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Ultra-rapid drug delivery in the oral cavity using ultrasound
Journal of Controlled Release 304 (2019) 1–6
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Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel
Ultra-rapid drug delivery in the oral cavity using ultrasound a
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Marion M. France , Tony del Rio , Hannah Travers , Erin Raftery , Katherine Xu , ⁎⁎ ⁎ Robert Langerd, Giovanni Traversoe,f,g, Jochen K. Lennerzf,h, , Carl M. Schoellhammera,
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Suono Bio, Inc., 501 Massachusetts Avenue, Cambridge, MA 02139, United States of America Northeastern University, 360 Huntington Avenue, Boston, MA 02115, United States of America Tufts University, 419 Boston Avenue, Medford, MA 02155, United States of America d David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02139, United States of America e Department of Gastroenterology, Brigham and Women's Hospital, 221 Longwood Ave., EBRC 111/113, Boston, MA 02115, United States of America f Harvard Medical School, Boston, MA, 02115, United States of America g Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, United States of America h Department of Pathology, Center for Integrated Diagnostics, Massachusetts General Hospital, Boston, MA 02114, United States of America b c
ARTICLE INFO
ABSTRACT
Keywords: Buccal delivery Drug delivery Oral mucositis Ultrasound
The delivery of therapeutics to the gastrointestinal (GI) mucosa remains primarily a function of diffusion and rapid delivery is a significant goal in drug delivery science. However, delivery is hindered by the molecular barrier properties of the mucosa, as well as environmental factors. We hypothesized that low-frequency ultrasound can overcome these barriers, achieving rapid delivery in an engineered, clinically-relevant system for buccal administration. The hand-held system enabled delivery of macromolecules in short, 60-s treatment times ex vivo. Tolerability of the prototype was demonstrated in awake, (unsedated) dogs. Finally, this technology enhanced the efficacy of the anti-inflammatory agent, budesonide, allowing for prophylactic treatment in a hamster model of oral inflammatory lesions in vivo. The capacity to deliver therapeutics in a targeted and rapid manner in a clinically-relevant form-factor presents an intriguing capability to expand the repertoire of therapeutics that can be applied topically in the mouth and beyond.
1. Introduction The ability to facilitate the rapid and targeted delivery of a therapeutic directly into tissue independent of diffusion and drug-tissue contact time has been a major aim of the drug delivery field. Indeed, enabling ultra-rapid, and targeted drug administration to the gastrointestinal (GI) tract has been a long-standing goal given the great potential for treating a myriad of diseases such as inflammatory bowel disease (IBD) [1,2]. In patients and in rodent models of IBD, better therapeutic outcomes correlate with high, local drug concentrations at the site of disease [3,4]. However, therapeutic delivery to the GI tract remains an area of intense research owing to the challenges presented by the physiology of the GI tract itself [5]. For example, the GI mucosa functions as a striking barrier to drug absorption, greatly limiting passive diffusion [6]. In addition, the wealth of proteases and nucleases present locally further complicates the mucosal delivery of therapeutics, particularly biologics or nucleic acids [4,6]. Current strategies
for targeted delivery of therapeutics focus on formulation-based approaches [5,7–9]. Such approaches are limited by the requirement for tedious drug-specific formulation, intravenous administration, potentially fatal side effects [10], and minimal long-term tissue retention [11]. We have recently reported on the use of ultrasound as a drug delivery method in the GI tract [4,12]. Ultrasound is a sound wave with a frequency greater than the audible range of humans (> 20 kHz) [13]. In the clinical setting, ultrasound is utilized for a broad range of applications including imaging, lithotripsy, liposuction, and tumor ablation [14]. Ultrasound has been proposed as an effective and safe transdermal drug delivery method [15] and has gained FDA clearance for transdermal delivery of lidocaine [16]. Specifically, low-frequency ultrasound (≤100 kHz) can be used as a physical enhancer to facilitate therapeutic delivery by leveraging the phenomenon of transient cavitation, in which micron-scale bubbles are nucleated and subsequently collapse, creating a jet of fluid, to physically propel therapeutics into the tissue [16,17]. We have
Correspondence to: C. M. Schoellhammer, 700 Main St., North, Cambridge, MA 02139, United States of America. Correspondence to: J. K. Lennerz, Department of Pathology, Center for Integrated Diagnostics, Massachusetts General Hospital/Harvard Medical School, 55 Fruit St., GRJ 1015, Boston, MA 02114, United States of America. E-mail address: [email protected] (C.M. Schoellhammer). ⁎
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https://doi.org/10.1016/j.jconrel.2019.04.037 Received 24 January 2019; Received in revised form 7 April 2019; Accepted 26 April 2019 Available online 27 April 2019 0168-3659/ © 2019 Elsevier B.V. All rights reserved.
Journal of Controlled Release 304 (2019) 1–6
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previously demonstrated the safety and tolerability of ultrasound in the GI tract in both pigs and mice [12]. In addition, we have demonstrated the capability to deliver naked nucleic acids for the treatment of acute colitis in mice [4]. In these studies, the therapeutic and ultrasound were simultaneously administered locally in the colon [4]. In the setting of IBD, the colon is filled with a medicated enema and ultrasound applied through the fluid. This mode would not be feasible in other areas of the GI tract, such as the oral cavity. Treating diseases localized to the oral cavity would require a technology to have a self-contained drug reservoir that could be applied locally to the oral mucosa. To the best of our knowledge, such an embodiment has not been engineered previously. Therefore, the goal of this work was to develop a system for ultrasound-mediated drug delivery for the treatment of diseases of the oral cavity. A technology that facilitates rapid, local administration in the oral cavity independent of drug-tissue contact time could have a profound positive impact on treatment paradigms for diseases of the oral cavity [18]. Mastication and salivation greatly diminish drug-tissue contact time, and therefore, drug absorption, complicating outcomes [19]. For example, in the setting of head and neck cancer, the treatment-limiting side effect of oral inflammation (oral mucositis) is common and management of this results in additional treatment costs on the order of $17,000 – $25,000 per patient [20,21]. Patients whose oral mucositis cannot be managed by this extensive intervention typically discontinue radiation for a week, which increases the risk of cancer recurrence, shortens recurrence-free survival, and results in inferior overall survival [22]. The oral component of Crohn's disease is another debilitating example where patients today resort to compounded mouth rinse solutions using corticosteroids [23]. Poorly controlled oral inflammation often requires emergency medical intervention and supplemented nutrition in a clinical setting because eating can become too painful for affected patients [24]. The efficacy of currently-utilized therapies, such as corticosteroids, might be dramatically enhanced by maximizing local uptake at the site of disease [18]. In the present study, we aimed to engineer a system for ultrasoundmediated rapid drug delivery in the mouth. Data on the usability and effectiveness of an ultrasound-based drug delivery device in treating inflammatory diseases in the oral cavity is relevant because the ability to rapidly deliver therapeutics to mucosal surfaces of the GI tract may have applications that go beyond regional mucosal injuries.
sizes generated from our preliminary ex vivo data. No samples were excluded from analysis in this study. 2.2. Chemicals and drug preparation Dextran, Alexa Fluor™ 680, 3 kDa (D34681) was purchased from Thermo Fisher Scientific (US). Cy5.5 labeled dextrans, 10 kDa (DX10S55-1) and 500 kDa (DX500-S55-1) were purchased from Nanocs Inc. (US). Budesonide (B7777), 2-hydroxypropyl-β-cyclodextrin (HP-β-CD, H107), and glacial acetic acid (ARK2183) were purchased from Sigma Aldrich. Budesonide (0.1% w/v) was prepared in deionized water containing HP-β-CD (7% w/v). Suspensions were vortexed and placed in an ultrasonic bath (42 °C) until solubilized. Budesonide:HP-β-CD solutions were prepared fresh for each experiment and were stored at −20 °C until use. Acetic acid (50%) solution was prepared fresh in deionized water for each experiment. 2.3. Ultrasound device The device developed for this study was based on a 40 kHz CV401 converter (Sonics & Materials, Newtown, CT) utilized for ultrasound generation that was coupled to a custom-built aluminum half-wave horn. The horn was a linear taper used to focus the acoustic energy while keeping the overall length amenable to hand-held use. The housing of the device secured the aluminum horn at a working distance of 3 mm from the oral surface. The bottom of the housing formed a fluid chamber that contained a maximum fill volume of 2 mL of fluid. Therapeutic aqueous solutions (1 mL) were introduced into the fluid chamber through a fluid channel in the housing that was fitted with a Luer fitting connected to a syringe. The housing (Fig. S1) was 3Dprinted using Formlabs Form2 using clear resin according to the manufacturer's instructions (Formlabs, Inc., Somerville, MA). Briefly, a zlayer thickness setting of 200 μm was used as recommended by the manufacturer. After curing, leftover resin was removed using the FormWash (10 min), and then the housing underwent post-curing with FormCure (15 min at 60 °C). The 3D acoustic pressure field produced by the device was mapped in a water tank using a needle hydrophone (Precision Acoustics, UK). The hydrophone position was set by motor-controlled stages (Velmex Inc., Bloomfield, NY). A three-dimensional space was mapped in 0.5 mm increments utilizing control software to move the hydrophone, trigger the ultrasound probe, and record measurements with VirtualBench and a custom LabVIEW program (National Instruments, Austin, TX). Three amplitude measurements were recorded at each position of the hydrophone and averaged. Resulting signals were converted to kilopascals and 3D pressure maps were plotted using MATLAB (MathWorks, Natick, MA).
2. Materials and methods 2.1. Study design The purpose of this study was to engineer, develop, and test the tolerability and efficacy of an ultrasound-based device for ultra-rapid delivery in the oral cavity. It was hypothesized that low-frequency ultrasound would enable formulation-independent delivery by inducing transient cavitation. Based on the physics of ultrasound-emitting devices, it was proposed that a hand-held system could be engineered to enable application of a self-contained drug reservoir to achieve local delivery in the mouth. The prototype device was tested in both dogs (n = 5) and hamsters (n = 17). Dogs were selected to investigate the tolerability and sensation associated with application of the device because dogs can be trained to accept treatment, eliminating the need for sedatives and allowing for monitoring of the animal's behavior during treatment. Dogs were selected at random for the study. Trained veterinary staff qualified stress levels as a surrogate for acceptability of the device treatment by observing behaviors, including state of alertness and tail and ear movements. The efficacy of the device was tested in combination with budesonide delivery in an acetic acid-induced model of oral inflammatory lesions in hamsters. Hamsters were randomly assigned to experimental groups and lesion areas were measured in a blinded fashion. Sample sizes were determined based on hypothesized effect
2.4. Ex vivo delivery and quantification of fluorescently-labeled dextrans to porcine intestine Fresh porcine small intestinal tissue was harvested from Yorkshire pigs after sacrifice and used within 4 h of procurement. Tissues were rinsed, trimmed, and cut into circular punches using a 1.25-in. biopsy punch. The ultrasound device was positioned firmly on top of the mucosal surface to prevent leakage. Subsequently, the fluid chamber was filled with a solution of fluorescently-labeled dextrans prepared in phosphate-buffered saline (PBS) at a concentration of 0.5 μg/mL (3 kDa) or 1 μg/mL (10 and 500 kDa). Ultrasound was applied for 60 s at an intensity of 5 W/cm2 (calibrated by calorimetry) and a 50% duty cycle to reduce thermal effects [25]. All experiments were performed at room temperature. During ultrasound treatment, the temperature was measured at the tissue surface. The temperature probe of a digital thermometer (Traceable® type K thermometer, VWR) was placed at the serosal tissue surface. Temperature measurements were recorded with a sampling 2
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rate of 2.5 times per second using a custom LabVIEW program (National Instruments, Austin, TX). Immediately following ultrasound delivery, tissues were thoroughly rinsed to remove residual dextran and imaged to quantify fluorescence intensity. The mucosal surface was imaged using an Azure c600 Imaging System (Azure Biosystems, Inc., Dublin, CA). The IR channel (660 nm) was utilized with an exposure time of 500 ms at a resolution of 60 μm. Autofluorescence was accounted for by measuring fluorescence from the periphery of the tissue and subtracting this from the fluorescence measured from the treated area of the tissue.
60 s using a liquid holder (4 mm diameter). After acetic acid application, the hamsters were placed back into their cages to recover. On subsequent days, the cheek pouches were everted to observe the lesion and an image was captured to quantify lesion size. Treatments were then applied as described above. Lesion areas were measured using ImageJ (National Institutes of Health, Bethesda, MD). 2.7. Statistical analysis All data analysis was performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). For ex vivo studies, twogroup comparisons were made using Student's unpaired t-test using Welch's correction where delivery with or without ultrasound groups were compared. For in vivo studies, multiple comparisons were made using one-way ANOVA followed by Tukey's post-test. For all comparisons, p < .05 was considered statistically significant. Data are reported as mean ± standard error of the mean (SEM) except for temperature measurements that are reported as mean ± standard deviation (SD).
2.5. Administration in dogs in vivo All animal-related research aspects were approved and conducted in accordance with the ethical standards and guidelines of the Institutional Animal Care & Use Committee (IACUC) at Tufts University (Boston, MA). The tolerability of the oral ultrasound device was tested in adult beagles. Beagles were chosen because they can be trained to accept various procedures, making anesthesia unnecessary. Behavior and auditory cues were used to assess potential tolerability of the device because the animals were conscious. Five adult beagles were selected at random by veterinary staff for this study. Dogs were brought into the procedure room individually and allowed to move around freely. The device was initially run in a beaker of fluid at the same power described above to monitor the animals' reactions to the ultrasonic noise. While running in the beaker, the device was slowly brought closer to the animal and their behavior monitored by trained veterinary staff. To administer the device in the oral cavity, animals were placed on a risen platform and lightly restrained by veterinary staff to encourage them to lie on their left sides, allowing for application of the device to the right cheek. Before treatment began, an image was captured of the mucosal region to be treated. The device was placed against the cheek and the fluid chamber filled with water. The device was turned on for 5 s at a duty cycle of 100% and the animal's reaction and behavior were monitored by the veterinary staff. After treatment, the device was removed and the treated area gently blotted dry. A second image was taken of the treated area. The animals were then offered treats and taken out of the room. The temperature rise during ultrasound treatment in vivo was not measured.
3. Results 3.1. Characterization of the oral device The handheld oral ultrasound device was designed to easily and rapidly deliver an aqueous therapeutic agent to a mucosal surface. The device developed for this study housed the ultrasound converter and horn. The base of the housing formed a fluid chamber that contained the aqueous solution (Fig. 1a). A linear-tapered horn at a power of 5 W/ cm2 delivered 40 kHz frequency ultrasound to the mucosal surface at a working distance of 3 mm. The power used is within the relevant intensity for energy-emitting devices follows international standards [29] that have also been adopted in the U.S. regulatory framework [30]. 3D pressure recordings were mapped in a water tank and were used to characterize both the range and depth of ultrasound-generated pressure for the device (Fig. 1b–c). Below the outer housing surface, pressure readings of approximately 150, 100, and 80 kPa were measured at 0.5, 2, and 3.5 mm distances, respectively. These distances are comparable to the working distance of the ultrasound horn when the device is applied at the mucosal surface. The pressures measured at these distances correspond with pressures that generate transient cavitation [31].
2.6. In vivo treatment of oral inflammatory lesions in hamster cheek pouches
3.2. The device significantly enhances the delivery of macromolecules ex vivo
Male Golden Syrian hamsters weighing 100–120 g (Charles River Laboratories, Wilmington, MA) were used as received and randomly assigned to experimental groups by the researchers performing the work. To limit pain and distress, all animals received buprenorphine sustained release (SR) (subcutaneous administration, 1 mg/kg body weight, compounded by ZooPharm) just prior to the application of acetic acid for the induction of the oral lesion on day 0. All manipulations occurred with the animal under general anesthesia by inhaled isoflurane. Experimental treatment groups included topical application of budesonide solution with (Budesonide + US) and without (Budesonide) ultrasound. A positive disease control receiving no treatment was also included (Disease). Drug treatments were administered on day 0 immediately prior to lesion induction using acetic acid. While under general anesthesia, the left cheek pouch was everted, the device was placed on the buccal surface, and the fluid chamber filled with solubilized budesonide solution (0.1%, ~1 mL). For the Budesonide + US treatment group, ultrasound was simultaneously applied for 3 s at an intensity of 5 W/cm2 and a 100% duty cycle. In experimental groups receiving budesonide only (Budesonide), drug was applied similarly using the device without turning it on. Immediately following budesonide treatment, oral lesions were induced by topical application of acetic acid (50% solution) as described in previous studies [26–28]. Briefly, acetic acid was applied to the buccal surface for
To determine the delivery capacity of the device, fluorescently-labeled dextrans with molecular weights spanning two orders of magnitude were utilized and delivered to porcine intestine ex vivo (Fig. 2). Short, 60-s treatments enhanced the delivery of all dextrans 4 to 5.5fold compared to delivery without ultrasound (Fig. 2b, c; 3 kDa, p = .0003; 10 kDa, p = .02; and 500 kDa, p = .0007). During a 60 s ultrasound treatment, the tissue temperature increased on average 12 °C (21.9 ± 1.04 °C to 34.0 ± 1.41 °C, n = 36, Fig. S2). However, a shorter treatment time (3 s) used for the subsequent in vivo experiments (see below) resulted in a temperature increase of only ~1.3 °C. 3.3. Application in dogs is well tolerated The tolerability and sensation of the device treatment was characterized in awake, unanesthetized dogs. Dogs were chosen for the study because of their similarity to humans [32] and they can be trained to accept treatment without the use of anesthesia. For our studies, the dogs' reactions to the device were observed. All animals appeared unperturbed by the ultrasonic noise generated by the device when powered in a beaker of water, regardless of the distance between the device and the animal. Next, the application of the device on the 3
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Fig. 1. The oral ultrasound device for local administration of therapeutics to mucosal surfaces. (A) A 3D rendering of the printed device consisting of an aluminum half-wave horn (5 mm diameter) fitted in a housing that includes a fluid chamber filled with a therapeutic solution (1 mL). The aluminum half-wave horn is used to generate 40 kHz ultrasound that is emitted through the solution that facilitates the delivery of an agent into a mucosal surface. (B) The pressure mapping region for the horn within the device housing as viewed from (i) the side, and (ii) the bottom. (C) 3D acoustic pressure map of the horn with the fluid chamber fully submerged in water operating at 60% driving amplitude. The color scale ranges from 50 to 260 kPa.
inside of the cheek was tested since the noise generated from the ultrasound did not elicit any adverse reactions. During ultrasound treatment with water, animals did not display any behaviors associated with stress as determined by the trained veterinary staff. Following ultrasound treatment, macroscopic changes in appearance of the oral mucosa were not observed (Fig. S3). Upon completion, all animals accepted treats ad libitum.
the presentation of clinically relevant signs, including ulceration, necrosis with suppurative inflammation, edema, and infiltration of neutrophils [27,33]. In our studies, oral lesions induced by topical application of an acetic acid solution (50%) for 60 s resulted in round, elevated white lesions approximately 24 h after application (Fig. S4). Immediately following application, the injury was characterized by vasoconstriction within the area of application whereas the surrounding vessels showed vasodilation after 1 day. Days 2 and 3 were characterized by mounting edema, and congestion of the blood vessels surrounding the lesion. Further, these changes were accompanied by at least partial central necrosis surrounded by granulation tissue at the interface zone towards the uninjured epithelium. By day 4, all lesions appeared pus-filled, constricted, and well-demarcated from the surrounding epithelium. It is noteworthy that the lesion remained wellcircumscribed and corresponded to the initial site of injury. Lesions nearly self-resolve by day 8 post injury (Fig. S4). In order to evaluate whether ultrasound-mediated delivery of
3.4. Ultrasound-mediated budesonide delivery lessens oral lesion severity in vivo Finally, the efficacy of enhanced delivery of budesonide using the oral ultrasound device was tested in an animal model of oral inflammatory lesions (Fig. 3a). The most frequently used model for oral lesions is the hamster buccal pouch model [27]. Further, the size of the buccal pouch allows for use of the full-sized prototype. Previous histological evaluation of oral lesions induced by acetic acid has validated
Fig. 2. Ultrasound-mediated delivery to porcine intestine ex vivo. (A) Schematic demonstrating delivery of fluorescently-labeled dextrans to small intestinal biopsy punches using the oral ultrasound device. For application, the device was applied with the opening of the fluid chamber positioned against the mucosal surface. The outer dashed black line outlines the tissue punch while the smaller dashed line indicates where the device was applied. The fluid chamber was loaded with solution containing fluorescently-labeled dextrans (1 mL). (B) Representative fluorescence images of the mucosal surface after delivery of fluorescently-labeled dextrans of various molecular weights (3, 10, and 500 kDa) with (+) and without (−) 40 kHz ultrasound application for 60 s. PBS with ultrasound served as a control. The entire tissue is encircled (thicker dashed white line) and delivery of labeled dextrans are observed in red at the site of application (smaller dashed white line). Scale bar is 1 cm. (C) Fluorescence intensity of the mucosal surface was measured immediately after delivery (n = 3–6). Data are presented as mean ± SEM. p values were determined by two-tailed, unpaired t-test using Welch's correction; * p < .05, ** p < .001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 4
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Fig. 3. Ultrasound-mediated budesonide delivery lessens oral lesion severity in hamster cheek pouches in vivo. (A) Schematic of the three experimental groups, respective disease induction, and treatment regimen. Note that on day 0, the respective treatments for the Budesonide and Budesonide + US groups were administered before applying acetic acid. (B) Oral lesion areas remain smaller in the Budesonide + US treatment group at 24 and 48 h following lesion induction with acetic acid (n = 17 for 24 and 48 h; n = 4 for 72 h). (C) Representative images of macroscopic oral lesion areas at 48 h post acetic acid application. P values were determined by One-way ANOVA followed by Tukey's post-test. * p < .05 vs. Disease and Budesonide. ** p < .05 vs. Budesonide.
budesonide compared to traditional topical application is more effective in the treatment of oral lesions, the first 72 h following injury induction were analyzed because peak severity occurred during this period. In animals not receiving any treatment, oral lesions reached a maximum size 48 h following topical application of the acetic acid and began to heal naturally after 72 h (Fig. 3b and S4). When compared to Disease and Budesonide groups, animals that received Budesonide + US exhibited smaller oral lesion sizes 24 h post-acetic acid induced injury (Disease n = 17, 20.5 +/− 1.1 mm2; Budesonide n = 17, 20.3 +/− 1.1 mm2; Budesonide + US n = 17, 15.7 +/− 1.3 mm2; p < .05 one-way ANOVA followed by Tukey's post-test). These results demonstrate that budesonide delivered with ultrasound reduces the overall severity and initial size of the lesion using a single dose in a prophylactic manner. 48 h after acetic acid-induced injury, oral lesions in hamster cheek pouches treated with Budesonide + US remained smaller than those treated topically with budesonide without ultrasound (Budesonide + US, 18.71 +/− 2.3 mm2; Budesonide, 25.5 +/− 1.6 mm2, p < .05). After 72 h, oral lesions began to heal naturally and oral lesions in all groups were similar in size. These results demonstrate that budesonide in combination with ultrasound confines the size of the oral lesion, thus preventing the lesion from attaining the severity observed in the Disease and Budesonide groups.
intensity coded to the therapeutic being applied, which eliminates the need for more complex devices and or instructions. In addition to usability, the device must be safe to use and well tolerated. With regards to safety, the design of the device must meet biocompatibility and electrical safety requirements outlined by international standards and adopted by the FDA [34]. The present formfactor has been designed with these standards in mind. For example, to ensure a biocompatible device, the current housing can be made from a wide-range of moldable, acrylonitrile butadiene styrene-like materials. In addition, the transducer employed for our studies meets the electrical safety testing standards set forth in specifications IEC 80601-2-60:2012. These standards are put in place to minimize potential risk of shock to users or for unintended operation of the device. Currently, we are in the process of completing minor modifications to the power supply to ensure compliance with these standards. One additional concern surrounding powered devices is energy emission. When applied to tissues, the energy transfer typically manifests as a temperature increase and we have investigated as part of the present study. Previous strategies to reduce the resulting temperature increase during treatment have focused on modulating the intensity of ultrasound treatment [16,35]. However, here it was not necessary to adjust the intensity or duty cycle in vivo to allow for an “off” period. The temperature rise noted ex vivo over a 60 s treatment was observed to be only 12 °C These results, representing a “worst-case” condition are encouraging given that validated temperature increases in vivo are lower due to a larger “heat-sink” and regulation by the body of temperature [12]. Regardless, if a temperature rise of 12 °C did occur in a user at physiological body temperature, the resulting temperature would be below temperatures of many commonly-consumed hot beverages (60 °C) and temperatures that can cause adverse events [36]. Initial tolerability was assessed in dogs. This is an important readout to ensure clinical use would be pain-free. The fit, sound and sensation in the canine mouth was not rejected by the awake animal. In addition, repeated daily use on sedated hamsters did not show any ill effects with or without ultrasound application. Indeed, the lack of any reaction in dogs when using the device is encouraging and supports further testing in large animal models. Indeed, low-frequency ultrasound has previously been validated as pain-free for transdermal drug delivery and approved for the topical administration of lidocaine [16]. Further testing will be required in two large animal systems to validate the lack of toxicity in support of an eventual investigational new drug application for a combination product leveraging the device with a therapeutic. We used budesonide to assess device efficacy in an in vivo model.
4. Discussion The objective of this study was to engineer a system for low-frequency ultrasound-mediated drug delivery and to demonstrate therapeutic efficacy in an experimental injury model. The form-factor and tolerability of the device was assessed in awake, unsedated dogs. We demonstrate the ability to deposit dextrans of various molecular weights ranging from 3 to 500 kDa into tissue. We show enhanced efficacy of the anti-inflammatory agent, budesonide, through comparison of US-enhanced mucosal delivery, allowing for prophylactic treatment in an established mucosal injury model of oral inflammatory lesions in vivo. The capacity to deliver therapeutics in a targeted and rapid manner in a clinically-relevant form-factor is compelling and can be applied topically in the mouth and beyond. There are many considerations with regards to translating such a technology to the clinic. The first of which is the form-factor and usability of the device. In the present study, the device was designed to be compact and amenable to holding with one hand. While such technology might be used in clinical settings, the flexibility for self-administration by patients directly requires a compact, and simple to use device. This can be accomplished by having the treatment time and 5
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Specifically, we used the well-stablished acetic acid-induced injury model characterized by mucosal injury composed of a central necrotic core with surrounding inflammation. Importantly, necrosis would not be prevented by budesonide administration; rather, budesonide likely modulates and reduces the lesional severity and size due to a reduction in infiltration of inflammatory cells, by altering the inflammatory response at the site of injury [37–39]. In addition, budesonide delivered using ultrasound could result in an improved outcome compared to topical application, due to enhanced delivery. Indeed, our results demonstrate that the severity of the oral lesion was significantly reduced in animals receiving budesonide in combination with ultrasound when compared to those without ultrasound. In other words, topical budesonide without ultrasound demonstrated no such effect. These results strongly support the hypothesis that the efficacy of commonly-utilized therapeutics can be enhanced by targeted, loco-regional delivery. In summary, we have demonstrated a novel low-frequency ultrasound device that can enhance mucosal drug delivery. The device is tolerable in an awake large animal model and the prophylactic application of the anti-inflammatory agent, budesonide, in an oral lesion buccal pouch model. Enhancing the regional application of therapeutics by maximizing penetration may include clinical applications going beyond regional mucosal injury.
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Gastroenterol. 19 (2013) 8571–8579. [24] P. Asquith, R.A. Thompson, W.T. Cooke, Oral manifestations of Crohn's disease, Gut 16 (1975) 249–254. [25] C.M. Schoellhammer, et al., Rapid skin permeabilization by the simultaneous application of dual-frequency, high-intensity ultrasound, J. Control. Release 163 (2012) 154–160. [26] S.T. Sonis, Mucositis: the impact, biology and therapeutic opportunities of oral mucositis, Oral Oncol. 45 (2009) 1015–1020. [27] S.T. Sonis, et al., The gene expression sequence of radiated mucosa in an animal mucositis model, Cell Prolif. 35 (Suppl. 1) (2002) 93–102. [28] S.T. Sonis, et al., Defining mechanisms of action of interleukin-11 on the progression of radiation-induced oral mucositis in hamsters, Oral Oncol. 36 (2000) 373–381. [29] I. E. Commission. (2012), (vol. 80601-2-60). 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Acknowledgements We thank the Tufts University veterinary staff for assistance with all animal studies. Funding This work was funded by US National Institutes of Health (NIH) grant DE013023 to RL. Author contributions M.M.F., R.L., G.T., J.L., and C.M.S. conceived and designed the research. M.M.F., H.T., E.R, K.X., and C.M.S. performed device development and characterization work. M.M.F., T.d.R., E.R., J.K.L., and C.M.S. designed and performed the in vivo experiments. J.K.L. analyzed tissues. M.M.F. and C.M.S. performed the statistical analysis. M.M.F., T.d.R., E.R., R.L., G.T., J.K.L., and C.M.S. analyzed the data and wrote the manuscript. Competing interests M.M.F., T.d.R., and C.M.S. are employed by Suono Bio, Inc. R.L., G.T., and C.M.S. are co-founders of Suono Bio, Inc., and co-inventors on multiple patent applications surrounding ultrasound-mediated drug delivery technologies. J.K.L has no conflicts of interest. Data and materials availability All data for this study is presented here and all materials are available. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jconrel.2019.04.037. References [1] G. Traverso, A.R. Kirtane, C.M. Schoellhammer, R. Langer, Convergence for translation: drug-delivery research in multidisciplinary teams, Angew. Chem. Int. Ed. Eng. 57 (2018) 4156–4163.
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Contents lists available at ScienceDirect
Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel
Ultra-rapid drug delivery in the oral cavity using ultrasound a
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Marion M. France , Tony del Rio , Hannah Travers , Erin Raftery , Katherine Xu , ⁎⁎ ⁎ Robert Langerd, Giovanni Traversoe,f,g, Jochen K. Lennerzf,h, , Carl M. Schoellhammera,
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Suono Bio, Inc., 501 Massachusetts Avenue, Cambridge, MA 02139, United States of America Northeastern University, 360 Huntington Avenue, Boston, MA 02115, United States of America Tufts University, 419 Boston Avenue, Medford, MA 02155, United States of America d David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02139, United States of America e Department of Gastroenterology, Brigham and Women's Hospital, 221 Longwood Ave., EBRC 111/113, Boston, MA 02115, United States of America f Harvard Medical School, Boston, MA, 02115, United States of America g Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, United States of America h Department of Pathology, Center for Integrated Diagnostics, Massachusetts General Hospital, Boston, MA 02114, United States of America b c
ARTICLE INFO
ABSTRACT
Keywords: Buccal delivery Drug delivery Oral mucositis Ultrasound
The delivery of therapeutics to the gastrointestinal (GI) mucosa remains primarily a function of diffusion and rapid delivery is a significant goal in drug delivery science. However, delivery is hindered by the molecular barrier properties of the mucosa, as well as environmental factors. We hypothesized that low-frequency ultrasound can overcome these barriers, achieving rapid delivery in an engineered, clinically-relevant system for buccal administration. The hand-held system enabled delivery of macromolecules in short, 60-s treatment times ex vivo. Tolerability of the prototype was demonstrated in awake, (unsedated) dogs. Finally, this technology enhanced the efficacy of the anti-inflammatory agent, budesonide, allowing for prophylactic treatment in a hamster model of oral inflammatory lesions in vivo. The capacity to deliver therapeutics in a targeted and rapid manner in a clinically-relevant form-factor presents an intriguing capability to expand the repertoire of therapeutics that can be applied topically in the mouth and beyond.
1. Introduction The ability to facilitate the rapid and targeted delivery of a therapeutic directly into tissue independent of diffusion and drug-tissue contact time has been a major aim of the drug delivery field. Indeed, enabling ultra-rapid, and targeted drug administration to the gastrointestinal (GI) tract has been a long-standing goal given the great potential for treating a myriad of diseases such as inflammatory bowel disease (IBD) [1,2]. In patients and in rodent models of IBD, better therapeutic outcomes correlate with high, local drug concentrations at the site of disease [3,4]. However, therapeutic delivery to the GI tract remains an area of intense research owing to the challenges presented by the physiology of the GI tract itself [5]. For example, the GI mucosa functions as a striking barrier to drug absorption, greatly limiting passive diffusion [6]. In addition, the wealth of proteases and nucleases present locally further complicates the mucosal delivery of therapeutics, particularly biologics or nucleic acids [4,6]. Current strategies
for targeted delivery of therapeutics focus on formulation-based approaches [5,7–9]. Such approaches are limited by the requirement for tedious drug-specific formulation, intravenous administration, potentially fatal side effects [10], and minimal long-term tissue retention [11]. We have recently reported on the use of ultrasound as a drug delivery method in the GI tract [4,12]. Ultrasound is a sound wave with a frequency greater than the audible range of humans (> 20 kHz) [13]. In the clinical setting, ultrasound is utilized for a broad range of applications including imaging, lithotripsy, liposuction, and tumor ablation [14]. Ultrasound has been proposed as an effective and safe transdermal drug delivery method [15] and has gained FDA clearance for transdermal delivery of lidocaine [16]. Specifically, low-frequency ultrasound (≤100 kHz) can be used as a physical enhancer to facilitate therapeutic delivery by leveraging the phenomenon of transient cavitation, in which micron-scale bubbles are nucleated and subsequently collapse, creating a jet of fluid, to physically propel therapeutics into the tissue [16,17]. We have
Correspondence to: C. M. Schoellhammer, 700 Main St., North, Cambridge, MA 02139, United States of America. Correspondence to: J. K. Lennerz, Department of Pathology, Center for Integrated Diagnostics, Massachusetts General Hospital/Harvard Medical School, 55 Fruit St., GRJ 1015, Boston, MA 02114, United States of America. E-mail address: [email protected] (C.M. Schoellhammer). ⁎
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https://doi.org/10.1016/j.jconrel.2019.04.037 Received 24 January 2019; Received in revised form 7 April 2019; Accepted 26 April 2019 Available online 27 April 2019 0168-3659/ © 2019 Elsevier B.V. All rights reserved.
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previously demonstrated the safety and tolerability of ultrasound in the GI tract in both pigs and mice [12]. In addition, we have demonstrated the capability to deliver naked nucleic acids for the treatment of acute colitis in mice [4]. In these studies, the therapeutic and ultrasound were simultaneously administered locally in the colon [4]. In the setting of IBD, the colon is filled with a medicated enema and ultrasound applied through the fluid. This mode would not be feasible in other areas of the GI tract, such as the oral cavity. Treating diseases localized to the oral cavity would require a technology to have a self-contained drug reservoir that could be applied locally to the oral mucosa. To the best of our knowledge, such an embodiment has not been engineered previously. Therefore, the goal of this work was to develop a system for ultrasound-mediated drug delivery for the treatment of diseases of the oral cavity. A technology that facilitates rapid, local administration in the oral cavity independent of drug-tissue contact time could have a profound positive impact on treatment paradigms for diseases of the oral cavity [18]. Mastication and salivation greatly diminish drug-tissue contact time, and therefore, drug absorption, complicating outcomes [19]. For example, in the setting of head and neck cancer, the treatment-limiting side effect of oral inflammation (oral mucositis) is common and management of this results in additional treatment costs on the order of $17,000 – $25,000 per patient [20,21]. Patients whose oral mucositis cannot be managed by this extensive intervention typically discontinue radiation for a week, which increases the risk of cancer recurrence, shortens recurrence-free survival, and results in inferior overall survival [22]. The oral component of Crohn's disease is another debilitating example where patients today resort to compounded mouth rinse solutions using corticosteroids [23]. Poorly controlled oral inflammation often requires emergency medical intervention and supplemented nutrition in a clinical setting because eating can become too painful for affected patients [24]. The efficacy of currently-utilized therapies, such as corticosteroids, might be dramatically enhanced by maximizing local uptake at the site of disease [18]. In the present study, we aimed to engineer a system for ultrasoundmediated rapid drug delivery in the mouth. Data on the usability and effectiveness of an ultrasound-based drug delivery device in treating inflammatory diseases in the oral cavity is relevant because the ability to rapidly deliver therapeutics to mucosal surfaces of the GI tract may have applications that go beyond regional mucosal injuries.
sizes generated from our preliminary ex vivo data. No samples were excluded from analysis in this study. 2.2. Chemicals and drug preparation Dextran, Alexa Fluor™ 680, 3 kDa (D34681) was purchased from Thermo Fisher Scientific (US). Cy5.5 labeled dextrans, 10 kDa (DX10S55-1) and 500 kDa (DX500-S55-1) were purchased from Nanocs Inc. (US). Budesonide (B7777), 2-hydroxypropyl-β-cyclodextrin (HP-β-CD, H107), and glacial acetic acid (ARK2183) were purchased from Sigma Aldrich. Budesonide (0.1% w/v) was prepared in deionized water containing HP-β-CD (7% w/v). Suspensions were vortexed and placed in an ultrasonic bath (42 °C) until solubilized. Budesonide:HP-β-CD solutions were prepared fresh for each experiment and were stored at −20 °C until use. Acetic acid (50%) solution was prepared fresh in deionized water for each experiment. 2.3. Ultrasound device The device developed for this study was based on a 40 kHz CV401 converter (Sonics & Materials, Newtown, CT) utilized for ultrasound generation that was coupled to a custom-built aluminum half-wave horn. The horn was a linear taper used to focus the acoustic energy while keeping the overall length amenable to hand-held use. The housing of the device secured the aluminum horn at a working distance of 3 mm from the oral surface. The bottom of the housing formed a fluid chamber that contained a maximum fill volume of 2 mL of fluid. Therapeutic aqueous solutions (1 mL) were introduced into the fluid chamber through a fluid channel in the housing that was fitted with a Luer fitting connected to a syringe. The housing (Fig. S1) was 3Dprinted using Formlabs Form2 using clear resin according to the manufacturer's instructions (Formlabs, Inc., Somerville, MA). Briefly, a zlayer thickness setting of 200 μm was used as recommended by the manufacturer. After curing, leftover resin was removed using the FormWash (10 min), and then the housing underwent post-curing with FormCure (15 min at 60 °C). The 3D acoustic pressure field produced by the device was mapped in a water tank using a needle hydrophone (Precision Acoustics, UK). The hydrophone position was set by motor-controlled stages (Velmex Inc., Bloomfield, NY). A three-dimensional space was mapped in 0.5 mm increments utilizing control software to move the hydrophone, trigger the ultrasound probe, and record measurements with VirtualBench and a custom LabVIEW program (National Instruments, Austin, TX). Three amplitude measurements were recorded at each position of the hydrophone and averaged. Resulting signals were converted to kilopascals and 3D pressure maps were plotted using MATLAB (MathWorks, Natick, MA).
2. Materials and methods 2.1. Study design The purpose of this study was to engineer, develop, and test the tolerability and efficacy of an ultrasound-based device for ultra-rapid delivery in the oral cavity. It was hypothesized that low-frequency ultrasound would enable formulation-independent delivery by inducing transient cavitation. Based on the physics of ultrasound-emitting devices, it was proposed that a hand-held system could be engineered to enable application of a self-contained drug reservoir to achieve local delivery in the mouth. The prototype device was tested in both dogs (n = 5) and hamsters (n = 17). Dogs were selected to investigate the tolerability and sensation associated with application of the device because dogs can be trained to accept treatment, eliminating the need for sedatives and allowing for monitoring of the animal's behavior during treatment. Dogs were selected at random for the study. Trained veterinary staff qualified stress levels as a surrogate for acceptability of the device treatment by observing behaviors, including state of alertness and tail and ear movements. The efficacy of the device was tested in combination with budesonide delivery in an acetic acid-induced model of oral inflammatory lesions in hamsters. Hamsters were randomly assigned to experimental groups and lesion areas were measured in a blinded fashion. Sample sizes were determined based on hypothesized effect
2.4. Ex vivo delivery and quantification of fluorescently-labeled dextrans to porcine intestine Fresh porcine small intestinal tissue was harvested from Yorkshire pigs after sacrifice and used within 4 h of procurement. Tissues were rinsed, trimmed, and cut into circular punches using a 1.25-in. biopsy punch. The ultrasound device was positioned firmly on top of the mucosal surface to prevent leakage. Subsequently, the fluid chamber was filled with a solution of fluorescently-labeled dextrans prepared in phosphate-buffered saline (PBS) at a concentration of 0.5 μg/mL (3 kDa) or 1 μg/mL (10 and 500 kDa). Ultrasound was applied for 60 s at an intensity of 5 W/cm2 (calibrated by calorimetry) and a 50% duty cycle to reduce thermal effects [25]. All experiments were performed at room temperature. During ultrasound treatment, the temperature was measured at the tissue surface. The temperature probe of a digital thermometer (Traceable® type K thermometer, VWR) was placed at the serosal tissue surface. Temperature measurements were recorded with a sampling 2
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rate of 2.5 times per second using a custom LabVIEW program (National Instruments, Austin, TX). Immediately following ultrasound delivery, tissues were thoroughly rinsed to remove residual dextran and imaged to quantify fluorescence intensity. The mucosal surface was imaged using an Azure c600 Imaging System (Azure Biosystems, Inc., Dublin, CA). The IR channel (660 nm) was utilized with an exposure time of 500 ms at a resolution of 60 μm. Autofluorescence was accounted for by measuring fluorescence from the periphery of the tissue and subtracting this from the fluorescence measured from the treated area of the tissue.
60 s using a liquid holder (4 mm diameter). After acetic acid application, the hamsters were placed back into their cages to recover. On subsequent days, the cheek pouches were everted to observe the lesion and an image was captured to quantify lesion size. Treatments were then applied as described above. Lesion areas were measured using ImageJ (National Institutes of Health, Bethesda, MD). 2.7. Statistical analysis All data analysis was performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). For ex vivo studies, twogroup comparisons were made using Student's unpaired t-test using Welch's correction where delivery with or without ultrasound groups were compared. For in vivo studies, multiple comparisons were made using one-way ANOVA followed by Tukey's post-test. For all comparisons, p < .05 was considered statistically significant. Data are reported as mean ± standard error of the mean (SEM) except for temperature measurements that are reported as mean ± standard deviation (SD).
2.5. Administration in dogs in vivo All animal-related research aspects were approved and conducted in accordance with the ethical standards and guidelines of the Institutional Animal Care & Use Committee (IACUC) at Tufts University (Boston, MA). The tolerability of the oral ultrasound device was tested in adult beagles. Beagles were chosen because they can be trained to accept various procedures, making anesthesia unnecessary. Behavior and auditory cues were used to assess potential tolerability of the device because the animals were conscious. Five adult beagles were selected at random by veterinary staff for this study. Dogs were brought into the procedure room individually and allowed to move around freely. The device was initially run in a beaker of fluid at the same power described above to monitor the animals' reactions to the ultrasonic noise. While running in the beaker, the device was slowly brought closer to the animal and their behavior monitored by trained veterinary staff. To administer the device in the oral cavity, animals were placed on a risen platform and lightly restrained by veterinary staff to encourage them to lie on their left sides, allowing for application of the device to the right cheek. Before treatment began, an image was captured of the mucosal region to be treated. The device was placed against the cheek and the fluid chamber filled with water. The device was turned on for 5 s at a duty cycle of 100% and the animal's reaction and behavior were monitored by the veterinary staff. After treatment, the device was removed and the treated area gently blotted dry. A second image was taken of the treated area. The animals were then offered treats and taken out of the room. The temperature rise during ultrasound treatment in vivo was not measured.
3. Results 3.1. Characterization of the oral device The handheld oral ultrasound device was designed to easily and rapidly deliver an aqueous therapeutic agent to a mucosal surface. The device developed for this study housed the ultrasound converter and horn. The base of the housing formed a fluid chamber that contained the aqueous solution (Fig. 1a). A linear-tapered horn at a power of 5 W/ cm2 delivered 40 kHz frequency ultrasound to the mucosal surface at a working distance of 3 mm. The power used is within the relevant intensity for energy-emitting devices follows international standards [29] that have also been adopted in the U.S. regulatory framework [30]. 3D pressure recordings were mapped in a water tank and were used to characterize both the range and depth of ultrasound-generated pressure for the device (Fig. 1b–c). Below the outer housing surface, pressure readings of approximately 150, 100, and 80 kPa were measured at 0.5, 2, and 3.5 mm distances, respectively. These distances are comparable to the working distance of the ultrasound horn when the device is applied at the mucosal surface. The pressures measured at these distances correspond with pressures that generate transient cavitation [31].
2.6. In vivo treatment of oral inflammatory lesions in hamster cheek pouches
3.2. The device significantly enhances the delivery of macromolecules ex vivo
Male Golden Syrian hamsters weighing 100–120 g (Charles River Laboratories, Wilmington, MA) were used as received and randomly assigned to experimental groups by the researchers performing the work. To limit pain and distress, all animals received buprenorphine sustained release (SR) (subcutaneous administration, 1 mg/kg body weight, compounded by ZooPharm) just prior to the application of acetic acid for the induction of the oral lesion on day 0. All manipulations occurred with the animal under general anesthesia by inhaled isoflurane. Experimental treatment groups included topical application of budesonide solution with (Budesonide + US) and without (Budesonide) ultrasound. A positive disease control receiving no treatment was also included (Disease). Drug treatments were administered on day 0 immediately prior to lesion induction using acetic acid. While under general anesthesia, the left cheek pouch was everted, the device was placed on the buccal surface, and the fluid chamber filled with solubilized budesonide solution (0.1%, ~1 mL). For the Budesonide + US treatment group, ultrasound was simultaneously applied for 3 s at an intensity of 5 W/cm2 and a 100% duty cycle. In experimental groups receiving budesonide only (Budesonide), drug was applied similarly using the device without turning it on. Immediately following budesonide treatment, oral lesions were induced by topical application of acetic acid (50% solution) as described in previous studies [26–28]. Briefly, acetic acid was applied to the buccal surface for
To determine the delivery capacity of the device, fluorescently-labeled dextrans with molecular weights spanning two orders of magnitude were utilized and delivered to porcine intestine ex vivo (Fig. 2). Short, 60-s treatments enhanced the delivery of all dextrans 4 to 5.5fold compared to delivery without ultrasound (Fig. 2b, c; 3 kDa, p = .0003; 10 kDa, p = .02; and 500 kDa, p = .0007). During a 60 s ultrasound treatment, the tissue temperature increased on average 12 °C (21.9 ± 1.04 °C to 34.0 ± 1.41 °C, n = 36, Fig. S2). However, a shorter treatment time (3 s) used for the subsequent in vivo experiments (see below) resulted in a temperature increase of only ~1.3 °C. 3.3. Application in dogs is well tolerated The tolerability and sensation of the device treatment was characterized in awake, unanesthetized dogs. Dogs were chosen for the study because of their similarity to humans [32] and they can be trained to accept treatment without the use of anesthesia. For our studies, the dogs' reactions to the device were observed. All animals appeared unperturbed by the ultrasonic noise generated by the device when powered in a beaker of water, regardless of the distance between the device and the animal. Next, the application of the device on the 3
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Fig. 1. The oral ultrasound device for local administration of therapeutics to mucosal surfaces. (A) A 3D rendering of the printed device consisting of an aluminum half-wave horn (5 mm diameter) fitted in a housing that includes a fluid chamber filled with a therapeutic solution (1 mL). The aluminum half-wave horn is used to generate 40 kHz ultrasound that is emitted through the solution that facilitates the delivery of an agent into a mucosal surface. (B) The pressure mapping region for the horn within the device housing as viewed from (i) the side, and (ii) the bottom. (C) 3D acoustic pressure map of the horn with the fluid chamber fully submerged in water operating at 60% driving amplitude. The color scale ranges from 50 to 260 kPa.
inside of the cheek was tested since the noise generated from the ultrasound did not elicit any adverse reactions. During ultrasound treatment with water, animals did not display any behaviors associated with stress as determined by the trained veterinary staff. Following ultrasound treatment, macroscopic changes in appearance of the oral mucosa were not observed (Fig. S3). Upon completion, all animals accepted treats ad libitum.
the presentation of clinically relevant signs, including ulceration, necrosis with suppurative inflammation, edema, and infiltration of neutrophils [27,33]. In our studies, oral lesions induced by topical application of an acetic acid solution (50%) for 60 s resulted in round, elevated white lesions approximately 24 h after application (Fig. S4). Immediately following application, the injury was characterized by vasoconstriction within the area of application whereas the surrounding vessels showed vasodilation after 1 day. Days 2 and 3 were characterized by mounting edema, and congestion of the blood vessels surrounding the lesion. Further, these changes were accompanied by at least partial central necrosis surrounded by granulation tissue at the interface zone towards the uninjured epithelium. By day 4, all lesions appeared pus-filled, constricted, and well-demarcated from the surrounding epithelium. It is noteworthy that the lesion remained wellcircumscribed and corresponded to the initial site of injury. Lesions nearly self-resolve by day 8 post injury (Fig. S4). In order to evaluate whether ultrasound-mediated delivery of
3.4. Ultrasound-mediated budesonide delivery lessens oral lesion severity in vivo Finally, the efficacy of enhanced delivery of budesonide using the oral ultrasound device was tested in an animal model of oral inflammatory lesions (Fig. 3a). The most frequently used model for oral lesions is the hamster buccal pouch model [27]. Further, the size of the buccal pouch allows for use of the full-sized prototype. Previous histological evaluation of oral lesions induced by acetic acid has validated
Fig. 2. Ultrasound-mediated delivery to porcine intestine ex vivo. (A) Schematic demonstrating delivery of fluorescently-labeled dextrans to small intestinal biopsy punches using the oral ultrasound device. For application, the device was applied with the opening of the fluid chamber positioned against the mucosal surface. The outer dashed black line outlines the tissue punch while the smaller dashed line indicates where the device was applied. The fluid chamber was loaded with solution containing fluorescently-labeled dextrans (1 mL). (B) Representative fluorescence images of the mucosal surface after delivery of fluorescently-labeled dextrans of various molecular weights (3, 10, and 500 kDa) with (+) and without (−) 40 kHz ultrasound application for 60 s. PBS with ultrasound served as a control. The entire tissue is encircled (thicker dashed white line) and delivery of labeled dextrans are observed in red at the site of application (smaller dashed white line). Scale bar is 1 cm. (C) Fluorescence intensity of the mucosal surface was measured immediately after delivery (n = 3–6). Data are presented as mean ± SEM. p values were determined by two-tailed, unpaired t-test using Welch's correction; * p < .05, ** p < .001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 4
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Fig. 3. Ultrasound-mediated budesonide delivery lessens oral lesion severity in hamster cheek pouches in vivo. (A) Schematic of the three experimental groups, respective disease induction, and treatment regimen. Note that on day 0, the respective treatments for the Budesonide and Budesonide + US groups were administered before applying acetic acid. (B) Oral lesion areas remain smaller in the Budesonide + US treatment group at 24 and 48 h following lesion induction with acetic acid (n = 17 for 24 and 48 h; n = 4 for 72 h). (C) Representative images of macroscopic oral lesion areas at 48 h post acetic acid application. P values were determined by One-way ANOVA followed by Tukey's post-test. * p < .05 vs. Disease and Budesonide. ** p < .05 vs. Budesonide.
budesonide compared to traditional topical application is more effective in the treatment of oral lesions, the first 72 h following injury induction were analyzed because peak severity occurred during this period. In animals not receiving any treatment, oral lesions reached a maximum size 48 h following topical application of the acetic acid and began to heal naturally after 72 h (Fig. 3b and S4). When compared to Disease and Budesonide groups, animals that received Budesonide + US exhibited smaller oral lesion sizes 24 h post-acetic acid induced injury (Disease n = 17, 20.5 +/− 1.1 mm2; Budesonide n = 17, 20.3 +/− 1.1 mm2; Budesonide + US n = 17, 15.7 +/− 1.3 mm2; p < .05 one-way ANOVA followed by Tukey's post-test). These results demonstrate that budesonide delivered with ultrasound reduces the overall severity and initial size of the lesion using a single dose in a prophylactic manner. 48 h after acetic acid-induced injury, oral lesions in hamster cheek pouches treated with Budesonide + US remained smaller than those treated topically with budesonide without ultrasound (Budesonide + US, 18.71 +/− 2.3 mm2; Budesonide, 25.5 +/− 1.6 mm2, p < .05). After 72 h, oral lesions began to heal naturally and oral lesions in all groups were similar in size. These results demonstrate that budesonide in combination with ultrasound confines the size of the oral lesion, thus preventing the lesion from attaining the severity observed in the Disease and Budesonide groups.
intensity coded to the therapeutic being applied, which eliminates the need for more complex devices and or instructions. In addition to usability, the device must be safe to use and well tolerated. With regards to safety, the design of the device must meet biocompatibility and electrical safety requirements outlined by international standards and adopted by the FDA [34]. The present formfactor has been designed with these standards in mind. For example, to ensure a biocompatible device, the current housing can be made from a wide-range of moldable, acrylonitrile butadiene styrene-like materials. In addition, the transducer employed for our studies meets the electrical safety testing standards set forth in specifications IEC 80601-2-60:2012. These standards are put in place to minimize potential risk of shock to users or for unintended operation of the device. Currently, we are in the process of completing minor modifications to the power supply to ensure compliance with these standards. One additional concern surrounding powered devices is energy emission. When applied to tissues, the energy transfer typically manifests as a temperature increase and we have investigated as part of the present study. Previous strategies to reduce the resulting temperature increase during treatment have focused on modulating the intensity of ultrasound treatment [16,35]. However, here it was not necessary to adjust the intensity or duty cycle in vivo to allow for an “off” period. The temperature rise noted ex vivo over a 60 s treatment was observed to be only 12 °C These results, representing a “worst-case” condition are encouraging given that validated temperature increases in vivo are lower due to a larger “heat-sink” and regulation by the body of temperature [12]. Regardless, if a temperature rise of 12 °C did occur in a user at physiological body temperature, the resulting temperature would be below temperatures of many commonly-consumed hot beverages (60 °C) and temperatures that can cause adverse events [36]. Initial tolerability was assessed in dogs. This is an important readout to ensure clinical use would be pain-free. The fit, sound and sensation in the canine mouth was not rejected by the awake animal. In addition, repeated daily use on sedated hamsters did not show any ill effects with or without ultrasound application. Indeed, the lack of any reaction in dogs when using the device is encouraging and supports further testing in large animal models. Indeed, low-frequency ultrasound has previously been validated as pain-free for transdermal drug delivery and approved for the topical administration of lidocaine [16]. Further testing will be required in two large animal systems to validate the lack of toxicity in support of an eventual investigational new drug application for a combination product leveraging the device with a therapeutic. We used budesonide to assess device efficacy in an in vivo model.
4. Discussion The objective of this study was to engineer a system for low-frequency ultrasound-mediated drug delivery and to demonstrate therapeutic efficacy in an experimental injury model. The form-factor and tolerability of the device was assessed in awake, unsedated dogs. We demonstrate the ability to deposit dextrans of various molecular weights ranging from 3 to 500 kDa into tissue. We show enhanced efficacy of the anti-inflammatory agent, budesonide, through comparison of US-enhanced mucosal delivery, allowing for prophylactic treatment in an established mucosal injury model of oral inflammatory lesions in vivo. The capacity to deliver therapeutics in a targeted and rapid manner in a clinically-relevant form-factor is compelling and can be applied topically in the mouth and beyond. There are many considerations with regards to translating such a technology to the clinic. The first of which is the form-factor and usability of the device. In the present study, the device was designed to be compact and amenable to holding with one hand. While such technology might be used in clinical settings, the flexibility for self-administration by patients directly requires a compact, and simple to use device. This can be accomplished by having the treatment time and 5
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Specifically, we used the well-stablished acetic acid-induced injury model characterized by mucosal injury composed of a central necrotic core with surrounding inflammation. Importantly, necrosis would not be prevented by budesonide administration; rather, budesonide likely modulates and reduces the lesional severity and size due to a reduction in infiltration of inflammatory cells, by altering the inflammatory response at the site of injury [37–39]. In addition, budesonide delivered using ultrasound could result in an improved outcome compared to topical application, due to enhanced delivery. Indeed, our results demonstrate that the severity of the oral lesion was significantly reduced in animals receiving budesonide in combination with ultrasound when compared to those without ultrasound. In other words, topical budesonide without ultrasound demonstrated no such effect. These results strongly support the hypothesis that the efficacy of commonly-utilized therapeutics can be enhanced by targeted, loco-regional delivery. In summary, we have demonstrated a novel low-frequency ultrasound device that can enhance mucosal drug delivery. The device is tolerable in an awake large animal model and the prophylactic application of the anti-inflammatory agent, budesonide, in an oral lesion buccal pouch model. Enhancing the regional application of therapeutics by maximizing penetration may include clinical applications going beyond regional mucosal injury.
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Acknowledgements We thank the Tufts University veterinary staff for assistance with all animal studies. Funding This work was funded by US National Institutes of Health (NIH) grant DE013023 to RL. Author contributions M.M.F., R.L., G.T., J.L., and C.M.S. conceived and designed the research. M.M.F., H.T., E.R, K.X., and C.M.S. performed device development and characterization work. M.M.F., T.d.R., E.R., J.K.L., and C.M.S. designed and performed the in vivo experiments. J.K.L. analyzed tissues. M.M.F. and C.M.S. performed the statistical analysis. M.M.F., T.d.R., E.R., R.L., G.T., J.K.L., and C.M.S. analyzed the data and wrote the manuscript. Competing interests M.M.F., T.d.R., and C.M.S. are employed by Suono Bio, Inc. R.L., G.T., and C.M.S. are co-founders of Suono Bio, Inc., and co-inventors on multiple patent applications surrounding ultrasound-mediated drug delivery technologies. J.K.L has no conflicts of interest. Data and materials availability All data for this study is presented here and all materials are available. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jconrel.2019.04.037. References [1] G. Traverso, A.R. Kirtane, C.M. Schoellhammer, R. Langer, Convergence for translation: drug-delivery research in multidisciplinary teams, Angew. Chem. Int. Ed. Eng. 57 (2018) 4156–4163.
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