Accepted Manuscript A Thermo-sensitive Delivery Platform for Topical Administration of Inflammatory Bowel Disease Therapies Sidhartha R. Sinha, Linh P. Nguyen, Mohammed Inayathullah, Andrey Malkovskiy, Frezghi Habte, Jayakumar Rajadas, Aida Habtezion PII: DOI: Reference:
S0016-5085(15)00452-7 10.1053/j.gastro.2015.04.002 YGAST 59709
To appear in: Gastroenterology Accepted Date: 1 April 2015 Please cite this article as: Sinha SR, Nguyen LP, Inayathullah M, Malkovskiy A, Habte F, Rajadas J, Habtezion A, A Thermo-sensitive Delivery Platform for Topical Administration of Inflammatory Bowel Disease Therapies, Gastroenterology (2015), doi: 10.1053/j.gastro.2015.04.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
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A Thermo-sensitive Delivery Platform for Topical Administration of Inflammatory Bowel Disease Therapies Sidhartha R. Sinha1, 2 Linh P. Nguyen1, 2, Mohammed Inayathullah3, Andrey Malkovskiy3, Frezghi Habte4, Jayakumar Rajadas3, Aida Habtezion1 1
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Stanford University School of Medicine, Division of Gastroenterology and Hepatology, Contributed equally, 3Stanford University School of Medicine, Biomaterials and Advanced Drug Delivery Lab, 4Stanford University School of Medicine, Molecular Imaging Program at Stanford
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Correspondence should be addressed to S.S. ([email protected]), J.R. ([email protected]), or A.H. ([email protected])
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Acknowledgments We thank Ravinder Pamnani for his research on drug formulation and clinical needs assessment, as well as assistance in grants procurement. We thank Vishal Sharma for his technical assistance with the animal work. We thank Robert J. Wong for providing guidance on statistical analysis. This project was supported in part by an institutional training grant from the National Institutes of Health (NIH) T32DK007056-37. This work was also supported by the Inflammatory Bowel Disease Working Group Research Scholarship, Stanford University Translational Research and Medicine (TRAM) grant, the Wallace H. Coulter Foundation Translational Research Grant, Stanford Clinical and Translational Science Award to Spectrum, NIH KL2 TR 001083, and the Division of Gastroenterology and Hepatology at Stanford University School of Medicine.
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Abbreviations IBD, inflammatory bowel disease; BL, budesonide liquid; BPL, budesonide polymer lipid; CD, Crohn’s disease; CT, computerized tomography; DSS, dextran sulfate sodium; ML, mesalamine liquid; MPL, mesalamine polymer lipid; PBMC, peripheral blood mononuclear cells; TDDP, thermo-sensitive drug delivery platform; TNBS, 2,4,6-Trinitrobenzenesulfonic acid solution; UC, ulcerative colitis
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Disclosures The authors declare no competing interests. Author Contributions S.S. conceived this research project and TDDP, performed animal studies (with L.N.), analyzed results, and guided the development of the thermo-sensitive delivery platform with Stanford BioADD group including selection of components included in platform. L.N. contributed equally to the work in this project as a co-first author and performed the animal studies (with S.S.), analyzed histologic sections and results. M.I, J.R., and S.S., created and optimized the TDDP formulations. A.M. performed all rheological experiments. F.H. performed radiology experiments and analyzed results (with L.N. and S.S). J.R. provided technical direction and oversight of the development of the thermo-sensitive formulations. A.H. provided overall supervision and direction for the research project. A.H. suggested and/or reviewed the design of
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all experiments and analyzed all results. S.S., L.N. and A.H. wrote the manuscript. All authors discussed the results, provided edits and commented on the manuscript.
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Abstract: Systemic therapies for inflammatory bowel disease are associated with increased risk of infections and malignancies. Topical therapies reduce systemic exposure, but can be difficult to retain or have limited proximal distribution. To mitigate these issues, we developed a thermosensitive platform, using a polymer-based system that is liquid at room temperature but turns into a viscous gel upon reaching body temperature. Following rectal administration to mice with dextran sulphate sodium-induced colitis, the platform carrying budesonide or mesalamine becomes more viscoelastic near body temperature. Mice given the drug-containing platform gained more weight and had reduced histologic and biologic features of colitis than mice given the platform alone or liquid drugs via enema. Image analysis showed that enemas delivered with and without the platform reached similar distances in the colons of mice, but greater colonic retention was achieved by using the platform.
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KEYWORDS: mouse model of IBD, drug delivery, TDDP, technology
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Topical therapies delivered rectally are safe and effective treatments for colitis. Composed of mesalamine (5-aminosalicylic acid) or corticosteroids, topical therapies can successfully treat acute flares as well as maintain remission for many patients. In fact, more than
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half of patients with ulcerative colitis (UC) and, based on expert opinion, a smaller fraction with Crohn’s colitis may benefit from topical therapy alone, as their disease is limited to the distal colon/rectum.1-3 However, patients with active distal colitis are often unable to tolerate enemas
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due in part to urgency and the associated inability to retain a liquid solution.4 Foams and
suppositories may be easier to retain, but have the marked disadvantage of being unable to reach
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proximal areas of the left colon that are often accessible with an enema.5 Despite the effectiveness of current topical therapies, adherence remains low due to the associated inconvenience and limitations; this lack of adherence leads to increased health risks and costs of care.6
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We have developed a novel thermo-sensitive drug delivery platform (TDDP) that has the advantages of liquid enema (more proximal delivery) and addresses retention issues associated with liquids. This is accomplished by a delivery platform that is a liquid near room temperature,
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but transitions into a viscous gel at body temperature. The platform is a non-ionic surfactant copolymer consisting of hydrophilic polyethylene glycol and hydrophobic polypropylene glycol
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blocks. (Supplementary Figure 1a and Supplementary Figure 2). In this report, we demonstrate the efficacy of this platform with two commonly used therapeutic agents, budesonide and mesalamine. Our first objective was to develop the TDDP with a therapeutic agent that transitions
from a liquid at approximately room temperature to a viscous gel near 37 °C. Such a formulation with budesonide, a corticosteroid with significant first pass metabolism, does become more
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viscoelastic near body temperature, as evidenced by the non-linear increase in storage modulus at 37 °C and 34 °C for the 18% (G’18) and 20% (G’20) polymer solution/gels, respectively (Supplementary Figure 1B).7
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We next tested the therapeutic potential of the TDDP with budesonide (“BPL” for
budesonide, polymer, lipid), in the dextran sulphate sodium (DSS) colitis model. The BPL group reproducibly demonstrated improvement versus water and polymer controls, as well as
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budesonide liquid (BL), as shown by the limited body weight loss (Figure 1A and 1B). In addition, BPL-treated mice had longer colons (with well-formed stool) and histologically
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reduced leukocyte infiltration and more preserved epithelial architecture (Figure 1C-E). Similar anti-inflammatory activity of BL and BPL was seen in cultured cells (Supplementary Figure 3), but animal studies demonstrated that TDDP increases the effectiveness of budesonide in vivo. We hypothesize that the BPL enema, as a liquid at instillation, would have a non-inferior
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distribution, and as a transitioned gel, would have greater retention compared to liquid enema. To determine the colonic distribution and retention kinetics of BPL, we gave standardized enemas with contrast to healthy and colitis mice, and imaged the mice at pre-determined
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intervals. The BPL and BL enemas did indeed reach a similar distance (at 0.25 h; Supplementary Figure 4), but interestingly, the distance of BPL was better maintained (Figure
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2A and Supplementary Figure 4A). Based on 3-D imaging analyses, the volume of BPL (or polymer) enema retained was substantially greater than that of BL (Figure 2B and Supplementary Figure 4B) in both healthy and inflamed colons, indicating possible mucoadhesiveness.
The TDDP did not appear to alter bowel function, as BPL-treated mice observed for 4 additional weeks continued to gain weight and have normal bowel movements. In addition,
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multiple BPL applications did not increase the risk of obstruction in a model of TNBS-induced strictures, as evidenced by imaging and the absence of mortality (Supplementary Figure 5, and data not shown)8. However, long-term studies are needed to confirm safety of its use in patients
drug delivery without the systemic exposure.
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with colonic strictures. As with topical drugs, a potential benefit of the BPL enema is localized
To determine if our formulation could be used as a platform for other rectally
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administered drugs, we formulated TDDP with mesalamine (“MPL” for mesalamine, polymer, lipid). Unlike the standard mesalamine enema (“ML” for mesalamine, liquid) or vehicle
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controls, MPL minimized DSS-associated weight loss, colon shortening, and histologic inflammation in multiple independent experiments (Supplementary Figure 6). The positive outcome of using TDDP is not model- or strain-specific, as BPL also treated trinitrobenzene sulfonic acid (TNBS)-induced colitis in Balb/c mice (Supplementary Figure 7).
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In summary, there is a clinical unmet need for better-targeted and localized therapies that can reach diseased areas and are easier to retain. In addition, major limitations in current IBD therapies include the side effects and intolerances associated with systemic therapies.9 Our
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studies herein use TDDP, a novel thermo-sensitive platform that addresses these limitations. Using two IBD models, we show that TDDP with therapeutic is superior to standard treatments
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by seemingly overcoming significant issues with current topical therapies. The majority of IBD patients with colitis and all UC patients can benefit from topical
therapies (particularly during acute flares for effective and more rapid treatment), and many can be effectively managed with topicals exclusively.10 Due to its greater proximal reach and retention, the TDDP could allow for less frequent administration, which is likely to improve patient compliance.
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While local mesalamine and steroid enemas are first line therapies in treating IBD, one can envision utilizing the TDDP with other therapies, including biologic or cell-based treatments. Animal studies, for example, suggest that topically applied inhibitors of tumor necrosis factor
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(TNF)-α ameliorate colitis severity in animal models.11 Additionally, this platform could be used to deliver therapeutics for other disease states. With the established safety profile of its
components, TDDP has enormous potential to improve the care of patients with IBD, as well as
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other diseases.
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Figure Legends Figure 1. Budesonide polymer (BPL) improves DSS-induced colitis. (A) Weight of mice
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given DSS starting day 0 and water, polymer, budesonide liquid (BL) or BPL enemas on days 4 and 6 (representative of 4 experiments). n = 5 BL, BPL and water, n = 3 polymer; mean ± SEM. Two-way ANOVA with Bonferroni posttests. ***P < 0.001 between BPL and other treatments,
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except polymer on day 8, where *P < 0.05. (B) Body weight by end of 4 combined experiments. n = 14 water, 22 polymer, 19 BL and 19 BPL; mean ± SEM. One-way ANOVA with Tukey’s
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posttests. **P < 0.01, ***P < 0.001. (C) Gross colon morphology and length. n = 5; mean ± SEM. One-way ANOVA with Tukey’s posttests. **P < 0.01, ***P < 0.001. (D) H&E staining of colon. Scale bar represents 20 µm. (E) Histopathology score from 3 combined studies. n = 14 water, 18 polymer, 14 BL and 14 BPL; mean + SEM. One-way ANOVA with Tukey’s posttests.
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**P < 0.001, ***P < 0.0001.
Figure 2. Budesonide polymer (BPL) has better retrograde distance and retention. Healthy
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or colitis mice were treated with 150 µL of budesonide liquid (BL) or BPL enema and imaged after 0.5, 1.5 and 3 h. (A) Distance and (B) retained enema volume. n = 4; mean ± SEM.
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Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Mice at 0.5, 1.5 and 3 h post-enema. Contrast is software-highlighted green in right panel of each image pair.
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References:
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Hanauer SB, ed. Gastroenterol Hepatol (N Y) 2010;6:309-16. Scheurlen C et al. Hepatogastroenterology 1998;45:1539-45. Travis SPL et al. Gut 2006;55:i16-i35. Prantera C and Rizzi M. World J Gastroenterol 2009;15:4353-5. Kornbluth A et al. Am J Gastroenterol 2010;105:501-23; quiz 524. Rubin DT et al. Aliment Pharmacol Ther 2014;39:1143-55. Hanauer SB et al. Gastroenterology 1998;115:525-32. Lawrance IC and Wu F et al. Gastroenterology 2003;125:1750-61. Mahadevan U. Clin Colon Rectal Surg 2004;17:7-19. Kam L et al. Am J Gastroenterol 1996;91:1338-42. Lopetuso LR et al. Dig Liver Dis 2013;45:1017-21.
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Author names in bold designate shared co-first authorship
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1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
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Materials and Methods Mice Male C57BL/6 mice were purchased from Taconic (Hudson, NY) and housed for one week
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before DSS experiments. Balb/c mice were purchased from Jackson Laboratory (Bar Harbor, MN) and bred in house, and male mice were used for acute TNBS colitis studies, while females were used for chronic TNBS stricture studies. Mice used were 6-8 weeks old. Care and use of
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University institutional animal care and use committees.
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animals were in accordance to National Institutes of Health guidelines and approved by Stanford
Budesonide and Mesalamine formulations
FDA generally regarded as safe (GRAS) phospholipids (such as 1,2-distearoyl-sn-glycero-3phosphocholine, DSPC, 0.4 mg/ml) and budesonide (0.1mg/mL) were dissolved in ethanol in a
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round bottom flask. The solvent was evaporated in a Heidolph rotary evaporator (Laborota 4011) to form a thin film in the inner surface of flask. The film was suspended in water and sonicated for 30 minutes to obtain a liposome solution. Selected thermo-sensitive GRAS
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polymers (typically used as inactive ingredients for different types of preparations (e.g., intravenous, topical, ophthalmic, and oral formulations)) were then added with water at 4 °C to
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reach the appropriate concentration. The mixture was stirred for 30 minutes and the trapped air bubbles were removed by centrifuging at 600 × g in Heraeus Labofuge - 400 centrifuge. Stirring was continued at 4 °C until a homogeneous solution (budesonide-polymer-lipid, BPL) was obtained. Mesalamine-polymer-lipid (MPL) was also prepared using the same procedure using mesalamine (67 mg/mL) and phospholipids (10 mg/mL). For the budesonide liquid (BL) formulation, commercially available Entocort® Enema (AstraZeneca) was obtained containing
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budesonide tablet and pre-made enema solution. The budesonide tablet was suspended in enough solution to obtain a 0.1 mg/ml concentration of BL. For the mesalamine liquid formulation (ML), commercially available Rowasa® (Perrigo) was obtained. Mesalamine was
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mixed with enough pre-made solution to obtain a concentration of 67 mg/mL.
Rheology
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The shear modulus and viscosity of copolymer thermogels have been measured with an Ares® rheometer (Rheometric Scientific®) equipped with an oven for precise temperature control in a
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parallel plate geometry. Frequency scan measurements (0.5 – 200 rad/s) have been performed for every temperature, while maintaining a constant strain of 1%.
DSS colitis
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C57BL/6 mice were given 2% (w/v) dextran sulfate sodium salt (36,000-50,000 M.W.; MP Biomedicals, LLC; Solon, OH) in drinking water for the duration of the experiment1. Mice were weighed daily and assessed for presence of bloody stool, diarrhea and general well being.
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Treatment (BPL, BL) and control (water, polymer only) enemas were given on day 4 and day 6. Isoflorane-anesthetized mice were rectally given 150 µL of indicated solution through a 1 mL
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Luer Lock syringe attached to 23G needle and polyethylene tubing (0.048” O.D.), with tubing inserted ~1.5 cm. Animals were sacrificed on day 9 or earlier if moribund or their weight loss exceeded 20%, per Stanford University institutional animal care and use committee requirements.
TNBS colitis
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Balb/c mice were anesthetized with ketamine and rectally treated with 2 mg of 2,4,6Trinitrobenzenesulfonic acid solution (Sigma-Aldrich; St. Louis, MO) in 100 µL volume, as previously described1. Control mice received 100 µL of vehicle (40% ethanol). The next day
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(day 1), mice were anesthetized with isoflurane and given treatment or control enemas of 150 µL, as with DSS colitis model. Animals were weighed and assessed daily and sacrificed at or
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before loss of 20% body weight.
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Chronic TNBS-induced stricture
Female Balb/c mice were rectally treated once weekly with increasing doses of TNBS for 6 weeks to induce colonic strictures as previously described.2 The mice received 0.5 mg TNBS (in 100 µL 30% ethanol) on weeks 1 and 2, 0.75 mg TNBS on weeks 3 and 4 (in 35% ethanol) and 1 mg TNBS (in 40% ethanol) weeks 5 and 6. One week later, they were given 150 µL of
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Cystografin iodine contrast (Bracco Diagnostics) per rectum and scanned by computed tomography (CT) to determine the presence of strictures. Strictures (between the cecum and rectum) were scored based on visible proximal lumen distension and immediately distal lumen
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narrowing. Mice with strictures were randomly divided into two groups (of 4 animals each) and
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given BL or BPL enemas 1, 3 and 5 days afterwards. At 24 h after the last treatment, the mice were again scanned and their colons collected for H&E and trichrome analyses. A second cohort received weekly enemas for 4 weeks after the 6 week TNBS treatment, as shown in the Supplementary Figure 7, and these mice were also assessed for signs of obstruction using imaging studies.
Computed Tomography (CT) imaging
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Male C57BL/6 mice were given water containing 2% (w/v) DSS and on day 6 (when bloody stool is apparent) were administered 150 µL BPL or BL enemas containing 5% (v/v) barium sulfate suspension (E-Z-EM, Inc.; Lake Success, NY). The enema tubing was inserted in a
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standardized manner with insertion of ~1.5 cm of tubing and expulsion of enema took 3-5 seconds. For CT imaging, we used Inveon MicroPET/CT (Preclinical Solutions, Siemens
Healthcare Molecular Imaging, Knoxville, TN) located at the Stanford Center for Innovation in
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In-Vivo Imaging, Stanford School of Medicine shared facility. In each scan, a group of four mice were imaged and retrograde distance and enema volume were calculated using Inveon Research
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Workspace (IRW) analysis software (Preclinical Solutions, Siemens Healthcare Molecular Imaging, Knoxville, TN) from the acquired whole body CT images of ~200 micron resolution.
Anti-inflammatory activity of BL and BPL in vitro
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Peripheral blood mononuclear cells (PBMC) isolated from healthy donors were cultured with 1 µg/mL each of anti–CD3 (clone HIT3a) and anti–CD28 antibodies (clone CD28.2) in RPMI1640 media containing 10% FBS and penicillin-streptomycin (1 µg/mL each). The cells (0.5 ×
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106 cells per well in a 24-well plate) were treated daily with 10 µL of PBS, 20% polymer solution, BL, or BPL (for a final budesonide concentration of 2 µg/mL).3 After 48 h, the cells
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were washed and activated with PMA and ionomycin in the presence of Brefeldin A at 37 °C for 4 h. Expression of TNF-α (clone MAb11; BD Biosciences) was assessed by flow cytometry using the Fortessa (BD Biosciences).
Histology
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For histological assessment of the DSS model, the most distal 1 cm colon segments were fixed in 10% formalin-buffered, paraffin-embedded and sectioned for hematoxylin & eosin staining. For the TNBS model, the 2nd and 3rd distal most cm were used. Histopathology was scored in
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blinded fashion as previously described.4
Statistics
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Prism software (GraphPad Software; San Diego, CA) was used to perform statistical analyses
significant.
Supplementary Reference
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Wirtz S et al. Nat Protoc 2007;2:541-6. Lawrance IC and Wu F et al. Gastroenterology 2003;125:1750-61. McKay DM et al. J Pharmacol Exp Ther 1996;277:403-10. Ostanin DV et al. Am J Physiol Gastrointest Liver Physiol 2009;296:G135-46.
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1. 2. 3. 4.
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and statistical tests employed are indicated in the figures, with P values of
S0016-5085(15)00452-7 10.1053/j.gastro.2015.04.002 YGAST 59709
To appear in: Gastroenterology Accepted Date: 1 April 2015 Please cite this article as: Sinha SR, Nguyen LP, Inayathullah M, Malkovskiy A, Habte F, Rajadas J, Habtezion A, A Thermo-sensitive Delivery Platform for Topical Administration of Inflammatory Bowel Disease Therapies, Gastroenterology (2015), doi: 10.1053/j.gastro.2015.04.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
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A Thermo-sensitive Delivery Platform for Topical Administration of Inflammatory Bowel Disease Therapies Sidhartha R. Sinha1, 2 Linh P. Nguyen1, 2, Mohammed Inayathullah3, Andrey Malkovskiy3, Frezghi Habte4, Jayakumar Rajadas3, Aida Habtezion1 1
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Stanford University School of Medicine, Division of Gastroenterology and Hepatology, Contributed equally, 3Stanford University School of Medicine, Biomaterials and Advanced Drug Delivery Lab, 4Stanford University School of Medicine, Molecular Imaging Program at Stanford
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Correspondence should be addressed to S.S. ([email protected]), J.R. ([email protected]), or A.H. ([email protected])
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Acknowledgments We thank Ravinder Pamnani for his research on drug formulation and clinical needs assessment, as well as assistance in grants procurement. We thank Vishal Sharma for his technical assistance with the animal work. We thank Robert J. Wong for providing guidance on statistical analysis. This project was supported in part by an institutional training grant from the National Institutes of Health (NIH) T32DK007056-37. This work was also supported by the Inflammatory Bowel Disease Working Group Research Scholarship, Stanford University Translational Research and Medicine (TRAM) grant, the Wallace H. Coulter Foundation Translational Research Grant, Stanford Clinical and Translational Science Award to Spectrum, NIH KL2 TR 001083, and the Division of Gastroenterology and Hepatology at Stanford University School of Medicine.
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Abbreviations IBD, inflammatory bowel disease; BL, budesonide liquid; BPL, budesonide polymer lipid; CD, Crohn’s disease; CT, computerized tomography; DSS, dextran sulfate sodium; ML, mesalamine liquid; MPL, mesalamine polymer lipid; PBMC, peripheral blood mononuclear cells; TDDP, thermo-sensitive drug delivery platform; TNBS, 2,4,6-Trinitrobenzenesulfonic acid solution; UC, ulcerative colitis
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Disclosures The authors declare no competing interests. Author Contributions S.S. conceived this research project and TDDP, performed animal studies (with L.N.), analyzed results, and guided the development of the thermo-sensitive delivery platform with Stanford BioADD group including selection of components included in platform. L.N. contributed equally to the work in this project as a co-first author and performed the animal studies (with S.S.), analyzed histologic sections and results. M.I, J.R., and S.S., created and optimized the TDDP formulations. A.M. performed all rheological experiments. F.H. performed radiology experiments and analyzed results (with L.N. and S.S). J.R. provided technical direction and oversight of the development of the thermo-sensitive formulations. A.H. provided overall supervision and direction for the research project. A.H. suggested and/or reviewed the design of
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all experiments and analyzed all results. S.S., L.N. and A.H. wrote the manuscript. All authors discussed the results, provided edits and commented on the manuscript.
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Abstract: Systemic therapies for inflammatory bowel disease are associated with increased risk of infections and malignancies. Topical therapies reduce systemic exposure, but can be difficult to retain or have limited proximal distribution. To mitigate these issues, we developed a thermosensitive platform, using a polymer-based system that is liquid at room temperature but turns into a viscous gel upon reaching body temperature. Following rectal administration to mice with dextran sulphate sodium-induced colitis, the platform carrying budesonide or mesalamine becomes more viscoelastic near body temperature. Mice given the drug-containing platform gained more weight and had reduced histologic and biologic features of colitis than mice given the platform alone or liquid drugs via enema. Image analysis showed that enemas delivered with and without the platform reached similar distances in the colons of mice, but greater colonic retention was achieved by using the platform.
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KEYWORDS: mouse model of IBD, drug delivery, TDDP, technology
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Topical therapies delivered rectally are safe and effective treatments for colitis. Composed of mesalamine (5-aminosalicylic acid) or corticosteroids, topical therapies can successfully treat acute flares as well as maintain remission for many patients. In fact, more than
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half of patients with ulcerative colitis (UC) and, based on expert opinion, a smaller fraction with Crohn’s colitis may benefit from topical therapy alone, as their disease is limited to the distal colon/rectum.1-3 However, patients with active distal colitis are often unable to tolerate enemas
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due in part to urgency and the associated inability to retain a liquid solution.4 Foams and
suppositories may be easier to retain, but have the marked disadvantage of being unable to reach
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proximal areas of the left colon that are often accessible with an enema.5 Despite the effectiveness of current topical therapies, adherence remains low due to the associated inconvenience and limitations; this lack of adherence leads to increased health risks and costs of care.6
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We have developed a novel thermo-sensitive drug delivery platform (TDDP) that has the advantages of liquid enema (more proximal delivery) and addresses retention issues associated with liquids. This is accomplished by a delivery platform that is a liquid near room temperature,
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but transitions into a viscous gel at body temperature. The platform is a non-ionic surfactant copolymer consisting of hydrophilic polyethylene glycol and hydrophobic polypropylene glycol
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blocks. (Supplementary Figure 1a and Supplementary Figure 2). In this report, we demonstrate the efficacy of this platform with two commonly used therapeutic agents, budesonide and mesalamine. Our first objective was to develop the TDDP with a therapeutic agent that transitions
from a liquid at approximately room temperature to a viscous gel near 37 °C. Such a formulation with budesonide, a corticosteroid with significant first pass metabolism, does become more
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viscoelastic near body temperature, as evidenced by the non-linear increase in storage modulus at 37 °C and 34 °C for the 18% (G’18) and 20% (G’20) polymer solution/gels, respectively (Supplementary Figure 1B).7
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We next tested the therapeutic potential of the TDDP with budesonide (“BPL” for
budesonide, polymer, lipid), in the dextran sulphate sodium (DSS) colitis model. The BPL group reproducibly demonstrated improvement versus water and polymer controls, as well as
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budesonide liquid (BL), as shown by the limited body weight loss (Figure 1A and 1B). In addition, BPL-treated mice had longer colons (with well-formed stool) and histologically
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reduced leukocyte infiltration and more preserved epithelial architecture (Figure 1C-E). Similar anti-inflammatory activity of BL and BPL was seen in cultured cells (Supplementary Figure 3), but animal studies demonstrated that TDDP increases the effectiveness of budesonide in vivo. We hypothesize that the BPL enema, as a liquid at instillation, would have a non-inferior
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distribution, and as a transitioned gel, would have greater retention compared to liquid enema. To determine the colonic distribution and retention kinetics of BPL, we gave standardized enemas with contrast to healthy and colitis mice, and imaged the mice at pre-determined
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intervals. The BPL and BL enemas did indeed reach a similar distance (at 0.25 h; Supplementary Figure 4), but interestingly, the distance of BPL was better maintained (Figure
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2A and Supplementary Figure 4A). Based on 3-D imaging analyses, the volume of BPL (or polymer) enema retained was substantially greater than that of BL (Figure 2B and Supplementary Figure 4B) in both healthy and inflamed colons, indicating possible mucoadhesiveness.
The TDDP did not appear to alter bowel function, as BPL-treated mice observed for 4 additional weeks continued to gain weight and have normal bowel movements. In addition,
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multiple BPL applications did not increase the risk of obstruction in a model of TNBS-induced strictures, as evidenced by imaging and the absence of mortality (Supplementary Figure 5, and data not shown)8. However, long-term studies are needed to confirm safety of its use in patients
drug delivery without the systemic exposure.
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with colonic strictures. As with topical drugs, a potential benefit of the BPL enema is localized
To determine if our formulation could be used as a platform for other rectally
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administered drugs, we formulated TDDP with mesalamine (“MPL” for mesalamine, polymer, lipid). Unlike the standard mesalamine enema (“ML” for mesalamine, liquid) or vehicle
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controls, MPL minimized DSS-associated weight loss, colon shortening, and histologic inflammation in multiple independent experiments (Supplementary Figure 6). The positive outcome of using TDDP is not model- or strain-specific, as BPL also treated trinitrobenzene sulfonic acid (TNBS)-induced colitis in Balb/c mice (Supplementary Figure 7).
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In summary, there is a clinical unmet need for better-targeted and localized therapies that can reach diseased areas and are easier to retain. In addition, major limitations in current IBD therapies include the side effects and intolerances associated with systemic therapies.9 Our
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studies herein use TDDP, a novel thermo-sensitive platform that addresses these limitations. Using two IBD models, we show that TDDP with therapeutic is superior to standard treatments
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by seemingly overcoming significant issues with current topical therapies. The majority of IBD patients with colitis and all UC patients can benefit from topical
therapies (particularly during acute flares for effective and more rapid treatment), and many can be effectively managed with topicals exclusively.10 Due to its greater proximal reach and retention, the TDDP could allow for less frequent administration, which is likely to improve patient compliance.
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While local mesalamine and steroid enemas are first line therapies in treating IBD, one can envision utilizing the TDDP with other therapies, including biologic or cell-based treatments. Animal studies, for example, suggest that topically applied inhibitors of tumor necrosis factor
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(TNF)-α ameliorate colitis severity in animal models.11 Additionally, this platform could be used to deliver therapeutics for other disease states. With the established safety profile of its
components, TDDP has enormous potential to improve the care of patients with IBD, as well as
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other diseases.
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Figure Legends Figure 1. Budesonide polymer (BPL) improves DSS-induced colitis. (A) Weight of mice
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given DSS starting day 0 and water, polymer, budesonide liquid (BL) or BPL enemas on days 4 and 6 (representative of 4 experiments). n = 5 BL, BPL and water, n = 3 polymer; mean ± SEM. Two-way ANOVA with Bonferroni posttests. ***P < 0.001 between BPL and other treatments,
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except polymer on day 8, where *P < 0.05. (B) Body weight by end of 4 combined experiments. n = 14 water, 22 polymer, 19 BL and 19 BPL; mean ± SEM. One-way ANOVA with Tukey’s
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posttests. **P < 0.01, ***P < 0.001. (C) Gross colon morphology and length. n = 5; mean ± SEM. One-way ANOVA with Tukey’s posttests. **P < 0.01, ***P < 0.001. (D) H&E staining of colon. Scale bar represents 20 µm. (E) Histopathology score from 3 combined studies. n = 14 water, 18 polymer, 14 BL and 14 BPL; mean + SEM. One-way ANOVA with Tukey’s posttests.
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**P < 0.001, ***P < 0.0001.
Figure 2. Budesonide polymer (BPL) has better retrograde distance and retention. Healthy
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or colitis mice were treated with 150 µL of budesonide liquid (BL) or BPL enema and imaged after 0.5, 1.5 and 3 h. (A) Distance and (B) retained enema volume. n = 4; mean ± SEM.
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Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Mice at 0.5, 1.5 and 3 h post-enema. Contrast is software-highlighted green in right panel of each image pair.
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References:
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Hanauer SB, ed. Gastroenterol Hepatol (N Y) 2010;6:309-16. Scheurlen C et al. Hepatogastroenterology 1998;45:1539-45. Travis SPL et al. Gut 2006;55:i16-i35. Prantera C and Rizzi M. World J Gastroenterol 2009;15:4353-5. Kornbluth A et al. Am J Gastroenterol 2010;105:501-23; quiz 524. Rubin DT et al. Aliment Pharmacol Ther 2014;39:1143-55. Hanauer SB et al. Gastroenterology 1998;115:525-32. Lawrance IC and Wu F et al. Gastroenterology 2003;125:1750-61. Mahadevan U. Clin Colon Rectal Surg 2004;17:7-19. Kam L et al. Am J Gastroenterol 1996;91:1338-42. Lopetuso LR et al. Dig Liver Dis 2013;45:1017-21.
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Author names in bold designate shared co-first authorship
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1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
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Materials and Methods Mice Male C57BL/6 mice were purchased from Taconic (Hudson, NY) and housed for one week
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before DSS experiments. Balb/c mice were purchased from Jackson Laboratory (Bar Harbor, MN) and bred in house, and male mice were used for acute TNBS colitis studies, while females were used for chronic TNBS stricture studies. Mice used were 6-8 weeks old. Care and use of
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University institutional animal care and use committees.
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animals were in accordance to National Institutes of Health guidelines and approved by Stanford
Budesonide and Mesalamine formulations
FDA generally regarded as safe (GRAS) phospholipids (such as 1,2-distearoyl-sn-glycero-3phosphocholine, DSPC, 0.4 mg/ml) and budesonide (0.1mg/mL) were dissolved in ethanol in a
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round bottom flask. The solvent was evaporated in a Heidolph rotary evaporator (Laborota 4011) to form a thin film in the inner surface of flask. The film was suspended in water and sonicated for 30 minutes to obtain a liposome solution. Selected thermo-sensitive GRAS
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polymers (typically used as inactive ingredients for different types of preparations (e.g., intravenous, topical, ophthalmic, and oral formulations)) were then added with water at 4 °C to
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reach the appropriate concentration. The mixture was stirred for 30 minutes and the trapped air bubbles were removed by centrifuging at 600 × g in Heraeus Labofuge - 400 centrifuge. Stirring was continued at 4 °C until a homogeneous solution (budesonide-polymer-lipid, BPL) was obtained. Mesalamine-polymer-lipid (MPL) was also prepared using the same procedure using mesalamine (67 mg/mL) and phospholipids (10 mg/mL). For the budesonide liquid (BL) formulation, commercially available Entocort® Enema (AstraZeneca) was obtained containing
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budesonide tablet and pre-made enema solution. The budesonide tablet was suspended in enough solution to obtain a 0.1 mg/ml concentration of BL. For the mesalamine liquid formulation (ML), commercially available Rowasa® (Perrigo) was obtained. Mesalamine was
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mixed with enough pre-made solution to obtain a concentration of 67 mg/mL.
Rheology
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The shear modulus and viscosity of copolymer thermogels have been measured with an Ares® rheometer (Rheometric Scientific®) equipped with an oven for precise temperature control in a
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parallel plate geometry. Frequency scan measurements (0.5 – 200 rad/s) have been performed for every temperature, while maintaining a constant strain of 1%.
DSS colitis
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C57BL/6 mice were given 2% (w/v) dextran sulfate sodium salt (36,000-50,000 M.W.; MP Biomedicals, LLC; Solon, OH) in drinking water for the duration of the experiment1. Mice were weighed daily and assessed for presence of bloody stool, diarrhea and general well being.
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Treatment (BPL, BL) and control (water, polymer only) enemas were given on day 4 and day 6. Isoflorane-anesthetized mice were rectally given 150 µL of indicated solution through a 1 mL
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Luer Lock syringe attached to 23G needle and polyethylene tubing (0.048” O.D.), with tubing inserted ~1.5 cm. Animals were sacrificed on day 9 or earlier if moribund or their weight loss exceeded 20%, per Stanford University institutional animal care and use committee requirements.
TNBS colitis
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Balb/c mice were anesthetized with ketamine and rectally treated with 2 mg of 2,4,6Trinitrobenzenesulfonic acid solution (Sigma-Aldrich; St. Louis, MO) in 100 µL volume, as previously described1. Control mice received 100 µL of vehicle (40% ethanol). The next day
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(day 1), mice were anesthetized with isoflurane and given treatment or control enemas of 150 µL, as with DSS colitis model. Animals were weighed and assessed daily and sacrificed at or
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before loss of 20% body weight.
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Chronic TNBS-induced stricture
Female Balb/c mice were rectally treated once weekly with increasing doses of TNBS for 6 weeks to induce colonic strictures as previously described.2 The mice received 0.5 mg TNBS (in 100 µL 30% ethanol) on weeks 1 and 2, 0.75 mg TNBS on weeks 3 and 4 (in 35% ethanol) and 1 mg TNBS (in 40% ethanol) weeks 5 and 6. One week later, they were given 150 µL of
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Cystografin iodine contrast (Bracco Diagnostics) per rectum and scanned by computed tomography (CT) to determine the presence of strictures. Strictures (between the cecum and rectum) were scored based on visible proximal lumen distension and immediately distal lumen
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narrowing. Mice with strictures were randomly divided into two groups (of 4 animals each) and
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given BL or BPL enemas 1, 3 and 5 days afterwards. At 24 h after the last treatment, the mice were again scanned and their colons collected for H&E and trichrome analyses. A second cohort received weekly enemas for 4 weeks after the 6 week TNBS treatment, as shown in the Supplementary Figure 7, and these mice were also assessed for signs of obstruction using imaging studies.
Computed Tomography (CT) imaging
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Male C57BL/6 mice were given water containing 2% (w/v) DSS and on day 6 (when bloody stool is apparent) were administered 150 µL BPL or BL enemas containing 5% (v/v) barium sulfate suspension (E-Z-EM, Inc.; Lake Success, NY). The enema tubing was inserted in a
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standardized manner with insertion of ~1.5 cm of tubing and expulsion of enema took 3-5 seconds. For CT imaging, we used Inveon MicroPET/CT (Preclinical Solutions, Siemens
Healthcare Molecular Imaging, Knoxville, TN) located at the Stanford Center for Innovation in
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In-Vivo Imaging, Stanford School of Medicine shared facility. In each scan, a group of four mice were imaged and retrograde distance and enema volume were calculated using Inveon Research
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Workspace (IRW) analysis software (Preclinical Solutions, Siemens Healthcare Molecular Imaging, Knoxville, TN) from the acquired whole body CT images of ~200 micron resolution.
Anti-inflammatory activity of BL and BPL in vitro
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Peripheral blood mononuclear cells (PBMC) isolated from healthy donors were cultured with 1 µg/mL each of anti–CD3 (clone HIT3a) and anti–CD28 antibodies (clone CD28.2) in RPMI1640 media containing 10% FBS and penicillin-streptomycin (1 µg/mL each). The cells (0.5 ×
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106 cells per well in a 24-well plate) were treated daily with 10 µL of PBS, 20% polymer solution, BL, or BPL (for a final budesonide concentration of 2 µg/mL).3 After 48 h, the cells
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were washed and activated with PMA and ionomycin in the presence of Brefeldin A at 37 °C for 4 h. Expression of TNF-α (clone MAb11; BD Biosciences) was assessed by flow cytometry using the Fortessa (BD Biosciences).
Histology
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For histological assessment of the DSS model, the most distal 1 cm colon segments were fixed in 10% formalin-buffered, paraffin-embedded and sectioned for hematoxylin & eosin staining. For the TNBS model, the 2nd and 3rd distal most cm were used. Histopathology was scored in
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blinded fashion as previously described.4
Statistics
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Prism software (GraphPad Software; San Diego, CA) was used to perform statistical analyses
significant.
Supplementary Reference
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Wirtz S et al. Nat Protoc 2007;2:541-6. Lawrance IC and Wu F et al. Gastroenterology 2003;125:1750-61. McKay DM et al. J Pharmacol Exp Ther 1996;277:403-10. Ostanin DV et al. Am J Physiol Gastrointest Liver Physiol 2009;296:G135-46.
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1. 2. 3. 4.
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and statistical tests employed are indicated in the figures, with P values of
