Vazula Bekkers

Needle-free jet injector-assisted drug delivery in dermatology Towards effective and minimally invasive treatment of severe keloids VAZULA BEKKERS

Needle-Free Jet Injector-Assisted Drug Delivery in Dermatology Towards effective and minimally invasive treatment of severe keloids Vazula Zulfra Bekkers

ISBN: 978-94-6506-661-5 Cover design: Loes Vos, Amsterdam, The Netherlands Printing: Ridderprint, Ablasserdam, The Netherlands

Needle-Free Jet Injector-Assisted Drug Delivery in Dermatology Towards effective and minimally invasive treatment of severe keloids Naaldvrije behandeling middels jet injectoren in de dermatologie Naar effectieve en minimaal invasieve behandeling van ernstige keloïden Proefschrift ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof.dr.ir. A.J. Schuit en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op dinsdag 28 januari 2025 om 10:30 uur door Vazula Zulfra Bekkers geboren te Rotterdam.

PROMOTIECOMMISSIE Promotoren Prof.dr. E.P. Prens Prof.dr. R. Rissmann Overige leden Prof.dr.ir. D. Fernandez Rivas Prof.dr. M.A.M. Mureau Prof.dr. M. Haedersdal Copromotor Dr. M.B.A. van Doorn Paranimfen I.T. Küçük L.C. Dommershuijzen

TABLE OF CONTENTS Chapter 1 General introduction 9 Section I Needle-free jet injectors in dermatology Chapter 2 Efficacy and safety of needle-free jet injector-assisted intralesional 37 treatments in dermatology—a systematic review Section II Biodistribution using different injection techniques in severe keloids and properties of severe keloids Chapter 3 Biodistribution of needle-injections and needle-free jet-injectors 73 visualized by a 3D- Fluorescent Imaging Cryomicrotome System Chapter 4 Effects of keloid properties on treatment efficacy 95 Section III Efficacy and safety of intralesional bleomycin treatment in severe keloids Chapter 5 Needle-free electronically-controlled jet injector treatment with 115 bleomycin is efficacious and well-tolerated in patients with severe keloids: results of a randomized, double-blind, placebo-controlled trial Chapter 6 Needle-free electronically-controlled jet injector treatment with 145 bleomycin and lidocaine is effective and well-tolerated in patients with recalcitrant keloids Section IV Needle-free jet injector assisted treatment in children with keloids and hypertrophic scars Chapter 7 Needle-free jet Injector-assisted triamcinolone treatment of keloids and 177 hypertrophic scars is effective and well tolerated in children

Chapter 8 General discussion 199 Chapter 9 English and Dutch summaries 219 Chapter 10 Appendices 229 Abbreviations List of co-authors List of publications About the author PhD portfolio Dankwoord

9 Chapter 1 General introduction

1. General introduction 10 Keloids The word keloid is derived from the Greek word ‘chele’, meaning crab’s claw because of its typical sideways growth into normal skin.1 Keloids are fibroproliferative scars that cause reduced quality of life, due to pain, itch, social stigma and restriction of movement.2 The mean Dermatology Life Quality Index (DLQI) in patients with keloids is 7.8  5.1, meaning that keloids have a ‘moderate effect’ on patients quality of life, comparable to patients with mild to severe psoriasis vulgaris.3 Keloids can develop due to inflammation such as inflammatory acne and folliculitis or after (minor) trauma.4 Keloid formation may be caused by a dysregulation in at least one of the four phases of wound healing: hemostasis, inflammation, proliferation and remodeling.5,6 The inflammatory response in wound healing may play a crucial role in keloid formation. In keloids proinflammatory factors including interleukin-1α, -1 -6 and tissue growth factor-beta 1 (TGF-1) are upregulated.7 The upregulation of these proinflammatory factors suggests that the formation of keloids can be considered as an inflammatory disorder of the reticular dermis.7 Moreover, recent studies have proposed that keloids possess features of an auto-immune disease, since anti-hnRNPA2B1 autoantibodies, Immunoglobulin A and M, and Complement components C1Q and C3 depositions were found in keloid tissue.8,9 Keloids vs. hypertrophic scars Keloidal scars differ from hypertrophic scars because keloids will expand beyond the borders of the original wound while hypertrophic scars do not.10 However, keloids also differ from hypertrophic scars in other aspects; keloids are histologically different, often cause more symptoms, and are more difficult to treat (Figure 1A-1D).11,12 On histological examination, keloids show thicker collagen bundles, without a distinct pattern, while hypertrophic scars show relative thinner collagen bundles in a regular pattern.13 The prevalence of keloids strongly differs among different ethnic populations. Keloids occur most frequently in the African, Asian and Hispanic population (prevalence up to 16%),

1. General introduction 11 1. while the European population is least affected (prevalence of <0.1%).14 However, hypertrophic scars develop much more frequently (prevalence of 32-72%).15 A. B. C. D. Figure 1. A. Keloidal scar: dermis with increased cellularity, particularly fibrohistiocytic cells with intervening vessels and keloidal collagen. B. A 34 year old female patient with multiple keloidal scars on the scapulae. C. Hypertrophic scar: dermis containing intersecting bundles formed by proliferation of fibrohistiocytic cells with a small amount of collagen consistent with hypertrophic scar tissue. D. A 12 year old male patient with a hypertrophic scar on the medial side of the left helix. *All patients have provided written informed consent for publication of their photographs. The histological images (A, C) are provided by the pathology department of Erasmus MC without scale, and do not correspond to the clinical pictures (B, D). Patients with keloids: a heterogeneous population The clinical presentation of keloidal scars highly varies among patients. Patients can have one small keloid that causes minimal symptoms, but patients can also be severely affected with >70 keloids spread over different anatomic locations causing extreme 1 cm 4 cm

1. General introduction 12 physical symptoms, and is also associated with depression.16 For this reason, keloids are a heterogenous group of fibroproliferative scars. Several genetic factors play a role in the development of keloids. A Japanese genome-wide association study (GWAS) was the first to reveal four single-nucleotide polymorphisms (SNPs) in three chromosomal regions: 1q41, 3q22.3-23 and 15q21.3.17 Subsequently, another study found that these SNPs, specifically the rs8032158 SNP, may influence clinical keloid severity and may be a used as a biomarker for the prevention and treatment of keloidal scars.18 Recently, also other genes such as TFCP2L1 have been identified by machine learning and RNA-sequence that may serve as potential biomarker for keloid development.19 Other risk factors for keloid development include local factors (e.g. tension on scar and local infection), systemic factors (e.g. hypertension and rheumatism) and lifestyle factors (e.g. smoking).20-22 With regards to the phenotypes of keloids, a distinction has been made between ‘mild’ and ‘severe’ keloids.9 Severe keloids are defined as multiple or large (>10 cm2)) keloids.23 The heterogeneity among keloids is often not considered in clinical studies and may explain differences in results between studies. Besides genetic factors, keloid severity and treatment response may be influenced by the location-, thickness-, and treatment history of keloids.22,24 Therefore, the ‘standard keloid’ patient does not exist. However, especially in the severely affected patient group the burden of disease seems very high. Keloid treatment Several factors limit the risk of developing keloidal scars. Preventive measures include minimalizing tension on wounds (e.g. with z-plasty), infection prevention, hydration of wounds with silicon gels or - plasters, and of course minimalizing trauma to the skin as much as possible.12,22,25 However, up to date, clinicians face difficulties in the treatment of keloidal scars. Several anti-inflammatory treatments have been used to improve keloidal scars, including intralesional treatment with corticosteroids, or chemotherapeutics e.g. bleomycin and 5-fluorouracil.26 Yet, unfortunately no ‘holy grail’ to treat this heterogeneous group of fibroproliferative scars has been found. Especially severe keloids are challenging to treat in clinical practice, which can severely affect

1. General introduction 13 1. patients’ lives. Therefore, there is a high need for new efficacious, safe and minimal invasive treatment options for patients with severe and/or recalcitrant keloids. Conventional hypodermic needles Injection with conventional hypodermic needles is among the most commonly used procedures in clinical practice, but is hampered by high procedure-related pain and cannot be used in patients with (acquired) needle-phobia.27 Previous literature shows that approximately 2 in 3 children and 1 in 4 adult patients fear needles.28 The exact cause of needle-phobia is not fully understood, but traumatic experiences associated with needles during childhood (or during treatment) may set the stage for some patients.7 Other disadvantages of conventional needle injections include a lack of standardization: volume, pressure and the depth of the injection varies and is highly operator dependent. Furthermore, the procedure related pain can significantly hamper the effectiveness of the treatment and some patients prematurely decide to discontinue treatment. In addition, conventional needle injections can technically be difficult to administer in keloids, because of the tough fibroproliferative scar tissue.29 Alternative dermal drug delivery techniques Dermal drug delivery facilitates targeting of active substances to the appropriate skin layer(s) for the treatment of dermatological diseases.30 However, the stratum corneum, the most superficial layer of the skin, plays a crucial role as the physical barrier against pathogens but also to the penetration of compounds into skin.30 Drugs can only be delivered passively via the skin if it has an adequate lipophilicity and a low molecular mass preferably <500 Da.31 Due to the physical barrier of the skin, medication containing larger drug molecules may not be absorbed via the skin at all, and therefore have no efficacy when applied topically.32 To overcome the main obstacle, the stratum corneum, drugs can be injected intradermally or intralesionally using conventional hypodermic needles. However, due to

1. General introduction 14 the aforementioned limitations conventional needle injections are far from optimal. Therefore, several energy-based devices have been developed which can increase the skin bioavailability of the administered drug, including ablative (fractional) lasers, microneedles, microdermabrasion, iontophoresis, electroporation, sonophoresis and needlefree jet injectors.33,34 Needle-free jet injectors Needle-free jet injectors (NFI) are devices that are used for non-invasive drug delivery.35 These devices operate by a high-velocity jet (between 100-200m/s) which punctures the skin and thereby enables delivery of therapeutics in the epidermis, dermis, subcutis or muscle.36,37 A conventional NFI consists of 3 key components: (1) a nozzle, (2) an injection chamber which holds the drug, and (3) a pressure source that generates a high-velocity jet.38 The pressure required to generate these high-velocity jets can be generated by a compressed gas such as CO2 or N2, or Lorentz- or piezoelectric actuators.36 The drug absorption and the injection-related pain of jet-injections is greatly influenced by the distribution pattern and penetration depth.39 How a therapeutic is distributed is affected by several characters, including the physical drug properties (influenced by the density, viscosity and formulation of the fluid), jet velocity (influenced by pressure, filling volume, stand-off distance and nozzle diameter), and skin characteristics (influenced by elasticity, epidermis thickness, porosity, density and hardness).36 NFI can overcome several limitations that conventional hypodermic needles are faced with. They can minimize treatment-related pain, are free of risk for needle stick injuries and cross-contamination, and can be an alternative treatment option for patients experiencing needle phobia. Moreover, in a previous ex-vivo study with normal skin it was shown that NFI offers a more even distribution of fluids in the skin compared to hypodermic needles.29

1. General introduction 15 1. Developing new NFI technologies Up to date several types of jet injectors have become available and are increasingly used for intralesional treatment of various dermatological indications (Figure 2).40 The first concept of a NFI was already developed in the 1860s. However, it then took several decades before the first spring loaded jet-injectors (SLI) was introduced in clinical practice in 1956. Figure 2. Timeline from the first prototype of a needle-free jet injector developed in the 19th century to the clinical application of electronic pneumatic jet injectors to treat patients with keloids in the 21st century. Various types of jet-injectors were subsequently used for mass vaccinations worldwide, to immunize people against e.g. smallpox and typhus (Figure 3).41 Although the results were convincing as no smallpox epidemics were reported in 1990, in the same year a hepatitis B outbreak occurred which was related to contamination via the NFI.42 This contamination was caused by splash-back of aerosols carrying hepatitis B viral particles when the same nozzle was used in multiple patients.38

1. General introduction 16 Figure 3. Since the 1950’s, needle-free jet injectors have been employed globally to provide mass vaccination in both adults and children. Consequently, innovative technological improvements such as the usage of disposable nozzles and the implementation of adjustable settings in NFI were made, that improved the safety and broadened the scope of their application.36 Around 2015 electronically pneumatic jet injectors (EPIs), i.e. electronically controlled NFIs, were introduced (Figure 4).27

1. General introduction 17 1. A. B. C. Figure 4. Illustration of an electronically-controlled pneumatic jet injector-assisted treatment with bleomycin and lidocaine in a keloid scar. (A) Before administering treatment, the electronically-controlled pneumatic jet injector (EPI) hand piece with the injector tip is placed perpendicularly on the keloid scar. (B) A crosssectioned illustration of the injector tip and nozzle of an EPI-device. The liquid container within the EPI contains a solution with the combination of bleomycin and lidocaine (depicted in blue). (C) Illustration during injection. The EPI device generates a high-velocity jet stream that punctures the epidermis of the keloid, disperses the combination of bleomycin and lidocaine in the mid-deep dermis and creates visible skin papule or blanching. Recently, also the first needle-free jet-injector that relies on laser energy, the Mirajet, was commercialized.43 This EPI uses an Erbium YAG laser that generates vapor bubbles to generate pressure. These NFIs are yet being used to deliver small volumes in the superficial layers of the skin,27 and therefore they are probably more appropriate for soft tissue rather than treatment of keloids or other rigid skin disorders. Currently, a smaller and more affordable NFI prototype is being developed for medical applications by the group of Prof. Fernandez Rivas from the University of Twente. These NFIs operate using lasers with microfluidic components. Up to date this prototype has been investigated in skin models such as porcine skin. Potentially, in the future these NFIs may be used as portable device in a home setting by patients for indications such as acne scars and alopecia areata. The choice of jet injector may depend on the treatment indication, because the penetration depth and distribution patterns vary among jet injectors (Table 1). Other 0.5 mm

1. General introduction 18 factors that may play a role in the choice of NFI in clinical practice are costs and ease of use, for example SLIs are relatively easy to use, mobile and cheap. In the more innovative EPIs the volume and pressure can be adjusted, enabling more controlled and reliable drug delivery. The downside is that they are not portable, and relative expensive compared to the first generation SLIs. However, EPIs have shown to facilitate more standardized and consistent drug delivery compared to SLIs. Clinical endpoints In order for a treatment to be effective for keloids, it is important that the reticular dermis is reached.44 A previous ex vivo study by Bik et al. has shown that the formation of a papule is an immediate skin response that is directly visible after NFI-assisted injections, and indicates successful dermal drug delivery in normal skin.29 Therefore, it can be used as clinical endpoint when using NFI. However, the direct skin responses in keloids have not yet been studied, and may differ from normal skin because the fibrous tissue can be very thick and rigid. Intralesional corticosteroid treatment The most commonly used therapy for keloids is intralesional corticosteroid treatment using conventional needle injections, of which triamcinolone acetonide (10 to 40 mg per ml) is most frequently used.45 It is easy to perform, widely available and relatively cheap.46,47 Corticosteroids induce keloid regression through several mechanisms: suppression of inflammation by inhibition of cytokines, vasoconstriction which induces hypoxia, and inhibition of keratinocytes and fibroblasts proliferation due to its antimitotic effect.48,49 Overall, intralesional corticosteroids offer an effective treatment for keloids, and response rates vary from 50 to 100% after one year.49 However, recurrence rates appear to be high, and range from 33%–50% after five years.50 Moreover, intralesional corticosteroids appears to be less effective in severe keloids, and local adverse events such as fat atrophy,

1. General introduction 19 1. hypopigmentation, and telangiectasia are frequently observed after treatment.51 Although uncommon with intralesional corticiosteroid delivery, there is also a risk of systemic adverse events such as immunosuppression, adrenal insufficiency, and Cushing's syndrome, particularly with long-term use of high doses. Other intralesional treatment options are cryotherapy, and chemotherapeutics such as 5-Fluorouracil and bleomycin.52 A meta-analysis showed that bleomycin treatment was associated with a lower risk of keloid recurrence compared to 5-Fluorouracil monotherapy or combination therapy with 5-Fluorouracil and triamcinolone.53 Intralesional bleomycin treatment Bleomycin is an antibiotic with chemotherapeutic properties, derived from Streptomyces verticillus bacteria.54 This antineoplastic agent inhibits collagen synthesis via a decrease in TGF-β and causes DNA damage and cell apoptosis.55 Moreover, it inhibits endothelial cell migration and hereby also exhibits anti-angiogenic activities.56 Bleomycin has been used for intravenous treatment of head and neck tumors, and offlabel as intradermal treatment for several dermatological indications, including recalcitrant warts, hemangioma and non-melanoma skin cancer.57 Its topical use is limited due to the large molecular mass (1415 Da) and high hydrophilicity.58 However, it has been used successfully as intradermal treatment for keloids using conventional needles. Treatment with intralesional bleomycin potentially may lead to low recurrence rates in keloids due to its antimitotic and anti-angiogenic effects.59 Clinical application of NFI-assisted treatment with bleomycin in keloids Bleomycin can be degraded by the enzyme bleomycin hydrolase. Since there is a low expression of this enzyme in the lungs, high dosages of bleomycin can result in lung fibrosis.60,61 In patients treated for dermatological indications, lung toxicity has not been reported, probably because of the use of low dosages (usually 2–4 U). However, potentially harmful aerosols can form when bleomycin is administered with NFIs.62

1. General introduction 20 Therefore, it is important to use adequate safety measures such as protective gloves, goggles, and a smoke evacuator or a FFP-2 mask to effectively capture these aerosols.62 Moreover, intralesional bleomycin administration harbors the risk of mild to moderate adverse effects, including necrosis and ulceration. To minimize the risk of superficial necrosis, it is important to avoid too superficial drug delivery, and target the reticular dermis using a standardized and consistent drug delivery technique. EPI could potentially facilitate a standardized, effective and safe drug delivery of bleomycin in keloidal scars.

1. General introduction 21 1. PRECLINICAL AND CLINICAL METHODOLOGY In this thesis the clinical applicability of EPI-assisted jet injections in severe and recalcitrant keloids was studied using a variety of research techniques and designs. Prior to starting clinical research in patients with keloids, preclinical studies were performed using cutting-edge imaging techniques to provide a better understanding of the deposition and distribution of drugs in severe keloids after EPI-assisted drug delivery. Previous work of Bik et al. has provided comprehensive information of how drugs are distributed in skin after EPI-assisted injection.29,63 However, these experiments were limited to in vivo animal- and ex vivo human studies with normal skin. To gain more insights in the distribution of EPI-assisted jet injections in diseased tissue such as keloids, an ex vivo study that visualized distribution of EPI-assisted jet injections using the 3DFluorescent Imaging Cryomicrotome System (3D-FICS) in severe keloids was performed. The 3D-FICS (Figure 5) is an innovative 3D imaging technique that enables visualization of fluorescent markers representing the biodistribution of fluorescent labeled-fluids. Various imaging techniques can be used to visualize the layers of the skin, including high frequency ultrasound, optical coherence tomography, and confocal microscopy. A. B. Figure 5. A. The 3D-Fluorescent Imaging Cryomicrotome System (3D-FICS), a custom-built innovative 3D imaging technique that enables visualization of fluorescent markers representing the biodistribution of fluorescent labeled-fluids. B. A 3D image representing the biodistribution of fluorescent labeled triamcinolone acetonide administered in a keloid sample. 0.5 cm

1. General introduction 22 In contrast to older imaging techniques, our custom-built 3D-FICS can be used for high resolution segmentation of larger volumes, for example to visualize the biodistribution of fluorescence-labeled drugs in frozen tissue in 3D images. The 3D-FICS allows examination of deeper tissue layers, and can reconstruct volumes in organs with high spatial resolution (8-32 micrometer).64 This novel 3D imaging technique has been used for the first time in cardiology to visualize monocytes in the coronary arteries.65 In this thesis we describe the first time utilization of the 3D-FICS to explore the distribution of EPI- and needle assisted injections in ex vivo keloid- and normal skin samples. In addition, a powerful 3D-camera to capture high resolution images was used in two studies in this thesis (Figure 6). Firstly, this technique was used to measure papule dimensions, directly after EPI-assisted jet injections in ex-vivo keloids. Secondly, a 3Dcamera was used in the BLEOJET study, a randomized controlled split lesion clinical trial comparing the effects of bleomycin with normal saline. The 3D-camera facilitates the measurement of objective changes including volume, height, and roughness, before and after treatment of keloids. A. B. Figure 6. A. A stereophotogrammetric three-dimensional camera (LifeViz® Micro, Quantificare, Sophia Antipolis, France). B. A three-dimensional reconstruction of a keloid lesion before EPI treatment, after image reconstruction with a heat map showing the height of the object which is used for the 3D analysis. 1 cm

1. General introduction 23 1. Furthermore, in the BLEOJET study, the Laser Speckle Contrast Imaging (LSCI) technique was used (Figure 7). This imaging technique is a highly sensitive technique to visualize the blood flow of the skin.66 Erythrocytes act as a virtual contrast agent, outlining blood vessels and hereby enable the highly sensitive measurement of blood flow in the skin, which may correlate with the extent of dermal inflammation in keloids. In skin, erythrocytes are the main source of moving scatters. When coherent laser light interacts with skin tissue containing moving erythrocytes, it produces a speckle pattern because of the interference of scattered light waves. This pattern changes over time as erythrocytes move, with regions of slower flow exhibiting less variability in intensity compared to faster flowing areas. A. B. Figure 7. A. The Laser Speckle Contrast Imaging (LSCI), a highly sensitive technique to visualize the blood flow of the skin. B. Visualization of the blood flow of a keloid lesion before EPI treatment using the LSCI, with a heat map showing the intensity of the blood flow. The final step in evaluating the clinical applicability of EPI-assisted jet injections in recalcitrant keloids was to examine the effectiveness, tolerability and patient satisfaction in a real-world setting. Tolerability, safety and patient satisfaction were assessed by daily photographs made by the patient using an innovative custom-made e-diary app and 1 cm

1. General introduction 24 routinely asking about injection-related pain and side effects. Two retrospective cohort studies that evaluated the effectiveness, tolerability and patient satisfaction in patients in a real-world setting were performed. Since patients with recalcitrant keloids have been treated successfully and safe according to the BLEOJET study, hereafter adult patients from the outpatient clinic of Erasmus MC with recalcitrant keloids were offered treatment with a combination of bleomycin and lidocaine according to regular care. Lidocaine was added to the bleomycin in a real-world setting, because according to the data from the BLEOJET study, EPI-assisted injections with bleomycin monotherapy still resulted in relatively high injection-related pain, although assessed as less painful than needle-assisted injections. Patient reported outcome measures including the Patient and Observer Assessment Scale (POSAS) were used to assess effectiveness.

1. General introduction 25 1. OBJECTIVES AND OUTLINE OF THIS THESIS The aim of this thesis was to investigate the efficacy, safety and patient satisfaction of an innovative needle-free drug delivery device (Enerjet) to treat patients with keloidal scars. In order to study this, various research techniques and designs were used. First, the available evidence for needle-free jet injectors in dermatology was studied according to a systematic review approach. Hereafter, an ex vivo study was performed to visualize the distribution patterns of EPI-assisted jet injections in keloids. A randomized controlled trial was performed to study the efficacy and safety of EPI-assisted bleomycin in patient with recalcitrant keloids. Subsequently, two retrospective real-world studies using EPI-assisted drug delivery were performed. The first study aimed to explore the effectiveness, tolerability and patient satisfaction of EPI-assisted bleomycin and lidocaine in adult patients with recalcitrant keloids. The second study was performed to explore the effectiveness, tolerability and patient satisfaction of EPI-assisted triamcinolone in children with keloids or hypertrophic scars. Chapter 2 The second chapter of this thesis describes the current evidence regarding the efficacy and safety of needle-free jet injectors to treat various dermatological indications. The available evidence for needle-free jet injectors in dermatology is summarized and critically appraised in a systematic review with risk of bias assessment. Chapter 3 The third chapter describes the distribution of EPI-assisted jet injections in severe keloids by performing pre-clinical experiments. In this chapter we performed an ex-vivo study, in which EPI-assisted jet injections and conventional needle injections in normal skin samples and recalcitrant keloid samples were visualized using the 3D-FICS, an innovative 3D-imaging technique. Chapter 4 The fourth chapter describes the clinical factors that may negatively influence the treatment response in keloids according to previous literature. In this chapter we

1. General introduction 26 performed a systematic review, in which we describe the available evidence for clinically relevant keloid properties. Chapter 5 The fifth chapter describes the clinical application of EPI-assisted bleomycin in in vivo severe and recalcitrant keloids. In this chapter we performed a double-blinded, randomized, placebo-controlled trial with split-lesion design, to investigate the efficacy and safety of EPI-assisted bleomycin compared to placebo in patients with severe keloids. Chapter 6 The sixth chapter describes the findings of a retrospective cohort study that investigated the effectiveness, tolerability and patient satisfaction of EPI-assisted bleomycin combined with lidocaine in patients with recalcitrant keloids in a real-world setting. Chapter 7 The seventh chapter describes treatment with the EPI in children, to explore a traumafree, minimally invasive treatment option in children with keloids and hypertrophic scars. In this chapter we performed a retrospective cohort study in children, to study the effectiveness, tolerability and patient satisfaction of EPI-assisted triamcinolone in children with keloids or hypertrophic scars.

1. General introduction 27 1. REFERENCES 1. Rockwell WB, Cohen IK, Ehrlich HP. Keloids and hypertrophic scars: a comprehensive review. Plast Reconstr Surg. Nov 1989;84(5):827-37. doi:10.1097/00006534198911000-00021 2. Sitaniya S, Subramani D, Jadhav A, Sharma YK, Deora MS, Gupta A. Quality-of-life of people with keloids and its correlation with clinical severity and demographic profiles. Wound Repair Regen. May 2022;30(3):409-416. doi:10.1111/wrr.13015 3. Balci DD, Inandi T, Dogramaci CA, Celik E. DLQI scores in patients with keloids and hypertrophic scars: a prospective case control study. J Dtsch Dermatol Ges. Aug 2009;7(8):688-92. doi:DDG07034 [pii] 10.1111/j.1610-0387.2009.07034.x 4. McGinty S, Siddiqui WJ. Keloid. Jan 2023;doi:NBK507899 [bookaccession] 5. Wallace HA, Basehore BM, Zito PM. Wound Healing Phases. Jan 2023;doi:NBK470443 [bookaccession] 6. Berman B, Maderal A, Raphael B. Keloids and Hypertrophic Scars: Pathophysiology, Classification, and Treatment. Dermatologic Surgery. 2017;43:S3-S18. doi:10.1097/dss.0000000000000819 7. Orenius T, LicPsych, Saila H, Mikola K, Ristolainen L. Fear of Injections and Needle Phobia Among Children and Adolescents: An Overview of Psychological, Behavioral, and Contextual Factors. SAGE Open Nurs. Jan-Dec 2018;4:2377960818759442. doi:10.1177_2377960818759442 [pii] 10.1177/2377960818759442 8. Jiao H, Fan J, Cai J, et al. Analysis of Characteristics Similar to Autoimmune Disease in Keloid Patients. Aesthetic Plast Surg. Oct 2015;39(5):818-25. doi:10.1007/s00266-0150542-4 9. Liu R, Xiao H, Wang R, et al. Risk factors associated with the progression from keloids to severe keloids. Chin Med J (Engl). Apr 5 2022;135(7):828-836. doi:00029330202204050-00012 [pii] CMJ-2021-2354 [pii] 1097/CM9.0000000000002093 10. Alster TS, Tanzi EL. Hypertrophic scars and keloids: etiology and management. Am J Clin Dermatol. 2003;4(4):235-43. doi:443 [pii] 10.2165/00128071-200304040-00003

1. General introduction 28 11. Arno AI, Gauglitz GG, Barret JP, Jeschke MG. Up-to-date approach to manage keloids and hypertrophic scars: a useful guide. Burns. Nov 2014;40(7):1255-66. doi:S03054179(14)00071-0 [pii] 10.1016/j.burns.2014.02.011 12. Betarbet U, Blalock TW. Keloids: A Review of Etiology, Prevention, and Treatment. J Clin Aesthet Dermatol. Feb 2020;13(2):33-43. 13. Carswell L, Borger J. Hypertrophic Scarring Keloids. Jan 2023;doi:NBK537058 [bookaccession] 14. Chike-Obi CJ, Cole PD, Brissett AE. Keloids: pathogenesis, clinical features, and management. Semin Plast Surg. Aug 2009;23(3):178-84. doi:10.1055/s-0029-1224797 15. Lawrence JW, Mason ST, Schomer K, Klein MB. Epidemiology and impact of scarring after burn injury: a systematic review of the literature. J Burn Care Res. Jan-Feb 2012;33(1):136-46. doi:10.1097/BCR.0b013e3182374452 16. Lu W, Chu H, Zheng X. Effects on quality of life and psychosocial wellbeing in Chinese patients with keloids. Am J Transl Res. 2021;13(3):1636-1642. 17. Nakashima M, Chung S, Takahashi A, et al. A genome-wide association study identifies four susceptibility loci for keloid in the Japanese population. Nat Genet. Sep 2010;42(9):768-71. doi:ng.645 [pii] 10.1038/ng.645 18. Ogawa R, Watanabe A, Than Naing B, et al. Associations between Keloid Severity and Single-Nucleotide Polymorphisms: Importance of rs8032158 as a Biomarker of Keloid Severity. Journal of Investigative Dermatology. 2014/07/01/ 2014;134(7):2041-2043. doi:https://doi.org/10.1038/jid.2014.71 19. Huang J, Gong Y, Lin J-M, et al. TFCP2L1 as a potential diagnostic gene biomarker of Keloid given its association with immune cells-a study based on machine learning and RNA sequence. Alexandria Engineering Journal. 2024/04/01/ 2024;93:360-370. doi:https://doi.org/10.1016/j.aej.2024.02.043 20. Dong X, Mao S, Wen H. Upregulation of proinflammatory genes in skin lesions may be the cause of keloid formation (Review). Biomed Rep. Nov 2013;1(6):833-836. doi:br01-06-0833 [pii] 10.3892/br.2013.169

1. General introduction 29 1. 21. Huang C, Ogawa R. Systemic factors that shape cutaneous pathological scarring. FASEB J. Oct 2020;34(10):13171-13184. doi:10.1096/fj.202001157R 22. Ogawa R. The Most Current Algorithms for the Treatment and Prevention of Hypertrophic Scars and Keloids: A 2020 Update of the Algorithms Published 10 Years Ago. Plast Reconstr Surg. Jan 1 2022;149(1):79e-94e. doi:00006534-990000000-00503 [pii] 10.1097/PRS.0000000000008667 23. Limandjaja GC, Niessen FB, Scheper RJ, Gibbs S. The Keloid Disorder: Heterogeneity, Histopathology, Mechanisms and Models. Front Cell Dev Biol. 2020;8:360. doi:10.3389/fcell.2020.00360 24. Long X, Zhang M, Wang Y, Zhao R, Wang Y, Wang X. Algorithm of chest wall keloid treatment. Medicine (Baltimore). Aug 2016;95(35):e4684. doi:00005792-20160830000056 [pii] 10.1097/MD.0000000000004684 25. Sidle DM, Kim H. Keloids: prevention and management. Facial Plast Surg Clin North Am. Aug 2011;19(3):505-15. doi:S1064-7406(11)00025-3 [pii] 10.1016/j.fsc.2011.06.005 26. Walsh LA, Wu E, Pontes D, et al. Keloid treatments: an evidence-based systematic review of recent advances. Syst Rev. Mar 14 2023;12(1):42. 27. Schoppink J, Fernandez Rivas D. Jet injectors: Perspectives for small volume delivery with lasers. Adv Drug Deliv Rev. Mar 2022;182:114109. doi:S0169-409X(21)00502-0 [pii] 10.1016/j.addr.2021.114109 28. Taddio A, Ipp M, Thivakaran S, et al. Survey of the prevalence of immunization noncompliance due to needle fears in children and adults. Vaccine. Jul 6 2012;30(32):4807-12. doi:S0264-410X(12)00686-X [pii] 10.1016/j.vaccine.2012.05.011 29. Bik L, van Doorn MBA, Boeijink N, et al. Clinical endpoints of needle-free jet injector treatment: An in depth understanding of immediate skin responses. Lasers Surg Med. Jul 2022;54(5):693-701. doi:LSM23521 [pii] 10.1002/lsm.23521

1. General introduction 30 30. Badilli U, Gumustas M, Uslu B, Ozkan SA. Chapter 9 - Lipid-based nanoparticles for dermal drug delivery. In: Grumezescu AM, ed. Organic Materials as Smart Nanocarriers for Drug Delivery. William Andrew Publishing; 2018:369-413. 31. Brown MB, Martin GP, Jones SA, Akomeah FK. Dermal and transdermal drug delivery systems: current and future prospects. Drug Deliv. May-Jun 2006;13(3):175-87. doi:N45362P167475H46 [pii] 10.1080/10717540500455975 32. Bos JD, Meinardi MM. The 500 Dalton rule for the skin penetration of chemical compounds and drugs. Exp Dermatol. Jun 2000;9(3):165-9. doi:10.1034/j.16000625.2000.009003165.x 33. Prausnitz MR, Langer R. Transdermal drug delivery. Nat Biotechnol. Nov 2008;26(11):1261-8. doi:nbt.1504 [pii] 10.1038/nbt.1504 34. Krizek J, De Goumoens F, Delrot P, Moser C. Needle-free delivery of fluids from compact laser-based jet injector. Lab Chip. Oct 21 2020;20(20):3784-3791. doi:10.1039/d0lc00646g 35. Mitragotri S. Current status and future prospects of needle-free liquid jet injectors. Nat Rev Drug Discov. Jul 2006;5(7):543-8. doi:nrd2076 [pii] 10.1038/nrd2076 36. Mohizin A, Kim JK. Current engineering and clinical aspects of needle-free injectors: A review. JOURNAL OF MECHANICAL SCIENCE AND TECHNOLOGY. DEC 2018;32(12):5737-5747. doi:10.1007/s12206-018-1121-9 37. Mohizin A, Imran JH, Lee KS, Kim JK. Dynamic interaction of injected liquid jet with skin layer interfaces revealed by microsecond imaging of optically cleared ex vivo skin tissue model. J Biol Eng. Feb 27 2023;17(1):15. doi:10.1186/s13036-023-00335-x [pii] 335 [pii] 10.1186/s13036-023-00335-x 38. Han HS, Hong JY, Kwon TR, et al. Mechanism and clinical applications of needle-free injectors in dermatology: Literature review. J Cosmet Dermatol. Dec 2021;20(12):3793-3801. doi:10.1111/jocd.14047 39. Schramm-Baxter J, Mitragotri S. Needle-free jet injections: dependence of jet penetration and dispersion in the skin on jet power. J Control Release. Jul 7 2004;97(3):527-35. doi:S0168365904001853 [pii]

1. General introduction 31 1. 10.1016/j.jconrel.2004.04.006 40. Bekkers VZ, Bik L, van Huijstee JC, Wolkerstorfer A, Prens EP, van Doorn MBA. Efficacy and safety of needle-free jet injector-assisted intralesional treatments in dermatology-a systematic review. Drug Deliv Transl Res. Jun 2023;13(6):1584-1599. doi:10.1007/s13346-023-01295-x [pii] 1295 [pii] 10.1007/s13346-023-01295-x 41. Barolet D, Benohanian A. Current trends in needle-free jet injection: an update. Clin Cosmet Investig Dermatol. 2018;11:231-238. doi:ccid-11-231 [pii] 10.2147/CCID.S162724 42. Canter J, Mackey K, Good LS, et al. An outbreak of hepatitis B associated with jet injections in a weight reduction clinic. Arch Intern Med. Sep 1990;150(9):1923-7. 43. JSK Biomed, the World’s First to Earn CE-MDD Mark for “Needle-Free Injector”. Accessed 19-09-2023, 2023. http://www.khealth.com/news/articleView.html?idxno=49849 44. Ogawa R. Keloid and Hypertrophic Scars Are the Result of Chronic Inflammation in the Reticular Dermis. Int J Mol Sci. Mar 10 2017;18(3)doi:ijms18030606 [pii] ijms-1800606 [pii] 10.3390/ijms18030606 45. Ojeh N, Bharatha A, Gaur U, Forde AL. Keloids: Current and emerging therapies. Scars Burn Heal. Jan-Dec 2020;6:2059513120940499. doi:10.1177_2059513120940499 [pii] 10.1177/2059513120940499 46. Triamcinolone Prices, Coupons and Patient Assistance Programs. Accessed 20-092023, 2023. https://www.drugs.com/price-guide/triamcinolone 47. Wong TS, Li JZ, Chen S, Chan JY, Gao W. The Efficacy of Triamcinolone Acetonide in Keloid Treatment: A Systematic Review and Meta-analysis. Front Med (Lausanne). 2016;3:71. doi:10.3389/fmed.2016.00071 48. Klomparens K, Simman R. Treatment of Keloids: A Meta-analysis of Intralesional Triamcinolone, Verapamil, and Their Combination. Plast Reconstr Surg Glob Open. Jan 2022;10(1):e4075. doi:10.1097/GOX.0000000000004075 49. Morelli Coppola M, Salzillo R, Segreto F, Persichetti P. Triamcinolone acetonide intralesional injection for the treatment of keloid scars: patient selection and

1. General introduction 32 perspectives. Clin Cosmet Investig Dermatol. 2018;11:387-396. doi:ccid-11-387 [pii]10.2147/CCID.S133672 50. Lemperle G, Schierle J, Kitoga KE, Kassem-Trautmann K, Sachs C, Dimmler A. Keloids: Which Types Can Be Excised without Risk of Recurrence? A New Clinical Classification. Plast Reconstr Surg Glob Open. Mar 2020;8(3):e2582. doi:10.1097/GOX.0000000000002582 51. Robles DT, Berg D. Abnormal wound healing: keloids. Clinics in Dermatology. 2007/01/01/ 2007;25(1):26-32. doi:https://doi.org/10.1016/j.clindermatol.2006.09.009 52. Trisliana Perdanasari A, Torresetti M, Grassetti L, et al. Intralesional injection treatment of hypertrophic scars and keloids: a systematic review regarding outcomes. Burns & Trauma. 2015/08/26 2015;3(1):14. doi:10.1186/s41038-015-0015-7 53. Kim WI, Kim S, Cho SW, Cho MK. The efficacy of bleomycin for treating keloid and hypertrophic scar: A systematic review and meta-analysis. J Cosmet Dermatol. Dec 2020;19(12):3357-3366. doi:10.1111/jocd.13390 54. Umezawa H, Maeda K, Takeuchi T, Okami Y. New antibiotics, bleomycin A and B. J Antibiot (Tokyo). Sep 1966;19(5):200-9. 55. Viera MH, Caperton CV, Berman B. Advances in the treatment of keloids. J Drugs Dermatol. May 2011;10(5):468-80. 56. Mabeta P, Pepper MS. A comparative study on the anti-angiogenic effects of DNAdamaging and cytoskeletal-disrupting agents. Angiogenesis. 2009;12(1):81-90. doi:10.1007/s10456-009-9134-8 57. Bik L, Sangers T, Greveling K, Prens E, Haedersdal M, van Doorn M. Efficacy and tolerability of intralesional bleomycin in dermatology: A systematic review. J Am Acad Dermatol. Sep 2020;83(3):888-903. doi:S0190-9622(20)30226-7 [pii] 10.1016/j.jaad.2020.02.018 58. Hendel K, Hansen ACN, Bik L, et al. Bleomycin administered by laser-assisted drug delivery or intradermal needle-injection results in distinct biodistribution patterns in skin: in vivo investigations with mass spectrometry imaging. Drug Deliv. Dec 2021;28(1):1141-1149. doi:1933649 [pii] 10.1080/10717544.2021.1933649

1. General introduction 33 1. 59. Wang XQ, Liu YK, Qing C, Lu SL. A review of the effectiveness of antimitotic drug injections for hypertrophic scars and keloids. Ann Plast Surg. Dec 2009;63(6):688-92. doi:10.1097/SAP.0b013e3181978753 60. Yamamoto T. Bleomycin and the skin. Br J Dermatol. Nov 2006;155(5):869-75. doi:BJD7474 [pii] 10.1111/j.1365-2133.2006.07474.x 61. Kawai K, Akaza H. Bleomycin-induced pulmonary toxicity in chemotherapy for testicular cancer. Expert Opin Drug Saf. Nov 2003;2(6):587-96. doi:10.1517/14740338.2.6.587 62. Bik L, Wolkerstorfer A, Bekkers V, et al. Needle-free jet injection-induced small-droplet aerosol formation during intralesional bleomycin therapy. Lasers Surg Med. Apr 2022;54(4):572-579. doi:LSM23512 [pii] 10.1002/lsm.23512 63. Bik L, van Doorn M, Hansen ACN, et al. In vivo dermal delivery of bleomycin with electronic pneumatic injection: drug visualization and quantification with mass spectrometry. doi: 10.1080/17425247.2022.2035719. Expert Opinion on Drug Delivery. 2022/02/01 2022;19(2):213-219. doi:10.1080/17425247.2022.2035719 64. Bloemen P, van Leeuwen T, Siebes M, et al. 3D Fluorescence Imaging cryomicrotome system for multispectral structural, functional and molecular imaging of whole organs (Conference Presentation). 2018:10. 65. Hakimzadeh N, van Horssen P, van Lier MG, et al. Detection and quantification methods of monocyte homing in coronary vasculature with an imaging cryomicrotome. J Mol Cell Cardiol. Nov 2014;76:196-204. doi:S0022-2828(14)00266-1 [pii] 10.1016/j.yjmcc.2014.08.019 66. Senarathna J, Rege A, Li N, Thakor NV. Laser Speckle Contrast Imaging: theory, instrumentation and applications. IEEE Rev Biomed Eng. 2013;6:99-110. doi:10.1109/RBME.2013.2243140

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35 Section I Needle-free jet injectors in dermatology

37 Chapter 2 Efficacy and safety of needle-free jet injector-assisted intralesional treatments in dermatology—a systematic review V.Z. Bekkers L. Bik J.C. van Huijstee A. Wolkerstorfer E.P. Prens M.B.A. van Doorn Drug Deliv Transl Res. 2023 Jun;13(6):1584-159

2. Efficacy and safety of needle free jet injector assisted intralesional treatments in dermatology—a systematic review. 38 ABSTRACT Needle-free jet injectors are used for the intralesional treatment of various dermatological indications. However, a systematic review that evaluates the efficacy and safety of these treatments has not been published. The objectives of this study are to evaluate the efficacy and safety of needle-free jet injections for dermatological indications and to provide evidence-based treatment recommendations. An electronic literature search was conducted in April 2022. Two reviewers independently selected studies based on predefined criteria and performed a methodological quality assessment using the Cochrane Collaborations risk-of-bias 2.0 assessment tool and Newcastle-Ottawa Scale. Thirty-seven articles were included, involving 1911 participants. Dermatological indications included scars, alopecia areata, hyperhidrosis, nail diseases, non-melanoma skin cancer, common warts, local anesthesia, and aesthetic indications. Keloids and other types of scars (hypertrophic, atrophic, and burn scars) were investigated most frequently (n = 7). The included studies reported favorable efficacy and safety outcomes for intralesional jet injector-assisted treatment with triamcinolone acetonide/hexacetonide, 5-fluorouracil, bleomycin, or hyaluronic acid. Two high-quality studies showed good efficacy and tolerability of intralesional jet injections with a combination of 5-fluorouracil and triamcinolone acetonide in hypertrophic scars and with saline in boxcar and rolling acne scars. No serious adverse reactions and good tolerability were reported in the included studies. Overall, the methodological quality of the included studies was low. Limited evidence suggests that needle-free jet injector-assisted intralesional treatment is efficacious and safe for hypertrophic and atrophic acne scars. More well-powered RCTs investigating the efficacy and safety of jet injector treatment in dermatology are warranted to make further evidence-based recommendations.

2. Efficacy and safety of needle free jet injector assisted intralesional treatments in dermatology—a systematic review. 39 2. 2. INTRODUCTION Intradermal drug delivery has many advantages over other routes of administration, especially high bioavailability in the skin.1,2 Over the past decades, a variety of needlefree devices that enable intradermal drug delivery has been developed, including fractional ablative lasers, iontophoresis, sonophoresis, and various types of mechanical and energy-based jet injectors.3-5 Jet injectors are commonly used for the intralesional treatment of several dermatological conditions such as keloids, hypertrophic scars, and recalcitrant viral warts.6,7 Traditional mechanical jet injectors act with a fixed pressure predetermined by spring size.8 Innovative electronically controlled pneumatic jet injectors are devices in which volume and pressure can be controlled by accelerated and compressed gas as pressure source, which dispense fluids into the skin.7,9 Other types of jet injectors are controlled by Lorentz or piezoelectric actuators, lasers, and shockwaves to pressurize the injected drug.10 In contemporary healthcare, we are moving towards more patient-centered care. It is important to improve patient comfort and avoid physical or psychological harm as much as possible. According to a previous study, 63% of children and 24% of the adult population in the USA fear needles.11 This is one of the reasons why jet injectors can be a viable alternative for conventional needles.Needle-free jet injectors can be an attractive alternative for hypodermic needles for patients experiencing needle phobia, minimize treatment-related pain, and are free of risk for needlestick injuries and crosscontamination. Additionally, jet injectors enable accurate and reproducible dermal delivery of liquid drugs and disperse the drug more evenly in the skin than conventional needle injections.7, 9, 12, 13 At present, there are a few overviews and narrative reviews describing the use of jet injector-assisted intralesional treatment for different dermatological indications.7, 10, 12, 14 However, a systematic and critical review that evaluates the efficacy and safety of jet injector-assisted intralesional treatment in dermatology is lacking. In this review, we aimed to systematically review and evaluate the quality of clinical evidence for intralesional treatment of dermatological indications using needle-free jet injector systems and provide evidence-based recommendations for clinical practice.

2. Efficacy and safety of needle free jet injector assisted intralesional treatments in dermatology—a systematic review. 40 MATERIALS AND METHODS A literature search was conducted in April 2022 using Embase, MEDLINE ALL Ovid, Web of Science, and Cochrane Central Register of Controlled Trials databases, to identify relevant publications. This systematic review was registered in the PROSPERO (CRD42021258278) and followed the Preferred Reporting Items for the PRISMA 2020 checklist.15 Studies were included if they were human studies, written in English, published from inception to April 2022, randomized controlled trials (RCTs), controlled clinical trials (CCTs), prospective or retrospective cohort studies, and case series and included patients of all ages with dermatological indications eligible for intralesional treatment using needle-free jet injectors. Exclusion criteria included studies with fewer than 10 patients and intramuscular or subcutaneous drug delivery. Selection of the articles, standardized data extraction, and methodological quality assessment of the included studies were performed independently by two authors (V.B. and J.V.H.). Articles were screened based on title and abstract. The primary outcome measure was efficacy, and the secondary outcome measure was safety. For data extraction, we converted pressure settings, total injection volume, and drug concentration to psi, ml, and mg/ml, respectively. If possible, efficacy measures were simplified to percentages in terms of clinical response compared to baseline. Methodological quality was assessed using the Cochrane Collaborations risk-of-bias 2.0 tool (ROB 2.0) for RCTs and CCTs, and the Newcastle–Ottawa Scale (NOS) for cohort studies and case series.16–19 Final selection of the articles was based on screening of full texts. Discrepancies between reviewers were discussed and resolved by consensus and involved a third author (L.B.) if necessary. Illustrations of the methodological quality assessments were created using Robvis.17

2. Efficacy and safety of needle free jet injector assisted intralesional treatments in dermatology—a systematic review. 41 2. 2. RESULTS Our literature search identified 1326 records. Duplicates were removed. Based on title and abstract, 985 articles were screened. Full texts of 71 articles were assessed for eligibility of which 37 studies were selected with a total of 1911 participants (Fig. 1). The included studies comprised 6 RCTs, 6 CCTs, 16 prospective cohorts, 5 retrospective cohorts, and 4 case series. The studies investigated needle-free jet injector-assisted intralesional treatments for atrophic and hypertrophic scars, keloids, alopecia areata, hyperhidrosis, nail diseases (psoriasis, lichen planus, and idiopathic onycholysis), nonmelanoma skin cancer (basal cell carcinoma (BCC), squamous cell carcinoma (SCC), Bowen’s disease, and Paget’s disease), common warts, granuloma annulare, lichen simplex chronicus, psoriasis, seborrheic dermatitis, aesthetic indications (wrinkles, rejuvenation, rhytides, facelift), and local anesthesia. Figure 1. Study flow diagram of exclusion process resulting in 37 included studies

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