpine into the skin. In vitro penetration studies were carried out as described in the section In Vitro Penetration Studies, and the amount of pilocarpine penetrated into the skin was estimated by analyzing the processed samples by HPLC.
positive terminal of the battery to act as an anode. The one fixed to the dermal surface was connected to the negative terminal of the battery to act as a cathode. A 0.5 mA of current was applied for 10 min using Chattanooga Ionto™. The potential created due to the supply of current drives the pilocarpine across the skin because of its positive polarity. The amount of pilocarpine penetrated after 10 min and 40 min was determined by drug extraction technique as mentioned in the section In Vitro Penetration Studies.
250
200
150
100
50
0 Topical Formula on Topical Formula on Iontophoresis (Skin (Skin temp - 32°C) (Skin temp - 40°C) temp - 32°C)
b Amount penetrated (μg/cm2)
phosphate buffer pH 7.4. The Franz diffusion assembly was maintained at 32 ± 1 C or 40 ± 1 C with the help of thermostatic water circulator. Topical pilocarpine formulations were uniformly spread across the porcine skin at a dose of 115 mL/cm2 and were left to remain in contact for either 10 or 40 min. After the study period, the epidermal and subcutaneous surfaces of the porcine skin were washed using a validated protocol. The washing protocol was validated to ensure removal of any superficial drug on skin. Five milliliter of methanol-water (50:50% v/v) solution was flushed on each of the epidermal and subcutaneous surfaces of the porcine skin for 3 consecutive times (total 15 mL). After washing the skin, additional 1 mL of methanol-water solution was pipetted onto the skin and collected. The amount of drug present in 1 mL of methanol-water solution was determined using HPLC, and pilocarpine was not found in these samples.
300
**
250 200 150 100 50 0
Topical Formula on Topical Formula on Iontophoresis (Skin (Skin temp - 32°C) (Skin temp - 40°C) temp - 32°C) Figure 1. Amount of pilocarpine delivered by lead topical formulation (S12), topical formulation under mild hyperthermic condition, and iontophoresis after (a) 10 min (b) 40 min. For iontophoresis, 0.28 mA/cm2 current was applied using Ag/AgCl electrodes. (p < 0.01)**.
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T. Fatima et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
pump and a photodiode array detector was used for separation and determination of pilocarpine in the samples. The mobile phase consisted of 10 mM of potassium phosphate solution (adjusted to pH 3 with orthophosphoric acid) and methanol in the ratio 90:10 maintained at a flow rate of 0.6 mL/min. Detection wavelength was set at 215 nm. The injection volume was 30 mL, and LUNA 5m C18 (250 460 mm) column (Phenomenex, USA) was used to separate pilocarpine from the samples. The retention time of pilocarpine was 4.6 min. Sample Preparation. To determine the amount of pilocarpine penetrated into the porcine skin, the epidermal and dermal surfaces
Concentration of chlorideðmMol=LÞ ¼
combined buffer. The silver ions are generated till the chloride ions are completely neutralized forming silver chloride which is detected by the detector electrodes. The chloride analyzer works on the principle that the chloride ions present in the sample is equal to the number of silver ions produced by the electrodes to form silver chloride. The filter paper with sweat was transferred to a petri dish containing combined buffer and was then agitated to extract the chloride ions into the combined buffer. The combined buffer was then titrated to determine the chloride concentration in the sweat. Chloride concentration (mMol/L) in the sweat was determined by using the formula as mentioned in the following:
½20 R Density of sweat Weight of sweat produced
were thoroughly washed by a validated cleaning procedure. The procedure involved mincing of the skin into small pieces and vortexing overnight in a tube containing 15 mL of water. The pilocarpine extracted from the skin was analyzed by HPLC. For HPLC analysis, 600 mL of basified acetonitrile (1000 parts of acetonitrile and 1 part of 10% W/W ammonium hydroxide) was added to an Eppendorf containing 300 mL of skin extract. The samples were vortexed and centrifuged at 13,000 revolutions per minute (rpm) for 15 min. The supernatant was collected and transferred to HPLC vials for analysis. This extraction procedure was validated, and the extraction method had a recovery of >90%.
ReReading on the analyzer, density of sweate1.0047 g/cm3.
Clinical Evaluation of Topical Formulation The clinical study was conducted at Bangabandhu Sheikh Mujib Medical University as per the approved protocol by the institutional review board. The topical pilocarpine formulation (S12) was evaluated in 20 healthy human volunteers for its ability to stimulate sweat production. The right and the left forearms of the volunteers were washed with distilled water, allowed to dry, and 2 circular areas of 117 cm2 were marked on each of the forearms. On the right forearm, 400 mL of topical pilocarpine formulation (S12) preheated to 32 C was applied uniformly on the demarcated skin surface and allowed to dry for 10 min. A preweighed filter paper was placed on the application site of the formulation and was then covered with a plastic wrap, heat bandage, and a parafilm for 30 min to mimic the hyperthermia condition. The plastic wrap, heat bandage, and parafilm were removed, and the filter paper was collected and weighed immediately to determine the amount of sweat produced. Similarly, on the left forearm, the same procedure was followed except that water (control) was used instead of pilocarpine formulation. At the end of the study, both the forearms were washed with soap and water.
The results of the in vitro penetration studies showed that the formulations delivered more pilocarpine than the control (aqueous pilocarpine solution). The formulation with salicylic acid at 10% w/w showed highest penetration, when compared with the formulations with a single penetration enhancer. Among the different formulations, the S12 formulation which had a combination of penetration enhancers, menthol, and salicylic acid resulted in higher delivery compared with all other formulations. In certain formulations, the amount of drug present in the skin at the end of 40 min was less compared with that of the drug present at the end of 10 min. This may be attributed to the permeation of the penetrated drug from the deeper layers of the skin into the receiver compartment. The results of the in vitro penetration studies are presented in Table 2. Based on the results of the screening studies, S12 formulation was selected as lead formulation for further studies. S12 had 10% w/w PEG 200, 4% w/w menthol, and 10% w/w salicylic acid. The PEG 200 would act as a hydrating agent for the skin, whereas the menthol exhibits a cooling action to counter the irritation caused by salicylic acid.
Determination of Chloride Concentration. The chloride concentration of the sweat collected from the filter paper was determined using a calibrated chloride analyzer (CORNING Chloride Analyzer 925). The chloride analyzer consists of a pair of detector electrodes, silver anode, cathode, and a stirrer immersed in a combined buffer. Sample to be analyzed was added to the combined buffer in which the electrodes were immersed. The silver anode generates silver ions in the presence of chloride ions in the
Statistical Analysis Statistical analysis of the results was performed using unpaired t-test. A p < 0.05 was considered to be statistically significant. The data in the results are represented as mean ± standard deviation (SD). Results and Discussion Screening of Topical Pilocarpine Solutions by In Vitro Penetration Studies
Comparison of Extent of Pilocarpine Penetration into the Skin by Topical Pilocarpine Formulation Under Normal Condition, Topical Pilocarpine Formulation Under Mild Hyperthermia and Iontophoresis of Aqueous Pilocarpine Solution The results of the in vitro studies indicate that there was increased penetration of pilocarpine with corresponding increase in the skin temperature. There was no significant difference in the amount of pilocarpine recovered from the skin maintained at 32 C and 40 C immediately after 10 min of application of topical
T. Fatima et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5 Table 4 Amount of Sweat Collected and the Chloride Concentration in Sweat After Application of Topical Pilocarpine Formulation (S12) (***p < 0.001) and Control Formulation Formulation
Sweat Collected (mg)
Chloride Concentration (mMol/L)
Test formulation Control
77.28 ± 18.97*** 16.75 ± 9.71
11.67 ± 6.22 e
The values represented are mean ± SD, (n ¼ 20).
formulation. The pilocarpine recovered from the skin maintained at 40 C was 210.27 ± 53.72 mg/cm2, which was higher than the drug recovered from the skin maintained at 32 C (138.02 ± 23.33 mg/ cm2) after 40 min of application, showing that the drug penetration increased with mild hyperthermia. The drug extraction studies also indicated that the total amount of penetrated pilocarpine after application of lead topical formulation was significantly higher than iontophoresis with 0.5% w/w aqueous pilocarpine solution. In case of S12 formulation, the amount of pilocarpine penetrated was 152.04 ± 52.23 mg/cm2 after 10 min of application and 210.27 ± 53.72 mg/cm2 after 40 min of application. However, iontophoresis of 0.5% aqueous pilocarpine solution for 10 min on porcine skin led to a drug penetration of 97.05 ± 27.93 mg/cm2, whereas the amount of pilocarpine penetrated into skin after 40 min of study (10 min of iontophoresis followed by 30 min of contact of aqueous pilocarpine solution to the porcine skin) was 140.56 ± 88.66 mg/cm2 (Table 3; Figure 1). Clinical Evaluation in Human Volunteers. The results of the in vitro studies were clearly translated in the clinical study. The study showed that the amount of sweat collected for the topical pilocarpine formulation (S12) was significantly higher compared with that of the control solution. The amount of sweat produced by the topical pilocarpine formulation (77.28 ± 18.97 mg) was sufficient to determine the chloride concentration in the sweat by a chloride analyzer. All the individuals were healthy and were found to have chloride concentration in the normal range. The results of the clinical studies are presented in Table 4. Conclusion Topical pilocarpine formulation was successful in inducing sweat formation by passive penetration of pilocarpine without the aid of any physical method of penetration enhancement. The sweat
5
generated by topical pilocarpine formulation as indicated by the in vivo study on human volunteers was sufficient to measure the chloride concentration. Thus, the topical pilocarpine formulation could be a safe and compliant alternative diagnostic tool for CF.
References 1. O'Sullivan BP, Freedman SD. Cystic fibrosis. Lancet. 2009;373:1891-1904. 2. Cystic Fibrosis Foundation. Cystic fibrosis foundation patient Registry. 2016 Annual Data Report. Available at: https://www.cff.org/Research/ResearcherResources/Patient-Registry/2017-Patient-Registry-Annual-Data-Report.pdf. Accessed February 27, 2020. 3. Katz KA, Yan AC, Turner ML. Aquagenic wrinkling of the palms in patients with cystic fibrosis homozygous for the △F508 CFTR mutation. Arch Dermatol. 2005;141:621-624. 4. Boucher RC. New concepts of the pathogenesis of cystic fibrosis lung disease. Eur Respir J. 2004;23:146-158. C. The ABC protein turned chloride channel whose 5. Gadsby DC, Paola V, L aszlo failure causes cystic fibrosis. Nature. 2006;440:477-483. 6. Rommens JM, lannuzzi MC, Kerem B, et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science. 1989;245:1059-1065. 7. Veeze HJ. Diagnosis of cystic fibrosis. Neth J Med. 1995;46:271-274. 8. Cheng SH, Gregory RJ, Marshall J, et al. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell. 1990;63: 827-834. 9. Flume PA. Pulmonary complications of cystic fibrosis. Respir Care. 2009;54:618627. 10. Abrams CK, Hamosh M, Hubbard VS, Dutta SK, Hamosh P. Lingual lipase in cystic fibrosis. Quantitation of enzyme activity in the upper small intestine of patients with exocrine pancreatic insufficiency. J Clin Invest. 1984;73:374-382. 11. Donaldson SH, Bennett WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. Mucus clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med. 2006;354:241-250. 12. Ramsey BW, Davies J, McElvaney NG, et al. A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med. 2011;365:1663-1672. 13. Wainwright CE, Baloch NU, Janahi IA. Lumacaftoreivacaftor in patients with cystic fibrosis homozygous for Phe508del CFTR. N Engl J Med. 2015;373:220-231. 14. Warwick WJ, Huang NN, Waring WW, et al. Evaluation of cystic fibrosis screening system incorporating a miniature sweat stimulator and disposable chloride sensor. Clin Chem. 1986;32:850-853. 15. Gartner S. Protocol for the diagnosis and follow up of patients with cystic fibrosis. An Pediatr (Barc). 2009;71:250-264. 16. Rattenbury JM, Worthy E. Is the sweat test safe? Some instances of burns received during pilocarpine iontophoresis. Ann Clin Biochem. 1996;33:456-458. 17. LeGrys VA, Yankaskas JR, Quittell LM, Marshall BC, Mogayzel PJ. Diagnostic sweat testing: the cystic fibrosis foundation guidelines. J Pediatr. 2007;151: 85-89. 18. Abdulhamid I, Kleyn M, Langbo C, et al. Improving the rate of sufficient sweat collected in infants referred for sweat testing in Michigan. Glob Pediatr Health. 2014;1, 2333794X14553625. 19. Panda A, Sharma PK, Murthy SN. Effect of mild hyperthermia on transdermal absorption of nicotine from patches. AAPS PharmSciTech. 2019;20(2):77.
Contents lists available at ScienceDirect
Journal of Pharmaceutical Sciences journal homepage: www.jpharmsci.org
Research Article
Topical Pilocarpine Formulation for Diagnosis of Cystic Fibrosis Tasnim Fatima 1, Srinivas Ajjarapu 1, Vijay Kumar Shankar 1, Srinath Rangappa 1, H. Nanjappa Shivakumar 2, 3, Subrata Kumar Biswas 4, Mozammel Hoque 4, S. Narasimha Murthy 1, * 1
Department of Pharmaceutics of Drug Delivery, University of Mississippi, University, Mississippi 38677 KLE College of Pharmacy, Bengaluru, Karnataka, India 3 Institute for Drug Delivery and Biomedical Research (IDBR), Pharmaceutics, Bengaluru, Karnataka, India 4 Department of Biochemistry, Bangabandhu Sheikh Mujib Medical University, Dhaka, Dhaka District, Bangladesh 2
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 January 2020 Revised 27 January 2020 Accepted 29 January 2020
Cystic fibrosis is diagnosed in infants by estimating the levels of chloride ions present in the sweat induced by iontophoresis of pilocarpine solution. Elevated levels of chloride (60 mMol/L) in sweat are indicative of cystic fibrosis. However, the iontophoretic method of delivering pilocarpine is cumbersome and usually is associated with several side effects such as skin burn, skin rashes, erythema, and so forth. The objective of this study was therefore to develop a topical formulation that delivers adequate amount of pilocarpine. The drug delivery of formulation was compared with iontophoresis of aqueous solution of pilocarpine nitrate in vitro using porcine skin model. The pilocarpine levels in the skin exposed to topical pilocarpine solution under mild hyperthermia was 152.04 ± 52.23 mg/cm2 after 10 min of application, whereas it was 97.05 ± 27.93 mg/cm2 in the skin after 10 min of iontophoresis. The topical formulation was subjected to clinical evaluation to assess the efficacy of the product to induce sweat production. The average amount of the sweat secreted on application of topical formulation was found to be 77.28 ± 18.97 mg. Based on these results, it was found that the topical formulation was successful in delivering pilocarpine and to stimulate sweat secretion. © 2020 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
Keywords: skin permeation enhancer(s) clinical trial(s) formulation iontophoresis
Introduction Cystic fibrosis (CF) is a progressive, genetic disease inherited in an autosomal recessive manner.1 According to the Cystic Fibrosis Foundation Patient Registry, there are around 30,000 people with CF in the United States alone and more than 70,000 people worldwide. Every year, approximately 1000 new cases of CF are diagnosed in the United States.2 CF manifests as a persistent lung infection, aquagenic wrinkling of the palms, exocrine pancreatic insufficiency, and presence of excessive amount of salt in the sweat.3,4
Abbreviations used: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; HPLC, high-performance liquid chromatography; PEG, polyethylene glycol; PDA, photodiode array; rpm, revolutions per minute; SD, standard deviation. Conflict of interest: None. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. * Correspondence to: S. Narasimha Murthy (Telephone: þ1-662-915-5164). E-mail address: [email protected] (S.N. Murthy).
CF is caused because of mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene located on chromosome 7, resulting in malfunctioning of CFTR proteins. CFTR is a membrane protein and chloride channel present in the lungs, pancreas, intestine, sweat glands, and other exocrine glands. CFTR regulates the transport of sodium, chloride, and bicarbonate ions maintaining transepithelial homeostasis.5,6 Defective CFTR proteins affect gland duct reabsorption potential of electrolytes, leading to excess of salt in the sweat.7,8 There are several complications associated with CF such as bronchiectasis, chronic infections, pneumothorax, respiratory failure, and others.9 Management of CF involves maintenance of lung function by clearing airway, administering bronchodilators, antibiotics, and pancreatic enzyme replacement therapy.10,11 Ivacaftor alone or in combination with lumacaftor is used for improvement of defective CFTR’s function.12,13 Although incurable, early diagnosis and close monitoring could help in management of CF. Methods for diagnosis of CF includes sweat testing or genetic test. The levels of chloride ions in sweat are estimated for diagnosis of CF. Elevated levels of chloride (60 mMol/L) in sweat is indicative of CF. The sweat in patients with CF is
https://doi.org/10.1016/j.xphs.2020.01.030 0022-3549/© 2020 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
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T. Fatima et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
Table 1 Formulation Composition of Pilocarpine Nitrate Topical Solutions Formulation
Penetration Enhancer
Penetration Enhancer Concentration (%w/w)
Pilocarpine Nitratea (%w/w)
Ethanol (%w/w)
Water (%w/w)
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11
Transcutol® HP
5% 5% 5% 5% 5% 5% 9% 9% 5% 5% 6%
27% 27% 27% 27% 27% 27% 45% 45% 59.5% 59.5% 45%
100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%
S12
PEG 200 þ menthol þ salicylic acid
5% 10% 5% 10% 5% 10% 2.5% 5% 5% 10% 10% 4% 10% 4% 10%
6%
45%
100% (q.s)
a
PEG 400 PEG 200 Menthol Salicylic acid PEG 200 þ menthol
(q.s) (q.s) (q.s) (q.s) (q.s) (q.s) (q.s) (q.s) (q.s) (q.s) (q.s)
Calculated on basis of pilocarpine base.
induced by stimulation of cholinergic muscarinic receptors. The iontophoretic delivery of pilocarpine (muscarinic receptor agonist) by applying a current of 2.5-4.0 mA over a gauge (5.1 cm 5.1 cm) placed on the forearm soaked in 3 mL of pilocarpine solution (0.2%0.5% w/w) followed by collection of sweat for 30 min with the use of gauze/filter paper or macroduct coils and chloride concentration in sweat is determined using a chloride analyzer.14,15 Sweat test is generally performed in neonates. Application of current for iontophoresis causes discomfort (tingling sensation), with reported incidences of skin burn, rashes, erythema, and so forth. To avoid burns occurring during application of current, an automatic current limiter incorporated will shut down the iontophoresis when current exceeds 1.75 mA. In spite of this safety feature, there still exist burn incidences.16 Hence, developing a compliant topical formulation to deliver sufficient quantity of pilocarpine, which could generate significant quantity of sweat to quantify the chloride concentration, could be beneficial over iontophoresis.17,18
glycol monoethyl ether) was obtained as a gift sample from Gattefosse, USA. Combined acid buffer and chloride standard (100 mmol/L of chloride) were purchased from Nelson Jameson Inc. (Marshfield, WI). Adult Electrodes (3M Red Dot™) and adhesive tape (Transpore™) were purchased from 3M Science, USA. Heat bandages (ThermaCare Heatwraps) were purchased from Walmart, USA. All solvents used in the analysis were of HPLC grade. Methods Preparation of Pilocarpine Topical Formulations Pilocarpine nitrate topical solutions were prepared by using 5 different penetration enhancers (Transcutol® HP, PEG 400, PEG 200, menthol, and salicylic acid) either alone or in combination. The composition of pilocarpine nitrate topical solutions is presented in Table 1. The solutions were prepared by dissolving all the ingredients in a hydroalcoholic solution of water and ethanol with the help of a vortex mixer. The pH of all the solutions was adjusted to ~4 using sodium hydroxide.
Experimental Materials Pilocarpine nitrate (98.0%), salicylic acid (99%), and polyethylene glycol 200 and 400 (PEG 200 and PEG 400) were purchased from TCI America (Portland, OR). Menthol was purchased from Ward’s science (Rochester, NY). Sodium hydroxide was procured from VWR Amresco lifescience. Transcutol® HP (diethylene
In Vitro Penetration Studies In vitro penetration studies were carried out using Franz diffusion apparatus of 1.77 cm2 active diffusion area. Fullthickness porcine skin obtained from a local slaughterhouse was refrigerated at 80 C till the time of study. Porcine skin was thawed and cleaned just before the study and mounted in between the donor and the receiver compartments of the Franz diffusion apparatus. The receiver compartment was filled with
Table 2 Results of the In Vitro Penetration Studies After Application of Pilocarpine Formulations and Control Solution Formulation
Amount Penetrated After 10 Min (mg/cm2)
Amount Penetrated After 40 Min (mg/cm2)
Control (Aq. pilocarpine solution 9% w/w) S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12
43.48 ± 6.94 108.23 ± 46.70 115.54 ± 54.08 79.81 ± 31.35 68.99 ± 0.57 69.81 ± 6.25 57.38 ± 47.65 68.76 ± 8.97 71.26 ± 19.82 106.29 ± 4.46 120.29 ± 27.54 105.45 ± 41.58 145.91 ± 43.07
58.07 ± 15.91 89.27 ± 35.63 82.58 ± 13.23 74.99 ± 13.04 80.20 ± 33.93 106.14 ± 22.42 69.54 ± 8.06 73.43 ± 15.94 78.92 ± 10.18 139.65 ± 34.39 158.97 ± 20.15 140.60 ± 38.46 138.02 ± 23.33
The values represented are mean ± SD (n ¼ 3).
T. Fatima et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
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Table 3 Amount of Pilocarpine Delivered by Lead Topical Formulation and Iontophoresis Mode of Delivery
Formulation
Skin Temperature ( C)
Amount of Pilocarpine Penetrated After 10 Min (mg/cm2)
Amount of Pilocarpine Penetrated After 40 Min (mg/cm2)
Topical Formulation Topical Formulation Iontophoresis
S12 S12 Aqueous Pilocarpine Solution
32 40 32
145.91 ± 43.07 152.04 ± 52.23 97.05 ± 27.93
138.02 ± 23.33 210.27 ± 53.72 140.56 ± 88.66
The values represented are mean ± SD (n ¼ 6).
Comparison of Extent of Pilocarpine Penetration into the Skin by Topical Pilocarpine Formulation Under Normal Condition, Topical Pilocarpine Formulation Under Mild Hyperthermia and Iontophoresis of Aqueous Pilocarpine Solution In vitro penetration studies of the lead topical formulation (S12) were performed under normal condition to determine whether the pilocarpine formulation was as effective as iontophoresis in terms of drug penetration into the skin. In addition, lead topical pilocarpine formulation under mild hyperthermic condition (40 ± 1 C) was also tested, as hyperthermic condition is known to increase the drug penetration into the skin.19 The experimental setup for in vitro penetration studies remained the same as detailed in the section In Vitro Penetration Studies except that the cells were divided into 3 groups such that the skin temperature of first and third groups were maintained at 32 C, whereas the second group was maintained at 40 C. On the first and second groups of the skin, topical pilocarpine solution (S12) was applied, and the third group was subjected to iontophoresis, applying 0.5% w/w aqueous pilocarpine solution. Iontophoresis was a control study in which aqueous pilocarpine solution was applied on epidermal area of the fullthickness porcine skin. A dose of 115 mL/cm2 was selected based on the total amount of pilocarpine solution applied for sweat test (3 mL applied over an area of 26 cm2). Two 3M Red Dot™ adult electrodes were connected to either side of the skin surface, the electrode fixed to the epidermal surface was connected to the
Analytical Method and Sample Preparation for Determination of Pilocarpine Content Pilocarpine in the samples was quantified using reversedphase HPLC technique. Shimadzu UFLC equipped with a LC-20AB
a Amount penetrated (μg/cm2)
Screening of Topical Pilocarpine Formulations by In Vitro Penetration Studies The objective of the study was to develop a formulation that is as efficient as the iontophoretic delivery of pilocarpine. Different topical pilocarpine formulations were prepared with varying concentrations of drug and penetration enhancers. These formulations were screened along with control (9% w/w aq. pilocarpine solution) by in vitro skin penetration studies to evaluate their efficiency to penetrate pilocarpine into the skin. In vitro penetration studies were carried out as described in the section In Vitro Penetration Studies, and the amount of pilocarpine penetrated into the skin was estimated by analyzing the processed samples by HPLC.
positive terminal of the battery to act as an anode. The one fixed to the dermal surface was connected to the negative terminal of the battery to act as a cathode. A 0.5 mA of current was applied for 10 min using Chattanooga Ionto™. The potential created due to the supply of current drives the pilocarpine across the skin because of its positive polarity. The amount of pilocarpine penetrated after 10 min and 40 min was determined by drug extraction technique as mentioned in the section In Vitro Penetration Studies.
250
200
150
100
50
0 Topical Formula on Topical Formula on Iontophoresis (Skin (Skin temp - 32°C) (Skin temp - 40°C) temp - 32°C)
b Amount penetrated (μg/cm2)
phosphate buffer pH 7.4. The Franz diffusion assembly was maintained at 32 ± 1 C or 40 ± 1 C with the help of thermostatic water circulator. Topical pilocarpine formulations were uniformly spread across the porcine skin at a dose of 115 mL/cm2 and were left to remain in contact for either 10 or 40 min. After the study period, the epidermal and subcutaneous surfaces of the porcine skin were washed using a validated protocol. The washing protocol was validated to ensure removal of any superficial drug on skin. Five milliliter of methanol-water (50:50% v/v) solution was flushed on each of the epidermal and subcutaneous surfaces of the porcine skin for 3 consecutive times (total 15 mL). After washing the skin, additional 1 mL of methanol-water solution was pipetted onto the skin and collected. The amount of drug present in 1 mL of methanol-water solution was determined using HPLC, and pilocarpine was not found in these samples.
300
**
250 200 150 100 50 0
Topical Formula on Topical Formula on Iontophoresis (Skin (Skin temp - 32°C) (Skin temp - 40°C) temp - 32°C) Figure 1. Amount of pilocarpine delivered by lead topical formulation (S12), topical formulation under mild hyperthermic condition, and iontophoresis after (a) 10 min (b) 40 min. For iontophoresis, 0.28 mA/cm2 current was applied using Ag/AgCl electrodes. (p < 0.01)**.
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T. Fatima et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5
pump and a photodiode array detector was used for separation and determination of pilocarpine in the samples. The mobile phase consisted of 10 mM of potassium phosphate solution (adjusted to pH 3 with orthophosphoric acid) and methanol in the ratio 90:10 maintained at a flow rate of 0.6 mL/min. Detection wavelength was set at 215 nm. The injection volume was 30 mL, and LUNA 5m C18 (250 460 mm) column (Phenomenex, USA) was used to separate pilocarpine from the samples. The retention time of pilocarpine was 4.6 min. Sample Preparation. To determine the amount of pilocarpine penetrated into the porcine skin, the epidermal and dermal surfaces
Concentration of chlorideðmMol=LÞ ¼
combined buffer. The silver ions are generated till the chloride ions are completely neutralized forming silver chloride which is detected by the detector electrodes. The chloride analyzer works on the principle that the chloride ions present in the sample is equal to the number of silver ions produced by the electrodes to form silver chloride. The filter paper with sweat was transferred to a petri dish containing combined buffer and was then agitated to extract the chloride ions into the combined buffer. The combined buffer was then titrated to determine the chloride concentration in the sweat. Chloride concentration (mMol/L) in the sweat was determined by using the formula as mentioned in the following:
½20 R Density of sweat Weight of sweat produced
were thoroughly washed by a validated cleaning procedure. The procedure involved mincing of the skin into small pieces and vortexing overnight in a tube containing 15 mL of water. The pilocarpine extracted from the skin was analyzed by HPLC. For HPLC analysis, 600 mL of basified acetonitrile (1000 parts of acetonitrile and 1 part of 10% W/W ammonium hydroxide) was added to an Eppendorf containing 300 mL of skin extract. The samples were vortexed and centrifuged at 13,000 revolutions per minute (rpm) for 15 min. The supernatant was collected and transferred to HPLC vials for analysis. This extraction procedure was validated, and the extraction method had a recovery of >90%.
ReReading on the analyzer, density of sweate1.0047 g/cm3.
Clinical Evaluation of Topical Formulation The clinical study was conducted at Bangabandhu Sheikh Mujib Medical University as per the approved protocol by the institutional review board. The topical pilocarpine formulation (S12) was evaluated in 20 healthy human volunteers for its ability to stimulate sweat production. The right and the left forearms of the volunteers were washed with distilled water, allowed to dry, and 2 circular areas of 117 cm2 were marked on each of the forearms. On the right forearm, 400 mL of topical pilocarpine formulation (S12) preheated to 32 C was applied uniformly on the demarcated skin surface and allowed to dry for 10 min. A preweighed filter paper was placed on the application site of the formulation and was then covered with a plastic wrap, heat bandage, and a parafilm for 30 min to mimic the hyperthermia condition. The plastic wrap, heat bandage, and parafilm were removed, and the filter paper was collected and weighed immediately to determine the amount of sweat produced. Similarly, on the left forearm, the same procedure was followed except that water (control) was used instead of pilocarpine formulation. At the end of the study, both the forearms were washed with soap and water.
The results of the in vitro penetration studies showed that the formulations delivered more pilocarpine than the control (aqueous pilocarpine solution). The formulation with salicylic acid at 10% w/w showed highest penetration, when compared with the formulations with a single penetration enhancer. Among the different formulations, the S12 formulation which had a combination of penetration enhancers, menthol, and salicylic acid resulted in higher delivery compared with all other formulations. In certain formulations, the amount of drug present in the skin at the end of 40 min was less compared with that of the drug present at the end of 10 min. This may be attributed to the permeation of the penetrated drug from the deeper layers of the skin into the receiver compartment. The results of the in vitro penetration studies are presented in Table 2. Based on the results of the screening studies, S12 formulation was selected as lead formulation for further studies. S12 had 10% w/w PEG 200, 4% w/w menthol, and 10% w/w salicylic acid. The PEG 200 would act as a hydrating agent for the skin, whereas the menthol exhibits a cooling action to counter the irritation caused by salicylic acid.
Determination of Chloride Concentration. The chloride concentration of the sweat collected from the filter paper was determined using a calibrated chloride analyzer (CORNING Chloride Analyzer 925). The chloride analyzer consists of a pair of detector electrodes, silver anode, cathode, and a stirrer immersed in a combined buffer. Sample to be analyzed was added to the combined buffer in which the electrodes were immersed. The silver anode generates silver ions in the presence of chloride ions in the
Statistical Analysis Statistical analysis of the results was performed using unpaired t-test. A p < 0.05 was considered to be statistically significant. The data in the results are represented as mean ± standard deviation (SD). Results and Discussion Screening of Topical Pilocarpine Solutions by In Vitro Penetration Studies
Comparison of Extent of Pilocarpine Penetration into the Skin by Topical Pilocarpine Formulation Under Normal Condition, Topical Pilocarpine Formulation Under Mild Hyperthermia and Iontophoresis of Aqueous Pilocarpine Solution The results of the in vitro studies indicate that there was increased penetration of pilocarpine with corresponding increase in the skin temperature. There was no significant difference in the amount of pilocarpine recovered from the skin maintained at 32 C and 40 C immediately after 10 min of application of topical
T. Fatima et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-5 Table 4 Amount of Sweat Collected and the Chloride Concentration in Sweat After Application of Topical Pilocarpine Formulation (S12) (***p < 0.001) and Control Formulation Formulation
Sweat Collected (mg)
Chloride Concentration (mMol/L)
Test formulation Control
77.28 ± 18.97*** 16.75 ± 9.71
11.67 ± 6.22 e
The values represented are mean ± SD, (n ¼ 20).
formulation. The pilocarpine recovered from the skin maintained at 40 C was 210.27 ± 53.72 mg/cm2, which was higher than the drug recovered from the skin maintained at 32 C (138.02 ± 23.33 mg/ cm2) after 40 min of application, showing that the drug penetration increased with mild hyperthermia. The drug extraction studies also indicated that the total amount of penetrated pilocarpine after application of lead topical formulation was significantly higher than iontophoresis with 0.5% w/w aqueous pilocarpine solution. In case of S12 formulation, the amount of pilocarpine penetrated was 152.04 ± 52.23 mg/cm2 after 10 min of application and 210.27 ± 53.72 mg/cm2 after 40 min of application. However, iontophoresis of 0.5% aqueous pilocarpine solution for 10 min on porcine skin led to a drug penetration of 97.05 ± 27.93 mg/cm2, whereas the amount of pilocarpine penetrated into skin after 40 min of study (10 min of iontophoresis followed by 30 min of contact of aqueous pilocarpine solution to the porcine skin) was 140.56 ± 88.66 mg/cm2 (Table 3; Figure 1). Clinical Evaluation in Human Volunteers. The results of the in vitro studies were clearly translated in the clinical study. The study showed that the amount of sweat collected for the topical pilocarpine formulation (S12) was significantly higher compared with that of the control solution. The amount of sweat produced by the topical pilocarpine formulation (77.28 ± 18.97 mg) was sufficient to determine the chloride concentration in the sweat by a chloride analyzer. All the individuals were healthy and were found to have chloride concentration in the normal range. The results of the clinical studies are presented in Table 4. Conclusion Topical pilocarpine formulation was successful in inducing sweat formation by passive penetration of pilocarpine without the aid of any physical method of penetration enhancement. The sweat
5
generated by topical pilocarpine formulation as indicated by the in vivo study on human volunteers was sufficient to measure the chloride concentration. Thus, the topical pilocarpine formulation could be a safe and compliant alternative diagnostic tool for CF.
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