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In vitro and in vivo characterization of pharmaceutical topical nanocarriers containing anticancer drugs for skin cancer treatment

Vandana Gupta, Ex-Women Scientist (WOS-A), Department of Science and Technology, New Delhi, India, Piyush Trivedi, Former Vice Chancellor, Rajiv Gandhi Proudyogiki Vishwavidyalaya (A State Technological University of Madhya Pradesh), Bhopal, India

15.1 INTRODUCTION

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Cancer is a disease with a multifactorial etiology, resulting mainly from genetic alterations, environmental factors, and lifestyle (Ferreira et al., 2011; Taveira and Lopez, 2011). Skin cancers are the most common human cancers. Annually, more than 400,000 people find out they have skin cancer. Its etiology is related to various predisposing factors, such as skin type, age, sun exposure, poor tanning capability, inherited disorders (e.g., xeroderma pigmentosa, albinism, etc.), and immunocompromise, etc. In addition, skin cancers are the most frequent cancers in organ transplant recipients (Leiter and Garbe, 2008; Norval et al., 2011; Nikolaou and Stratigos, 2014; Gordon, 2013). Furthermore, despite growing public awareness of the harmful effects of sun exposure, incidence and morbidity continue to rise, which has generated great interest in unraveling of its etiology and in the search for new noninvasive treatment strategies. In general, the skin is composed of two layers: the epidermis and the dermis, which are separated by an irregular border (D’Orazio et al., 2013). Skin cancers (skin neoplasms) are named after the type of skin cell from which they arise (Diepgen and Mahler, 2002). Most skin cancers develop in the topmost layer of the skin (the epidermis). Nonmelanoma skin cancers are the most common human cancers and, despite growing public awareness of the harmful effects of sun exposure,

Lipid Nanocarriers for Drug Targeting. https://doi.org/10.1016/B978-0-12-813687-4.00015-3 Copyright © 2017.

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incidence continues to rise (Madan et al., 2010). A 3%–8% yearly increase in the incidence of nonmelanoma skin cancer has been reported since 1960, worldwide. Squamous-cell carcinoma (SCC) and basal-cell carcinoma (BCC) are the terms used under common umbrella (Lomas et al., 2012). Melanoma, arisen from the invasive transformation of melanocytes, is one of the most vigorous cutaneous malignancies, notorious for its high multidrug resistance, easy recurrence, and low vitality rate. Approximately 76,100 newly diagnosed cases of melanoma were observed in the United States in 2014, with 9710 subsequent deaths measured (Nikolaou et al., 2012; Jun et al., 2015). There are some well-established treatments for nonmelanoma skin cancer, such as curettage, surgery, cryotherapy, and chemotherapy. However, these conventional treatments are invasive and lead to severe inflammation, pain, and unappealing scars (Lopez et al., 2004). Treatments for melanoma, in turn, are primarily surgical because these tumors can be resistant to conventional chemo- and radiotherapies (Davids and Kleemann, 2010). Nonsurgical treatments for melanomas are limited to adjuvant therapies, such as immunotherapy, bio-chemotherapy, gene therapy, and photodynamic therapy (Martinez and Otley, 2001). To render the patient compliant and to reduce surgical costs and undesirable scars, particularly in cases where the cancer has spread over large areas of the body, the topical administration of anticancer drugs has been recommended. The topical administration of anticancer drugs is an interesting option for reducing side effects and for increasing drug targeting and therapeutic benefits. The major challenge of this kind of treatment is to increase penetration of the anticancer drug in sufficient levels to kill tumor cells. Several techniques and drug delivery systems have therefore been developed to successfully overcome skin barriers and to reach skin malignancies by favoring drug penetration into the deep layers of the epidermis. The use of chemical penetration enhancers is the simplest strategy, causing temporary and reversible disruption of the stratum corneum (SC) bilayers and leading to increased anticancer drug penetration into the tumor. This chapter will briefly discuss skin anatomy, the skin cancer, the most studied topical nanocarriers and their characterization for topical skin cancer treatment. The aim of this chapter is to provide a basic understanding and description of the strategies that can be used to overcome the skin cancer. These modalities will be discussed in the context for their characterization and application for promoting and targeting the delivery of skin tumor drugs following topical, noninvasive administration.

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15.2 SKIN The skin or the integument is the external organ that protects against mechanical trauma, UV light, and infection. In addition, the skin is concerned with thermoregulation, conservation and excretion of fluid, sensory perception, and has esthetic role (Harsh, 1998; Ferreira et al., 2011). In general, skin is composed of two layers, the

15.3 Skin Cancer

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15.3 SKIN CANCER

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epidermis and the dermis (Fig. 15.1). The epidermis performs an essential part in the penetration of substances into the skin. It is the outer avascular layer of the skin, primarily composed of keratinocytes at various stages of differentiation. Because of cellular differentiation, the epidermis is divided into different layers, which are produced by the subdivision of basal cells from the inner part of the body toward the surface. Consequently, basal cells undergo progressive maturation, differentiation, and migration toward the surface, giving rise to the spinous layer or squamous cells. These cells also differentiate, forming the granular layer and finally the SC, which is the outermost layer of the skin. The dermis is composed of fibro-collagenous tissue containing blood vessels, lymphatics, and nerves. Besides these structures, the dermis contains cutaneous appendages or adnexal structures. These are sweat glands, sebaceous glands, hair follicles, arrectores pilorum, and nails (Beers et al., 2006).

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Skin cancer is a tumor or growth of abnormal cells in the skin. Most experts agree that the primary cause of skin cancer is over exposure to sunlight. However, repeated exposure to X-ray radiation, certain chemical toxins, and a family history of skin cancer can also increase the risk for skin cancer. Skin cancers (skin neoplasms) are named after the type of skin cell from which they arise. Most skin cancers develop in the topmost layer of the skin (the epidermis). BCC originates from the lowest layer of the epidermis and is the most common, but least dangerous skin cancer. Squamous-cell cancer originates from the middle layer and is less common, but more likely to spread, and if untreated becomes fatal. Melanoma, which originates in the pigment-producing cells (melanocytes) (Fig. 15.2) is the least common

FIGURE 15.1 Illustrative representation of the skin layers (epidermis/dermis) and cells (basal cells, squamous cells, and the melanocytes).

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FIGURE 15.2

Illustration of the pathophysiology of skin. Cancer and cancer like lesions of squamous cell, basal cell and melanocytes (A), squamous cell carcinoma (B), basal-cell carcinoma (C), and melanoma (D).

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but most aggressive, most likely to spread, and if untreated becomes fatal (Russo et al., 2005).

15.3.1 NONMELANOMA SKIN CANCERS

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The term nonmelanoma skin cancers (or keratinocyte carcinomas) encompasses cutaneous lymphomas, adnexal tumors, Merkel cell carcinomas, and other rare primary cutaneous neoplasms, but is mainly used to define SCC and BCC. Grouping of these two carcinomas under a common umbrella term creates challenges, because clear differences exist in their aetiopathogenesis, clinical course, and management strategies. Nonmelanoma skin cancers are the most common human cancers and, despite growing public awareness of the harmful effects of sun exposure, incidence continues to rise (Hoey et al., 2007). A 3%–8% yearly increase in incidence of nonmelanoma skin cancer has been reported since 1960, worldwide (Madan et al., 2010; Freedberg et al., 2004). Tables 15.1 and 15.2 summarize the environmental risk factors and syndromes predisposing nonmelanoma skin cancer, respectively.

15.3.2 MELANOMA SKIN CANCER Cutaneous melanoma is the noxious form of skin cancer. Assumed to arise from epidermal melanocytes, melanoma is often a treatment refractory and metastasis-prone malignancy, whose incidence has amplified gradually and notably in the past

15.3 Skin Cancer

Table 15.1 Environmental Risk Factors for NMSC (Madan et al., 2010) Type of Skin Cancer

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Risk Factors Solar UV radiation Human papilloma virus Iatrogenic immunosupression HIV/AIDS and non-Hodgkin lymphoma PUVA therapy Photosensitizing drugs (e.g., fluoroquinolone antibiotics) UVB radiation Ionizing radiation Occupational factors Arsenic Tobacco smoking

BCC, SCC SCC, BCC SCC, BCC BCC, SCC SCC, BCC SCC, BCC BCC BCC BCC, SCC SCC, BCC SCC

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UV, ultraviolet; BCC, basal-cell carcinoma; SCC, squamous-cell carcinoma; PUVA, psoralen and UVA.

Table 15.2 Syndromes Predisposing to NMSC (Madan et al., 2010) Syndromes

Features

Xeroderma pigmentosum

Autosomal recessive, multiple epidermal skin cancers in childhood, increased susceptibility to DNA damage, abnormal DNA base excision repair. Part or complete failure to produce melanin in the skin and eyes. Autosomal dominant, germline mutations in genes involved in DNA mismatch repair, sebaceous neoplasms in association with internal malignancy. Development of invasive squamous-cell carcinoma within dysplastic lesions

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Albinism Muir-Torre syndrome

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KID (keratosis, icthyosis, deafness) Dystrophic Autosomal recessive mechano-bullous disorder, mutations in the human epidermolysis type VII collagen gene (COL7A1) bullosa Fanconi anemia Autosomal recessive, congenital malformations, bone marrow failure, development of squamous- cell carcinoma, and other cancers. RothmundAutosomal recessive, progressive poikiloderma including alopecia, Thompson dystrophic teeth and nails, juvenile cataracts, short stature, hypogonadism, syndrome and bone defects. Werner Early aging, excess cancer risk, high incidence of type 2 diabetes mellitus, syndrome early atherosclerosis, ocular cataracts, osteoporosis. Others Hereditary nonpolyposis coli, dyskeratosis congenital, Huriez syndrome, Li-Fraumeni syndrome, chronic mucocutaneous candidiasis.

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few decades (D’Orazio et al., 2013; Narayanan et al., 2010). Melanoma is frequently detected on the trunk of men and the lower legs of women, although it can be found on the head, neck, or elsewhere (Narayanan et al., 2010; Gloster and Neal 2006). Early detected melanomas can be cured by surgical excision alone. Nonetheless, melanomas are fast to invade and metastasize, with the long-term survival being poor for advanced disease. Progressions have been made in the treatment of melanoma with targeted therapy (Flaherty et al., 2010; Sosman et al., 2012; Nikolaou et al., 2012; Flaherty, 2012; Ji et al., 2012) and immunotherapy (Hodi et al., 2008, 2010), but melanoma is still particularly problematic to manage once proliferated in other organs (D’Orazio et al., 2013). The epidemiology of melanoma is more documented than nonmelanoma skin cancers (NMSC). It is estimated that, in Europe in 2000, 37,000 deaths were caused by melanoma (Boyle et al., 2004). Moreover, it is estimated that 132, 000 new cases of melanoma occur worldwide each year (Narayanan et al., 2010; WHO, 2014). Incidence rates are at least 16 times greater in Caucasians than African Americans and 10 times higher than Hispanics (Narayanan et al., 2010; Gloster and Neal, 2006). The World Health Organization (WHO) also estimates that as many as 65,000 people a year worldwide die from malignant skin cancer, approximately 48,000 of whom are registered (Narayanan et al., 2010; AAD, 2014). Melanoma represents less than 5% of all skin cancers in the United States, but accounts for the vast majority of all skin cancer deaths (AAD, 2014; David and Wang, 2014).

15.4 TREATMENT OPTIONS

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The industry standard for treatment of skin cancers is excision biopsy (Fig. 15.3). As mentioned in the literature (Clarke, 2012), for NMSC not easily treated with elliptical excision, treatment options include curettage and diathermy, liquid nitro

FIGURE 15.3 Illustrative representation of the conventional treatment options. Various standards of excision are excision biopsy (A), Mohs surgery (B), and cryosurgery (C).

15.5 Topical Drug Delivery

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gen, imiquimod or 5-fluorouracil (5-FU), radiotherapy or excision, and flap repair/ graft. Nonetheless, it is recommended that the only therapeutic options that should be used on the face are excision or radiotherapy. The treatment of melanomas in situ has been a controversial topic in the literature for over a decade. Surgical excision with 5 mm margins is the standard of care in the US; however, margins larger than 5 mm may be required when treating larger or indistinct lesions. For clinically ill-defined melanomas arising on UV-damaged skin, especially in regions of esthetic concern, some forms of Mohs surgery may provide the highest cure rate and create the smallest surgical defect. Topical imiquimod therapy appears to provide relatively low cure rates for melanomas. IFN-α treatment may be employed in patients with stage II and III melanoma as an adjuvant therapy. For some metastatic patients, systemic chemotherapy (dacarbazine, temozolomide, or carboplatin/paclitaxel) continues to play an important role. BRAF inhibitors, such as vemurafenib for BRAF mutated patients, as well as the CTLA-4 antibody ipilimumab (recombinant, fully human IgG1 monoclonal antibody against cytotoxic T lymphocyte-associated antigen 4) offer fresh therapeutic prospects, apart from conventional chemotherapy. Limited progress has been made in the treatment of skin cancer over the past four decades, through the use of immunotherapy, chemotherapy, radiotherapy, and combinations because of reduced response rates and low median survival associated with significant toxic profiles. Various topical chemotherapeutic agents (i.e., 5-FU, tretinoin, imiquimod, mechlorethamine HCl, carmustine, potent glucocorticoids, etc.) are available in the market for the treatment NMSC (Taveira et al., 2011). Apart from that, targeting of active moiety to the site of interest is very important, especially in case of cancer treatment which may be achieved by the use appropriate drug carrier for the onsite delivery of the drug. The purpose of using carriers for vectorization of active moiety is to obtain a controlled release of drug and maintaining therapeutic drug levels over a specified time period, while reducing systemic absorption. The entrapped drug is associated in the microenvironment of the system and may even affect it due to molecular interplay, particularly if the drug comprised of amphipathic and/or mesogenic properties (Prow et al., 2011). Colloidal carriers have attracted increasing attention during recent years. They are vesicular or particulate forms of nanometer size, required for effective carriage of loaded drug to the target. Investigational approaches include nanoparticles, nanoemulsions, nanosuspensions, liposomes, micelles, vesicles, soluble polymer–drug conjugates, and liquid crystal dispersions.

15.5 TOPICAL DRUG DELIVERY Drug application to the skin as topical route of administration has increased potential in therapy because it indicates large surface. Moreover, topical delivery allows controlled release of therapeutics into internal surroundings, since it is easy for application practical management. Topical delivery of drugs offers many advantages,

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convenient and safe, increased patient acceptability (noninvasiveness), avoid GIT degradation and first pass effect of drugs, avoid GIT disturbances due to drugs, minimize side effects, and avoiding fluctuations in drug levels.

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compared to traditional drug delivery systems, including oral and potential systems. Advantages claimed are:

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Topical drug delivery is the term used for localized treatment of dermatological conditions where the medication is not targeted for systemic delivery. One or more of several pathways could be involved in the process of topical drug delivery. The passage of drug molecules by the intercellular route could occur via the lipoidal pathway or the aqueous pathway, whereas the transcellular route (a polar pathway) involves the penetration of drug by swelling of the intracellular protein matrix and/or alteration of protein structure within the corneocytes. The transport of such molecules across the SC barrier is mainly by passive diffusion (Fig. 15.4) (Jain et al., 2003). However, this limits the basic potential of topical drug delivery systems. As SC is the most formidable barrier to the passage of the drugs, except for highly lipophilic, low molecular weight drugs. Therefore, the most prominent disadvantage of dermal drug delivery is lower SC permeability to drugs (Mezei and Gulasekharam, 1980; Rahimpour and Hamishehkar, 2012). Although skin is one of the major sites for noninvasive delivery of active moiety into the body, this task can be relatively challenging, owing to the impermeability of the skin. The penetration of small moieties into or through the skin has been investigated widely and the factors that consider in the absorption of these molecules by the skin are well understood. However, the topical delivery to deep skin strata or across the skin for macromolecules is controversial, especially in light of

FIGURE 15.4 Topical routes of penetration. Skin cross-section showing topical routes for drug delivery (A), and simplified representation of skin showing routes of drug transport (B) (Rahimpour and Hamishehkar, 2012).

15.6 Topical Nanocarriers for Skin Cancer

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the old dogma concerning the 500 rule, which states that molecules with a molecular weight >500 Da cannot cross the skin. Recent literature suggests that a revision of this rule might be necessary, owing to the availability of novel methods that might improve the transport of large molecular weight moieties into or through the skin (Cevc and Blume, 1992; Egbaria and Weiner, 1991; Foldvari et al., 1999a,b; Baca-Estrada et al., 2000; Foldvari and Moreland, 1997). The barrier function of the skin is created by lamellar granules, which are synthesized in the granular layer and later become organized into the intercellular lipid bilayer domain of the SC (Sharma et al., 2012; Pandey et al., 2013). Barrier lipids are tightly controlled and any impairment to the skin results in active synthetic processes to restore them. The skin’s barrier function appears to depend on the specific ratio of various lipids. Studies in which nonpolar and relatively polar lipids were selectively extracted with petroleum ether and acetone, respectively, indicate that the relatively polar lipids are more crucial to skin barrier integrity (Kalpana et al., 2010; Pandey et al., 2013; Williams and Barry, 2012; El-Nabarawi et al., 2013). Because of its highly organized structure, the SC is the major permeability barrier to external materials and is regarded as the rate limiting factor in the penetration of therapeutic agents through the skin. The ability of various agents to interact with the intercellular lipid therefore dictates the degree to which absorption is enhanced. The role of the viable epidermis in skin barrier function is mainly related to the intercellular lipid channels and to several partitioning phenomena. Depending on their solubility, drugs can partition from layer to layer after diffusing through the SC. Several other cells (melanocytes, Langerhans cells, dendritic T cells, epidermotropic lymphocytes, and Merkel cells) are also scattered throughout the viable epidermis, which also contains a variety of active catabolic enzymes (esterases, phosphatases, proteases, nucleotidases, and lipases) (Naegel et al., 2013; Bolzinger et al., 2012).

15.6 TOPICAL NANOCARRIERS FOR SKIN CANCER

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Intralesional and site-specific applications of chemotherapeutic agents have been reported to be effective in selected cases of actinic keratosis, BCC, and SCC. The major limiting factor to the effectiveness of topical application of anticancer drugs appears to be the percutaneous absorption of therapeutic levels of drugs to the sites where invasive tumors are found (Raaf et al., 1976; Strange and Lang, 1992; Brenner et al., 1993; Mir et al., 1998; Sersa et al., 1998; Gibbs et al., 2001). To overcome the problems associated with topical delivery of anticancer drugs, strategies toward developing adequate drug delivery vehicles have been shouted (Katarzyna and Jerzy, 2016). Ideally, sustained drug release, cutaneous accumulation for localized effect in different strata of skin, and low extent of permeation of a drug are required. Therefore, vesicular carriers are widely explored as value-added carriers for successful topical delivery, which ensures selective, effective delivery of bioactives to the skin (Schreier and Bouwstra, 1994).

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Moreover, for the topical delivery of anticancer drugs, various formulations, such as emulsions, liposomes, niosomes, lipid sponges, and nanocolloid lotions (Lopez-Davila et al., 2012; Grazu et al., 2012), were studied for cutaneous malignancies. Furthermore, some novel formulations, including microemulsions (Pepe et al., 2013), gel formulation (Batheja et al., 2011), lecithin-based organogel (Yosra et al., 2014), and hydrogels (Alhaique et al., 2016) were reported. Vesicular carriers facilitate the transport of drug and increase its concentration in various skin layers and consequently potentiate efficacy (Deli, 2009; Montenegro et al., 2016). They may help in localizing the drug at the application site by serving as a regional depot or reservoir and reducing the effective dose, dosing frequency, as well as systemic side effects associated with conventional topical therapy (Beloqui et al., 2016; Mao et al., 2016; Wolinsky et al., 2012).

15.6.1 LIPOSOMES AND NIOSOMES

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Liposomes have been used as delivery vesicles for both systemic and topical administration of drugs (Cereda et al., 2013). Structurally, liposomes are made of cholesterol and phospholipids (Sinico et al., 2005). The classifications consist of unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes (Fig. 15.5). It has been shown that in vitro tetracaine delivery into human skin was higher from a specific multilamellar liposome formulation, which also increased the deposition of liposomal phospholipid within the skin layers (Foldvari, 1994).

FIGURE 15.5

Classifications of liposome consist of unilamellar liposomes, multilamellar liposomes (100–2000 nm) and multivesicular liposomes (100–2000 nm). The unilamellar liposomes are usually distinguished into small unilamellar liposomes (10–100 nm) and large unilamellar liposomes (100–1000 nm) (Zhai and Zhai, 2014).

15.6 Topical Nanocarriers for Skin Cancer

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The liposomal delivery system could enhance capillary permeability of drugs and localize to the target tissue. Moreover, phospholipids are nontoxic, biodegradable, and prolong the half-life of the drug to attain a sustained release effect (El-Nabarawi et al., 2013). In addition, previous reports have demonstrated that liposomal formulations can increase efficiency in tumor organ culture and in animal studies (Manosroi et al., 2004; Baruah and Surin, 2012). Niosomes are analogous to liposomes and offer higher chemical stability, lower costs, and much more variability of surfactants, compared to phospholipid vesicle-based liposomes (Rahimpour and Hamishehkar, 2012; Moghassemi and Hadjizadeh, 2014). Until now, there have been numerous studies and patents on the topical delivery of lipid-based colloid systems, which were designed into different structures for various disease treatments, either local function or topical therapeutic system. For examples, a liposome was prepared to provide a localized depot in the skin, minimizing systemic effects or target to skin appendages (hair follicles and sweat glands). Other than the localizing effects, liposome can achieve targeting to skin appendages, especially to the pilosebaceous units (hair follicles with their associated sebaceous glands). Plessis et al. (1992) prepared γ-interferon-loaded liposome and evaluated the in vitro permeation by employing human skin and the skin of hairless mice and hamsters as models. The results showed that the greatest deposition accumulation was achieved in hamster skin, which also possessed the highest follicular density, suggesting that the follicular pathway could be a route for drug deposition from liposome. Achieving hair follicle targeting may serve as an alternative pathway for dermal absorption of liposomes since it contributes significantly to the absorption of small molecules within the lag time after application. Moreover, for some hair follicle-related diseases such as acne and alopecia, the hair follicle itself is the target site. Some researchers hypothesize that lipid coating or lipophilic material properties may favor higher uptake into hair follicles because the hair follicles are filled with sebum and provide relatively lipophilic environment. In order to prove the hypothesis, a quantification research of uptake into hair follicles was performed by Raber et al. (2014). Previous studies advocated that liposomes and niosomes are capable of incorporating tretinoin (TRA) and maximizing the accumulation of drug into the skin (Ourique et al., 2011; Manconi et al., 2006). In addition, they deliver the drug to deep skin strata (Zhai and Zhai, 2014), with a higher deposition of drug in the follicular region (Tabbakhian et al., 2006). Simultaneously, drug systemic toxicity is reduced, due to the modification of drug pharmacokinetics and bioavailability (Sou et al., 2000). The most beneficial effect is their amphiphilic nature, which allows the incorporation of a wide variety of hydrophilic and hydrophobic drugs (Cereda et al., 2013). These carriers may serve as a solubilizing matrix and a local depot of drug for sustained release (Manconi et al., 2011). Nonetheless, they suffer drawbacks, ascribed to their liquid nature and wash-out problems.

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15.6.2 DEFORMABLE VESICLES

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Self-regulating, lipid-based drug carriers, so-called transfersomes (carrying bodies) for targeted and noninvasive delivery of agents into or through the skin (Cevc et al., 1998; Qiu et al., 2008; Mishra et al., 2007; Pandit et al., 2014; Modi and Bharadia, 2012) have been proposed. Cevc and Blume (1992) constructed transfersomes, which are liposomes such as vesicles of a specific composition (8.7 wt% soybean phosphatidylcholine, with varying amounts of sodium cholate and ethanol) (Fig. 15.6). The lipid penetrating deep into the dermis represented up to 10% of the total lipid (2.5 mg/cm2) applied. The use of these transfersomes was associated with increased penetration of drug molecules. Recent studies using electron paramagnetic resonance (EPR) imaging suggest that multilamellar liposomes prepared from phosphatidylcholine and 30–50 mol% cholesterol interact and mix with deeper layers of the skin (Vrhovnik et al., 1998). The prerequisites are: carrier stability, self-deformation under stress (Fesq et al., 2000; Ning et al., 2005), and virtual pathways opening through the organ (Pavelic et al., 2001; Pandey et al., 2010). The mechanism relies on local adjustment of the ultradeformable vesicle composition to the surrounding stress (Dhiman et al., 2008) and transepidermal water concentration gradient (Wang et al., 1998), which pushes the vesicles through the natural hydrophilic passages of the skin (Cevc and Gebauer, 2003), through which normally water evaporates. Ultradeformable vesicles consequently transport drugs spontaneously into and across the nonoccluded skin barrier better than the previously tested vehicle systems (Zhai and Zhai, 2014; Mishra et al., 2007; Fesq et al., 2000; Schatzlein and Cevc, 1998). As per previously reported research, they also offer a means for controlling drug deposition into the skin (Cevc et al., 1997). Another specially tailored

FIGURE 15.6

Schematic representation of lipid vesicles showing differences between conventional vesicles (liposomes) and elastic vesicles (transfersomes). Liposome comprised of lecithin–cholesterol and transfersome made up of lecithin-surfactant (Jain et al, 2006).

15.6 Topical Nanocarriers for Skin Cancer

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delivery carrier, ethosomes, were designed and characterized, with the goal of developing a potential effective treatment for deep dermal and intracellular cutaneous disease, such ethosomal delivery systems. The delivery properties and mechanism of permeation enhancement of ethosomal systems were further tested in vitro and in vivo (Zhang et al., 2012). Transfersomes, deformable liposomes, elastic liposomes, ultradeformable liposomes, nanotransfersomes, and protransfersomes all are elastic vesicles, and these differ from more conventional vesicles in several respects. The most important is the extremely high and stress-dependent adaptability of such elastic vesicles. Said aggregates are thus ultradeformable and can squeeze themselves between the cells in the SC, despite the large average vesicle size. These are categorized under phospholipid-based type elastic vesicles. Nanotransfersomes have reduced vesicle size, in comparison to others, and protransfersomes are liquid crystalline proultraflexible lipid vesicles, which will be converted into the ultraflexible vesicles transfersome in situ by absorbing water from the skin. Previous studies reveal that a protransfersome carrier is more stable, having higher entrapment efficiency. Furthermore, such type of vesicles can be incorporated into commonly used dermal vehicles, such as hydrogels, in order to have an appropriate semisolid consistency to facilitate convenient dermal application (Elnaggar et al., 2014; Ashara et al., 2014; Sharma and Pathak, 2011). Padamwar and Pokharkar (2006) have prepared carbopol (CP) 980 NF gels containing liposomal dispersion and have reported a sixfold and fourfold increase in drug deposition, compared to control gel and marketed cream, respectively.

15.6.3 NANOPARTICLES

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Production and evaluation, as well as topical use of lipid nanoparticles for skin diseases have been reviewed in detail (Marepally et al., 2014; Raber et al., 2014; Fang et al., 2008). Solid lipid nanoparticles (SLNs) are made of solid lipids at room temperature and nanostructured lipid carriers (NLC) are mixtures of solid and fluid lipids (Pardeike et al., 2009). These carriers are alternatives to polymeric carriers (Prow et al., 2011; Avgoustakis et al., 2002; Craig et al., 2012; Puglia and Bonina, 2012; Sun et al., 2012), liposomes, niosomes (Cereda et al., 2013; Rahimpour and Hamishehkar, 2012), and nanoemulsions (Hoeller et al., 2009; Contente et al., 2014; Primo et al., 2008), as well as microemulsions (Barot et al., 2012; Fouad et al., 2013). SLN and NLCs, developed to overcome stability problems of liposomes, may result in approved drugs with improved stability, high and constant cutaneous absorption, or even drug targeting in the near future. Given this, carrier systems of this type will present a remarkable therapeutic progress. Furthermore, the study compared the hair follicle uptake efficiency of plain nanoparticles and nanoparticles incubated with large, unilamellar vesicles consisting of phospholipid. The results showed that the phospholipid-coated particles achieved higher hair follicle uptake (6.95%±2.30%), compared to the plain PLGA nanoparticles (3.15%±1.23%). Drawing some inspirations, liposome may achieve the goal of targeting skin appendages by using relatively lipophilic materials (Zhai and Zhai, 2014). Topical

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and transdermal delivery of cyclosporine A is being explored as a therapy for various inflammatory skin diseases, such as psoriasis, atopic dermatitis, and diseases of hair follicles such as alopecia areata (Wang et al., 1998). By combining bio-responsive NPs with internal or external stimuli (such as pH gradient, hyperthermia, alternating magnetic field, light, and acoustic), stimuli-triggered drug release can be successfully achieved. These stimuli-responsive NPs are designed to only release the encapsulated therapeutic drugs upon applying locally confined triggers, thereby maximizing drug release at the pathological sites of tumors (Fig. 15.7) (Jun et al., 2015).

15.6.4 SOLID LIPID NANOPARTICLES

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In 1991, SLNs were introduced to challenge traditional colloidal carriers (Ram et al., 2012). SLNs are prepared by using physiologically tolerated solid lipid components. The solid matrix of nanoparticles can protect entrapped drugs against degradation and modify release rate (Pallerla and Prabhakar, 2013). SLNs were considered to be the most suitable lipid-based colloidal carriers when they came on the scene in the early 1990s. They have many merits, such as good biocompatibility, low toxicity, and feasibility to scale up and sterilize (Dolatabadi et al., 2014). Due to the structural similarity between the lipid matrix of SLNs and the epidermal lipids in skin, SLNs were outstanding as topical drug carriers in the rapidly increasing nanotechnology field. During the past several years, SLNs were claimed to act as carriers for cosmetic products (Muller et al., 2002a,b; Wissing et al., 2004). It

FIGURE 15.7 Schematic illustration of the stimuli-triggered drug release from the NPs (Martinez and Otley, 2001).

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had been reported that SLNs could increase miniaturization via an occlusive effect by preventing transepidermal water loss (Fang et al., 2008). SLNs have also been claimed to give improved UV absorbance, which is of great significance in the cosmetics industry. Regretfully, they are not yet in use in commercial sunscreen systems. The most likely reasons are the complex manufacturing processes, such as high temperatures and high pressure homogenization requirement (Uner and Yener, 2007). The low loading of UV sunscreen in the final SLN dispersions also restricts the prospect. Even so, SLNs are still playing a role in topical drug delivery, since the clear advantage such as better controlled release kinetics, and feasibility of commercial sterilization procedures. Top-drawer, the tiny size, and narrow size distribution of SLNs permit facilitating drug penetration into deeper skin or achieving skin targeting (Katsambas and Papakonstantinou, 2004). Isotretinoin has been clinically used for the first-line therapy of severe acne and the other dermatological diseases (Queille-Roussel et al., 2001). However, the obvious adverse effects induced by oral dosage form limit its application. Unfortunately, the launched topical formulations, such as cream, showed inevitable skin irritation. Accordingly, it is urgent to prepare an innovative formulation to achieve skin target and reduce the systemic absorption. Liu et al. (2007) constructed an isotretinoin-loaded SLNs (IT-SLNs) formulation by selecting PRECIROL ATO 5 as solid lipid, and Tween 80 and soybean lecithin as surfactants. The in vitro permeation study indicated that isotretinoin was not available in receptor chambers, proving that all IT-SLN formulations were unable to penetrate through skin. Conversely, the tincture showed a steady permeation rate (0.76±0.30 μg/cm2/h1). This should indicate a clear reduction in systemic side effects and an achievement of skin targeting. Perioral dermatitis is commonly seen. It presents as red papules that form superficial plaques around the perioral area, nasolabial folds, and/or lower eyelids (Lipozencic and Hadzavdic, 2014; Hengge et al., 2006). Long-term topical glucocorticoid treatment can result in skin atrophy due to the inhibition of fibroblasts. Therefore, looking for newly developed drug carriers with function of epidermal targeting may contribute to the reduction of this risk. Santos et al. (2002) incorporated prednicarbate (PC) into SLNs and evaluated the drug penetration as well as local tolerability in excised human skin. The reports showed an improved PC uptake if applied as SLN dispersion or SLNs dispersed in cream, compared to conventional PC cream and ointment. More interestingly, PC-SLNs were claimed to induce a localizing effect within the epidermis. Further experiments were done to explore the targeting mechanism. It was found that the targeting effect was unabated if diluted in the PC-SLNs with cream (1:9), whereas the targeting effect disappeared when adding blank SLNs into PC-containing cream. It can be concluded that the targeting effect only relates to the PC-SLN colloid particles but not a specific lipid. Accordingly, SLNs could increase the benefit/risk ratio of topical therapy by inducing epidermal targeting (Porter et al., 2013).

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15.6.5 NANOSTRUCTURED LIPID CARRIERS

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As mentioned earlier, SLNs were made by solid lipid, especially in the case of highly pure lipids. The particles could form relatively perfect lipid crystals and then trend to recrystallize. Accordingly, SLNs have major limitations of low drug loading and expulsion, due to advancing lipid crystallization or transformation during stored procedures (Muller et al., 2002a,b; Radtke and Muller, 2001). To overcome this, NLCs were introduced. NLCs are mixtures of solid and liquid lipids. It was investigated that (Williams et al., 2013) the liquid lipid can embed into solid lipid matrix or localize at the interface of solid matrix and the surfactant layer. These spatially different lipids led to generally imperfectible crystal structure, accordingly providing more space for accommodating the encapsulated drugs (Uner, 2006). Consequently, compared to SLNs, NLCs were characterized by higher drug-loading rate and better stabilization (Obeidat et al., 2010). Many attempts were applied to explore the potential of NLCs in skin care (Chen et al., 2013; Kawadkar et al., 2013). Xia et al. (2007) produced cosmetic product (sunscreen-loaded NLCs) by hot high pressure homogenization technique and evaluated the loading capacity and storage stability of the formulations. The results showed that all NLC formulations were stable within 30 days at room temperature. It is worth emphasizing that the loading capacity of NLCs was 70%, achieving breakthroughs, compared to that for molecular sunscreens previously reported (10%–15%). The author suggested that NLCs may provide an advantageous alternative to conventional sunscreen formulations. Furthermore, Guo et al. (2012) evaluated pharmaceutical products of quercetin-loaded NLCs in vitro and in vivo for transdermal delivery. The results showed that NLCs could improve the permeation of quercetin and increase the level of quercetin retention within epidermis and dermis, but the level of quercetin in plasma was less than 0.5 μg/mL (Table 15.3). Lipid nanoparticles have distinct occlusive characteristics which could facilitate drug vectorization through the SC by decreasing transepidermal water loss. In addi

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Table 15.3 Dermal Responses Scale

Response

0 1 2 3 4 5 6 7

No evidence of irritation Minimal erythema, barely perceptible Definite erythema, readily visible; minimal edema or minimal popular response Erythema and papules Definite edema Erythema, edema, and papules Vesicular eruption Strong reaction spreading beyond test site

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tion, the drug penetration might also be affected by carriers themselves; the species and the lipid content in the dispersion affect the interaction with skin. Correspondingly, it controls whether the drug be detained within the skin or into blood through skin. One thing is clear, lipid-based carriers could attach themselves onto the skin surface, making close contact with the superficial junction of corneocyte clusters and channels between corneocyte islands. Finally, it makes drug permeation easier, since the lipid cover could reduce corneocyte packing and widen the intercorneocytes spaces.

15.6.6 CHARGED COLLOIDAL PARTICLES

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Charged colloidal particles are also having some impact on topical drug delivery. Since topical drug carriers have constrained to choose the drugs in appropriate merger of molecular weight, lipophilicity, and charge. Compounds with positive charge can also have a promising effect on skin penetration, since the skin exhibits negative surface charge due to lipid and carbohydrates (Campbell, 2007; Hoeller et al., 2009). Nanoemulsions with positive charge comprised of phytosphingosine (PS) were found to be more permeable for drugs through porcine skin than the system with negative charge. As investigated, there are number of variables which influence the interaction of nanoemulsions with skin, including electrical charge of the systems. The findings of earlier research reported that nanoemulsions with positive charge are capable of carrying the active moieties, fludrocortisone acetate and flumethasone pivalate, efficiently into and across the skin. The level of skin interaction is apparently more crucial with the systems with positive charge, in comparison to systems with negative charge as the dermal charge is negative at neutral pH. Furthermore, Song and Kim (2006) showed that in vitro skin permeation and in vivo penetration into the deeper skin layers of the low molecular weight drug were significantly higher from cationic flexible liposomes than from anionic and neutral flexible liposomes.

15.6.7 POLYMERIC GEL

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The strategies for vectorization of drug across the skin have been extended to polymeric gels, such as pluronic lecithin organogels (Pandey et al., 2010), bioadhesive gels (Rigogliuso et al., 2012; Mangalathillam et al., 2012), protransfersome gel (Gupta et al., 2011), etc. Pluronic lecithin organogel is a new formula for vectoring the topical delivery of drugs. The innovation and versatility of this vesicular lipidic system lies in the attainment of direct access of the therapeutics in the area of activity to rapidly and efficiently stimulate the effect (Pandey et al., 2010). Barot et al. (2012) reported a higher retention of terbinafine in the human cadaver skin after topical application of microemulsion-based gel, when compared to microemulsion containing the same drug. Rao and Murthy (2000) reported that HPMC gels containing CP-loaded liposomes applied topically showed lower absorption of the drug in the bloodstream, when compared to the same formulation containing free drug.

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Zhu et al. (2009) showed that penciclovir-loaded microemulsion-based gel has excellent sustained release capability; it enhanced skin permeation and retention due to viscosity imparted by carbomer 940.

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Topical nanocarriers for skin cancer are generally evaluated for physicochemical properties such as pH, viscosity, size, zeta potential, surface morphology, in vitro release studies, etc.

15.7.1 DETERMINATION OF PH

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The pH of various topical nanocarriers can be determined by a digital pH/mV meter. The pH of the saturated solution should be in between 5 and 9. Application of topical formulation whose pH values are very high or very low can be destructive to the skin. With moderate pH values, the flux of the ionizable drugs can be affected by changes in pH that alter the ratio of charged and uncharged species and their dermal permeability. In the evaluation of topical nanocarriers, the pH of the formulation should be between 5 and 7, accordingly lying in the normal pH range of skin; consequently, the preparation will potentially be nonirritating. The formulation comprised such pH, consistent with the desired functional attributes of the site-specific application which may be suitable for topical application without any discomfort (Verma Pathak, 2011; Chen-yu et al., 2012). Skin irritation is associated with variables which include factors that associated to dermal irritation include variation in the dermal pH of the skin, disturbance in the SC barrier (i.e., lipid disruption, moistening, and disordering of lipid packing), physiological and immunological sequences, microbial generations at the site of delivery, and physicochemical characteristics of the drug or drug delivery system. The pH in formulations, particularly how it reacts to pH factors in different layers of skin, is very important. The epidermal layer is more acidic, acting as a defense and barrier, so creating a more acidic environment for the formulation allowed this particular peptide to be delivered to the target site (Vertuani et al., 2011; Campanini et al., 2013).

15.7.2 RHEOLOGICAL CHARACTERIZATION Viscosity of a topical drug delivery system can affect the delivery of drug. Diffusion of the drug from the drug delivery system might be directly affected by the viscosity. Semisolid topical formulations comprised of high viscosity can use high diffusion rates, compared to semisolid products of relatively lower viscosity. These investigations highlight the influence of rheological properties of topical carriers, especially viscosity, on the performance of pharmaceutical formulation. Most topical formulations, when sheared, reveal non-Newtonian behavior. Topical semisolid drug formulation during development phase can show different behaviors, includ

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ing shear thinning viscosity, thixotropy, and structural disturbance that may be invariable or only partially variable. Moreover, the viscosity of semisolid topical formulations is highly affected by such parameters, including inbuilt physical structure of the formulation, formulation sampling technique, environmental conditions for viscosity testing, container size and shape, and particular methodology carried out in the determination of viscosity (Ueda et al., 2009). Various methods can be used to represent the consistency of semisolid formulations, such as viscometry, rheometry, and penetrometry. Among all the methods, remarkable attention is assured to the shear history of the sample. Viscometer geometries for the determination of rheological properties in the case of semisolids typically fall into the following heads: concentric cylinders, cone-plates, and spindles. Concentric cylinders and spindles are especially applicable for less viscous, flowable semisolid formulations. Cone-plate geometries are especially applicable for the small sample size or the high viscous samples and less flowable. At the time of rheological measurement, it is very important to consider the characteristics of the drug formulation both “at rest” (without agitation) and sheared (with agitation) during application. The rheological characteristics of the drug formulation at rest may have an impact on product shelf-life. Its characteristics under comprehensive shear can affect its spreadability and, accordingly, its application rate that will influence the safety and efficacy of the drug formulation. Moreover, although it is important to optimize the temperature of the test sample at the viscosity determination, one should correlate the specific choice of the temperature to the intended application of the drug formulation (e.g., skin temperature for dermal application effects) (Al-Khamis et al., 1986; Chawla and Saraf, 2012). Since semisolid formulations frequently exhibit non-Newtonian flow properties, formulators should accordingly give proximate consideration to the shear history of the sample being tested, such as the shear applied during the filling operation, dispensing the product from its container, and introducing the sample into the instrument for rheological measurement. The point of highlighting this aspect is that appreciable variability and many failures to meet requirement can be directly contributed to a lack of consideration to this description rather than a change in viscosity (or fluidity) in the drug product (Contreras et al., 2001).

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15.7.3 MICROMERITICS MEASUREMENTS Particle size, zeta potential, and morphology are three critical parameters for micromeritics characterization in nanotechnology.

15.7.3.1 Size determination The term “nano-range” refers to a particle size range from ~1 to 100 nm, but for the purpose of drug delivery, nanoparticles in the range of 50–500 nm are acceptable, depending on the route of administration (Abdulkarim et al., 2010). Light scattering is an important way of characterizing colloidal and macromolecular nanocarriers and could be useful in assessing properties of particulate topical drug delivery sys

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tem. The particle size and size distribution are primarily measured using wet laser diffraction sizing, otherwise called dynamic light scattering (DLS) (Attari et al., 2016). The size of nanoformulation can also be determined using DLS (e.g., Zetasizer). This is necessary to ascertain the possible effect of the size on drug release and penetration across barriers in transdermal and dermal delivery, as well as to monitor stability over time. Particle size and shape affect drug release, physical stability, and cellular uptake of the nanocarrier materials. The transport characteristics of the drug are determined primarily by its size and by its level of interaction with the media or carrier through which diffusion is takes place, i.e., delivery system, SC, or viable epidermis. Smaller particle size thereby enhances drug flux through the skin.

15.7.3.2 Determination of zeta potential

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The zeta potential of nanocarriers is very important. It is determined by using a Zetasizer or by other means, and gives information on the charge of the particles and the tendency of the particles in a formulation to aggregate or to remain discrete. Particles with zeta potentials of more than +30 mV or more than −30 mV are normally considered stable (Honary and Zahir, 2013a,b). The stability of emulsions and colloids, according to DLVO electrostatic theory, is a balance between the attractive van der Waals forces and the electrical repulsion because of the net surface charge. If the zeta potential falls below a certain level, the emulsion droplets or colloids will aggregate as a result of the attractive forces. Conversely, a high zeta potential (either positive or negative), typically more than 30 mV, maintains a stable system. Zeta potential is an important physicochemical parameter that influences the stability of nanoformulations. Extremely positive or negative zeta potential values cause larger repulsive forces, whereas repulsion between particles with similar electric charge prevents aggregation of the particles and accordingly ensures easy redispersion (Beck-Broichsitter et al., 2011; Harush-Frenkel et al., 2010). In the case of a combined electrostatic and steric stabilization, a minimum zeta potential of ± 20 mV is desirable.

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15.7.3.3 Morphological analysis The morphological analysis of nanocarrier was carried out by electron microscopy [transmission electron microscopy and freeze-fracture electron microscopy, confocal laser scanning microscope (CLSM)]. Morphological characteristics to be taken into account are: flatness, sphericity, and aspect ratio. Appropriate morphology is related to the particles which have spherical shapes and uniform distributions (Adolfina et al., 2008; Gupta et al., 2010). Morphology of nanocarrier is becoming a task to be performed not only at a transmission electron microscope (TEM) but also more and more at modern, high-resolution scanning electron microscopes (SEMs). A SEM is probably the most widespread analytical instrument available in analytical laboratories destined to characterize physical properties such as morphology, shape, size, or size distribution of materials at the micro and nanoscale. The

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performance of a modern, high-resolution SEM, in particular its spatial resolution, can reach to enable identification and even morphology characterization of nanocarriers down to below 10 nm (Hodoroaba et al., 2014).

15.7.4 IN VITRO RELEASE STUDIES

Drug release is assessed by using various techniques including sample and separate (SS), continuous flow (CF), dialysis membrane (DM) techniques, and their combinations, as well as novel methods include voltametry and turbidimetry. The in vitro drug release for topical nanocarriers was evaluated by Franz diffusion cell using Spectra/Por regenerated cellulose membrane of MWCO 10,000 g/mol (surface area of 2.1 cm2). Primary purpose of in vitro release study is one or more of the mention below:

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(1) determination of the influence of product variables and production methods on the drug formulations, (2) routine monitoring of quality control to favor the batch release, (3) insure the product label claims, (4) optimization of an in vitro in vivo correlation (ivivc), (5) measuring the level of change as per the supac guidelines, and (6) as a pharmacopoeial requirement (Abdel-Mottaleb and Lamprecht, 2011; Bhardwaj and 2010).

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Among all the methods applied to measure drug release from nano-ranged drug delivery systems, the dialysis technique is the most widely used. In this technique, physical separation of the formulations is takes place by the help of a DM which admit for ease of sampling at predetermined intervals. According to other techniques, several alterations of the DM have been published in literature with primary variations in set-up, compartment dimension, and molecular weight cut-off (MWCO). Besides various DM set-ups used, the most widely used is the dialysis bag or regular dialysis, other changes being the reverse dialysis and side-by-side dialysis arrangements (Chidambaram and Burgess, 1999; Yan et al., 2010; Calvo et al., 1996) (Figs. 15.8–15.10). With the dialysis bag method, the nano-vehicles are kept into a dialysis bag containing media (inner compartment) that is further sealed and placed in a wide mouth vessel containing release media (outer compartment), stirred to minimize unstirred water layer effects. Generally, the release media placed in a dialysis bag (inner compartment) is comparatively smaller than the outer media. As per reported literature, inner media comprised of 1–10 mL, whereas the outer media comprised much greater, around 40–90 mL (Yan et al., 2010; Kumar et al., 2011; Muthu and Singh, 2009). Accordingly, compartment dimension will base on the total volume of release media needed for the in vitro release testing. In the dialysis bag technique, drug released from the nano-vehicles release through the DM to the outer compartment from where it is sampled for analysis.

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FIGURE 15.8

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Regular dialysis set-up (D’Souza and DeLuca, 2006).

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In the reverse dialysis arrangement, the nano-vehicles are placed in the outer compartment (stirred) and the inner compartment is sampled for drug release (Fig. 15.9) (Levy and Benita, 1990; Xu et al., 2012). Other alterations of the DM is the side-by-side dialysis arrangement where donor and receiver compartments, both comprised of equal volumes of media stirred with a magnetic bar, are distinct by a DM, and sampling takes place from the receiver compartment and a Franz diffusion cell (Fig. 15.10) (Kilfoyle et al., 2012; Uprit et al., 2013). As with other methods, dissolution in the release media is crucial to its transit across the DM. Besides from release media, the significance of selecting favorable MWCO for the DM cannot be avoided. Noteworthy, the basic organization of the DM is that drug that is released from the system will enter quickly from one compartment, via membrane, followed by the second compartment from where it is sampled for analysis. Accordingly, high MWCO membranes are usually chosen for in vitro release studies so that drug release is not a limiting factor. Generally, the MWCO should be adequately large to allow drug transport. The simplicity of arrangement and sampling with the DM demonstrate it a straight forward technique to study drug transport from a broad variety of nano-ranged formulations include liposomes, niosomes, nanoemulsions, nanospheres, nanoparticles, and so forth (Yan et al., 2010; Calvo et al., 1996; Muthu and Singh, 2009). Though, problems have been associated with the regular dialysis method. If inappropriately sealed, leakage of media and formulation may take place from two

FIGURE 15.9

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Reverse dialysis set-up (D’Souza, 2014).

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FIGURE 15.10

Schematic illustration of various parts of Franz diffusion cell (Beriro et al., 2016).

ends of the regular dialysis set-up. Deficient release profile may be reported if nonsink conditions occur or equilibration times are more (Heng et al., 2008). Moreover, the variation in equilibration times can be imposed as a discriminatory tool to differentiate release properties between fast and sustained releasing formulations (Kim et al., 1997). Another parameter to be notified is that the DM cannot be applied with drugs that interact to the dialyzing membrane.

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15.7.5 DETERMINATION OF DRUG CONTENT

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The method of the choice for the drug content determination is separation of the nanocarriers by the ultracentrifugation and following quantitative analysis of the drug after dissolution of the pelleted sediment. Other useful separation methods for nanocarriers are ultrafiltration and gel filtration. The major disadvantage for ultracentrifugation as well as ultrafiltration is that undissolved or secondarily precipitated drug particles may be removed from the liquid phase together with the carrier particles. This problem can in most cases be avoided by using gel filtration as the separation process. The disadvantage of latter method is that some drug release may occur during passage over the gel column. The relatively short separation time of 10–35 min necessary for this method, however reduces the possible error caused by this drug release. Alternatively, the drug content can be determined in the supernatant or in the filtrate. The amount of drug bound to the carriers can then be calculated by subtraction of this amount from the total amount of drug exist in the suspension. Generally, the construction of drug-loading sorption isotherms is relatively time consuming. A more rapid determination of the plateau of the isotherm is the measurement of the electric parameters of drug carrier suspensions at high frequencies. So far, however, the applicability of this method has only been demonstrated for a limited number of drugs and polymers (Lau et al., 2003). Another, very interesting alternative drug content analysis methods for drug delivery systems that allowed the determination of bound drug without prior separation. These methods utilized the bathochromic shift or the quenching of the fluorescence caused by that amount of drug that is bound to the drug carriers. These methods, however, are drug specific and therefore not applicable for every drug (Ajima and Onah, 2015).

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15.7.6 IN VITRO STABILITY STUDIES

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Pharmaceutical storage stability studies are a major investigation that varies in the quality of any drug formulation with respect to environmental condition and duration, such as temperature, humidity, and light. Stability studies are generally recommended during the product development and optimization of new drugs in terms of shelf-life assurance for the drug product and to propose a suitable storage condition. For all novel drug formulations, including nanocarriers, stability testing should comprise the testing of all parameters that are prone to vary during transportation and storage and are probable to affect the safety, efficacy and quality of these drug products (Craig et al., 2012). The International Conference on Harmonisation (ICH) of scientific requirements for enrolling of therapeutics for human use is an exclusive project that furnishes together the regulatory authorities of Europe, Japan, and the United States, and authorities from the pharmaceutical organization in these three vicinities to explore scientific and technological perspective of drug-product registration (Grimm

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and Krummen, 1993). In the pharmaceutical industry, stability studies for new drug products are commonly carried out following the ICH guidelines. The procedures for stability study of new drug products, new dosage forms and on biotechnological-based drug products are practicable in the ICH guidelines Q1A (R2), Q1C, and Q5C, respectively. Now days, various novel drug delivery systems (e.g., polymeric nanoparticles, SLNs, micelles, dendrimers, liposomes, gold nanoparticles, fluorescent nanoparticles, polymer conjugates, and magnetic nanoparticles) and multifunctional drug delivery systems (e.g., nanomicelles, dendrimers, and magnetic nanoparticles) have been observed for drug delivery and imaging (Manosroi et al., 2004). Nontargeted drug delivery systems are free from the regulations for biotechnological products, which can follow the regulations for the procedures of ICH guidelines Q1A (R2) and Q1C. The targeted drug delivery system, however, are widely associated with biotechnological products (e.g., cancer-targeting antibodies, proteins and peptides, etc.). Accordingly, stability study of these types of drug products can adopt the procedures of ICH guideline Q5C (Bai et al., 2011). A reported literature reveals that limited attention has been given to the thermal stability study of these novel drug delivery systems produced for drug delivery and imaging, the stability study of some nano-vehicles, such as polymeric nanoparticles, SLNs, and liposomes have been well investigated (Liang et al., 2005; Lemoine et al., 1996).

15.7.6.1 Stability testing of nanomedicines

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Nontargeted drug delivery systems are generally produced without the incorporation of biotechnological products. These nanomedicines are incorporated only with an active pharmaceutical agents and/or tracing agent. Stability testing of such drug products follows the ICH guidelines Q1A (R2) and Q1C (for new dosage forms). Lemoine and colleagues investigated the stability of nanoparticles comprised of poly (ε-caprolactone), poly(D,L-lactide) and poly(D,L-lactide-co-glycolide) polymers, which were stored at different temperatures, such as 16°C, 4°C, and 37°C (room temperature). The authors finally suggested suitable storage conditions of 4°C and 37°C for these polymeric nano-vehicles. Nano drug delivery system comprised of copolymers, such as poly (D,L-lactide-co-glycolide)-monomethoxy poly (ethylene glycol) also proved their stability at 4°C and 37°C (Mulik et al., 2009). In another study, lyophilized formulations of SLNs were stored under three different sets of environmental conditions as per ICH guidelines: 25°C/60%RH, 30°C/ 65%RH, and 40°C/75%RH. Results indicated that stability SLNs were increased at 25°C/60%RH and 30°C/65%RH, compared to conditions at 40°C/75%RH (Pallerla et al., 2013). Mulik et al. (2009), reported curcuminoid (a light-sensitive drug)-loaded poly (butyl cyanoacrylate) nanoparticles and stored them at 40°C/75%RH in the presence and absence of light for 6 months for stability testing. No appreciable changes were recorded with respect to the stability of nanoparticles (except drug content).

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The percentage of drug content in curcuminoid-loaded poly (butyl cyanoacrylate) nanoparticles after 6 months at 40°C/75%RH in the absence of light was observed to be in in the range of 87% and 93%, and was reduced to between 79% and 89% in the presence of light, indicating the influencet of light on the stability of curcuminoids. In the reported literature, the thermal stability of various liposomes at 4°C and 25°C have been proven, as per the ICH guidelines, by various authors (Muppidi et al., 2012).

15.8.1 SKIN MODELS

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On account of inconsistent human skin availability, however, pig skin (Simon et al., 2016; Peira et al., 2014; Pazourekova et al., 2013) and preferentially pig-ear skin (Luo et al., 2016) is used as a relevant anatomical site for ex vivo permeation studies. Excised human skin is properly regarded as the first choice for ex vivo penetration experiments related to human dermal risk assessment. However, it is often neither timely nor sufficiently available and there is variability among samples due to differences in gender, race, age, and anatomical site of the donor (Caputo and Cametti, 2016; Pyatski et al., 2016; Ishida et al., 2015). Numerous animal skin models from various mammals, rodents, and reptiles have been developed as surrogates for human skin (Barbero and Frasch, 2009; Flaten et al., 2015). For ethical reasons, primate research is restricted, accordingly pig and goat skin is preferred as it can be readily obtained as waste from animals slaughtered for food. As per various researchers (Wester and Maibach 1993; Schmook et al., 2001) the skin of pigs and goats is composed of an epidermis and dermis with characteristics such as those of human skin. However, the skin of laboratory animals, for example, mice, rat, guinea pig, rabbits, etc., shows marked anatomic differences from human skin. In particular, the epidermis of these animals is too thin and the flat epidermal–dermal interface does not have rete ridges and papillary projections. For ethical reasons, primate research is restricted and pig skin is preferred and based on morphological and functional data, domestic pig skin seems to be the closest to human skin (Ponec 2002). Furthermore, in these animals, dermal structures are relatively loose, and the vascular system is underdeveloped, therefore, the skin of most animals presents a much weaker barrier than human skin.

15.8.2 THE USE OF TISSUE CULTURE-DERIVED SKIN EQUIVALENTS IN TOPICAL RESEARCH A number of tissue culture-derived skin equivalents such as living skin equivalent models (LSEs) and human reconstructed epidermis (HRE) have been used to measure percutaneous absorption. These models generally are comprised of human cells grown as tissue culture and matrix equivalents normally present in skin, and are utilized as alternatives to animal skins. LSEs resemble human skin, having a

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dermis, epidermis, and partially differentiated SC, but are lacking in dermal appendages including pilosebaceous systems, hair follicles, and sweat glands (Kong and Bhargava 2011). These tissues provide much lower barrier properties than the whole skin due to their structure and lipid composition. For this reason, the pharmacokinetic parameters of skin penetration occur when using LSEs usually highly overestimate flux across human skin. For example, in a study by Schmook et al. (2001), the permeation characteristics of human, porcine, and rat skins with the Graftskin LSE and the Skinethic HRE models were compared using four low molecular weight dermatological drugs with various hydrophilicities. The permeation of more hydrophobic compounds (clotrimazole and terbinafine) through the skin equivalents resulted in an 800–900 fold higher flux than through split-thickness human skin. On the other hand, transdermal flux of a less hydrophobic compound, salicylic acid, was in the same order of magnitude as fluxes obtained with human skin. In this study porcine skin performed as the most appropriate model for human skin and they concluded that reconstituted skin models are not suitable for in vitro penetration studies (Kong and Bhargava, 2011). A similar conclusion was drawn from results of another study in which researchers (Roy et al., 1994) evaluated the in vitro permeabilities of alkyl p-aminobenzoates through LSE and human cadaver skin. In the case of cadaver skin, the permeability coefficient increased as the carbon chain length increased. However, this relationship was not observed in the permeability coefficients of these esters across LSE. Moreover, LSE showed very low resistance to flux compared to cadaver skin as the permeability coefficients of these esters through LSE were an order of magnitude higher than through cadaver skin. On the other hand, numerous reports support the use of skin equivalents for evaluation of skin irritation (Zhang and Michniak-Kohn, 2012). In a study by Monteiro-Riviere and Riviere (1996) EpiDerm LSE was found to be morphologically and biochemically comparable to normal human epidermis, providing a model in toxicological and skin metabolism studies. Ponec et al. (1997) reported that architecture, homeostasis, and lipid composition of reconstructed human skin models (EpiDerm, SkinEthic, Episkin, and REDED) were comparable to native human tissue. It is noteworthy that Colipa, the European Trade Association for cosmetic and toiletry industry, recommends the use of in vitro reconstructed skin equivalents as the preferred testing model for skin irritation studies (Park et al., 2010). However, the overall use of skin cultures is likely to be limited due to questionable performance as a barrier in skin permeation studies, as well as due to their cost and data reproducibility.

15.8.3 PERMEATION STUDIES The skin permeation kinetics of drug from topical therapeutic carriers can be evaluated, using a two-compartment diffusion cell assembly, under identical conditions. The diffusion cells of either vertical (Franz) or horizontal (Chein-Keshary). The cells consist of (a) donor and (b) receptor compartments (Fig. 15.11). The temperature in the bulk of solution is maintained by circulating thermostated water through

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FIGURE 15.11

Sampling in the skin by Microdialysis (Okoro et al., 2014).

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a water jacket that surrounds the receptor compartment. It is carried by mounting individually the full thickness of skin, which has been freshly excised (Manconi et al., 2011; Gupta and Vyas, 2012). The drug delivery systems are applied with their drug-releasing surface in intimate contact with the SC surface of the skin (Patel et al., 2013). The contents of receptor compartments are stirred using magnetic stirring bar at 600–800 rpm. The skin permeation profile of drug is followed by sampling the receptor solution at predetermined intervals for duration of up to 24 h and assaying drug concentrations in the sample by a sensitive analytical method, such as high-performance liquid chromatography (HPLC) (Klimich and Chandra, 1986). The release profile of drug from these topical therapeutic systems can also be investigated using the same experimental set-up without the skin (Gupta and Vyas, 2012).

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15.8.4 SKIN RETENTION/DEPOSITION STUDIES There are currently a number of methods available for quantifying drugs localized within the skin or various layers of the skin. Skin extraction measurements represent an easy, rapid, and inexpensive methodology for quantifying drugs in whole skin or possibly in a particular skin tissue. The disadvantage of this method is that it does not provide any information on drug localization. From horizontal sectioning, it is possible to determine the penetrants concentration–depth profile throughout the integument. However, since the technique does not yield data about concentrations in the glands or hair follicles, it is helpful to apply it in conjunction with either qualitative autoradiography or a follicle-isolation method. Of the latter, the

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scraping method in the Syrian Hamster model could perhaps be duplicated in human skin derived from tumors or cadavers. More work needs to be conducted on validating the induced follicle-free rodent skin model. It should be noted that skin extraction and horizontal sectioning are “two step” approaches while quantitative autoradiography and the spectroscopic methods are “single step” approaches in which analysis is included within the technique. Quantitative autoradiography is a novel methodology which allows quantification and visualization of the penetrants throughout an entire vertical cross-section of the investigated skin. Since the computerized system measures drug concentrations throughout the whole tissue depth, no horizontal sectioning is required. This technique is advantageous in that quantification can be performed down to the depth of the lower dermis as well as within the pilosebaceous structures. The system requires adequate equipment and it is limited to the use of radiolabelled molecules. Although the autoradiography exposure period was 4–7 weeks long when drugs were employed, this process would be accelerated with the use of C-labeled penetrants. The spectroscopic methods represent noninvasive, rapid assay systems. However, although widely employed, ATR-FTIR spectroscopy is relatively invasive as the technique has to be employed together with tape stripping for the acquisition of in depth measurements (Lee et al., 2007). Consequently, the technique is associated with all the limitations of tape stripping such as variable adherence and incomplete removal of the cornified layer. Out of the remaining spectroscopic methods, fluorescence spectroscopy is generally the most sensitive. Direct fluorescence spectroscopy can only be used to assay self-fluorescent compounds. Indirect fluorescence spectroscopy involves the measurement of UV-absorbing penetrants, and it is therefore much more widely applicable. It still requires further research. One promising avenue of research is to measure the photo-thermal effect by examining changes in the temperature-dependent air refraction index. In preliminary in vivo experiments with a so-called mirage detector, fluctuations in the air refractive index were recorded from the changed position of a helium–neon laser beam. This technique permits the measuring distance to be extended down to 30 mm which is well into the viable epidermis. This represents the only spectroscopic technique which can detect drugs at this depth. Clearly, photo-thermal spectroscopy and the generally more reproducible remittance spectroscopy require further methodological development. To this end, contrasting methodologies should be employed in order to quantify drugs localized within the same transdermal penetration model. By this means, it will be possible to more effectively evaluate each measuring technique (Fabin and Touitou, 1972).

15.8.5 DERMATOPHARMACOKINETICS Dermatopharmacokinetics describe the pharmacokinetics of topically applied drugs in the SC with pharmacodynamic effects. The smart techniques (tape stripping and microdialysis) use in dermatopharmacokinetic (DPK) methodology assesses the cutaneous drug accumulation at application site. Various studies have shown DPK to

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be a reliable and reproducible method for determining bioequivalence, and have indicated that it is applicable for all topical dermatological drug products. Dermatopharmacokinetics refer to the determination of SC concentration-time curves for topical actives. This is analogous to plasma/urine concentration-time curves for systemically or orally administered drugs, and the concept is clearly adaptable to microdialysis, where drug is determined in the skin compartment in which the microdialysis fiber is positioned (Fig. 15.11) (Muller, 2012). Although, this procedure is invasive, it is a method of great potential offering information of high value and relevance. There could be sampling in a compartment within the skin. It is a technically demanding procedure, however, requiring experimental dexterity of high order. The potential for use on diseased skin is a unique and considerable advantage over other techniques, but real challenges remain with respect to reproducibility, sensitivity, applicable drugs, etc. Different techniques and methods exercised in DPK. There are number of techniques for pharmacokinetic assessment of the dermal formulations, of which the most essential and simple method is in vivo tape stripping technique.

15.8.5.1 Tape stripping technique

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The technique comprised of the standardized procedure of multiple applications and removal of adhesive tape on the skin surface, whereby successive layers of SC cells are sampled. As reported by Lademann et al. (2012) tape stripping is a standard method for the determination of the DPK of dermally applied isoconazole nitrate. These tape strips are appropriately applied and removed from the skin surface after application and permeation of topically applied components; accordingly, the layers of the SC and definite amount of topically applied components are removed. The amount of the components and the amount of corneocytes layers removed with the single tape strip is to be measured for calculation of the permeation profile. Dermal application of the components removed from the skin can be accordingly measured by various analytical techniques such as HPLC, Mass spectroscopy, and other spectroscopic measurements (Pople and Singh, 2013; Shah et al., 1998).

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15.8.5.2 Microdialysis Microdialysis method has been applied to investigate the amount of drug after topical drug administration. The method comprised of placing an ultrathin selective permeable hollow fiber known as the marker in the dermis and perfusing this fiber with a tissue suited sterile buffer at a very low rate with a Microdialysis pump. The marker functions as an artificial vessel in the dermis and accordingly interchanges small, diffusible molecules from the marker to tissue and vice versa. The retrieval of the given component closely reflects the concentration of unbound, that is, pharmacologically active component in the intracellular fluid of the tissue surrounding the marker.

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15.8.5.3 Pharmacodynamic aspects

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Phamacodynamic perspectives for definite selected corticosteroid drugs have already proved applicable to document bioequivalence, which is associated with the well-known skin blanching effects of corticosteroids. Also another terminal point which evident useful is the increase in transepidermal water loss and desquamation rate of the SC following the application of retinoic acid dose. This occurs over the duration of several days and the process is willingly followed with respect to time.

15.8.5.4 In vitro permeation determination

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In vitro experiments are carried out using artificial membranes or excised skin (from humans or an animal model) to scrutinized and optimize topical formulations. The artificial membranes such as silicone membrane or even pig-ear skin are utilized to serve the purpose. As mentioned by Shah et al. (1998) the proof exist suggests that the rate of penetration of therapeutics from their formulations and the spatial profiles of such permeation may be similar as long as the formulation themselves are the same. Although there are variations in clinical terminal points the permeation rates have shown to vary and these finding still need investigation. In this technique all comparisons must be carried out with skin membranes cut from the same section of unblemished skin ex vivo.

15.8.5.5 Confocal laser scanning

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15.8.5.6 Validation procedures

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DPK technique should be validated and verified. Validation should include all features of sampling, e.g., SC stripping and determination of drug concentration in SC, or any other investigation. At every crucial step in the method development and optimization methodology should be established. The methods used for assessing the validity of SC tape stripping technique are as follows:

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• Cadaver skin permeation As reported in literature, this method validation procedure is done by choosing multiple sections of dermatome human trunk skin and placed on Franz diffusion cells comprising of dermal receptor solution which is constantly stirred and maintained at optimal temperature. Each specimen integrity was verified by measuring its permeability to titrated water. Subsequently test sample was applied to a required number of sections and multiple donors were applied for each section.

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At appropriate time intervals the solution is replaced with fresh solution, and aliquot is used for assay by HPLC. • Vasoconstrictor assay

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The potential of vasoconstrictor is tested on normal human volunteers. The test sample and the control were applied and after a definite time it was removed and sites skin color was characterized using Minolta Chroma Meter. The change in scale value between pre-dosing and postdosing after the specified time was determined for each site.

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15.8.6 NANOCARRIER–SKIN INTERACTION STUDIES

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Nanocarrier–skin interaction studies generally carried out using CLSM equipped with a white laser. Fluorescent markers generally used as probe during nanocarrier preparation and examined under CLSM to investigate the fluorescent probe distribution in the different skin strata. The instrument allowed simultaneous fluorescence and differential interference contrast imaging, which enabled the acquisition of images showing the distribution of the markers among skin structures. The composition of skin lipids is unique and varies greatly from SC to the basal layer (Simoes et al., 2015). Specifically, the very low phospholipid content of the SC lipids which consist, to approximately 50%, of ceramides (Mojumdar et al., 2016) is indicative for a particular role of protective properties of the SC. Abraham and Downing (1992) have investigated the formation of the lipid lamellae in the SC. Small unilamellar liposomes prepared from ceramides (40%), cholesterol (25%), cholesterol sulfate (10%), and free fatty acids (25%) were shown to transform to large unilamellar liposomes and finally to lamellar lipid sheets when calcium chloride was added to the dispersion. Gay et al. (1994) characterized the physical properties of these skin lipids and found a transition temperature of the mixture around 60–80°C accompanied by an abrupt change in permeability at about 70°C. Consequently, it was documented that the highly structured lipid lamellae in skin are the main barrier and controlling parameter for water flux across skin. Freeze-fracture electron microscopic techniques for the visualization of skin ultrastructure have been developed by Holman et al. (1990) and Bodde et al. (1990). Following exposure of liquid state niosomes to skin, Pazos et al. (2015) have demonstrated the appearance of structural changes deeper in the SC, resembling multilamellar vesicular structures. The authors speculate that either intact niosomes migrated into the SC, or that molecularly dispersed high local concentrations of nonionic surfactants could form curved lamellar structures within the lipid interstitial spaces of the SC. One-dimensional EPR imaging has been employed to monitor the fate of a liposome-associated spin probe following dermal application of various types of liposomes (Burks et al., 2011). Although the technique might provide valuable information, an independent validation is mandatory before it can be accepted as a valid experimental method.

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15.8.7 HISTOLOGICAL ASSESSMENT OF SKIN FOR ANTITUMOR EFFECT

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Histologically, NMSC (SCC) in situ is composed of atypical keratinocytes, which can be identified throughout the full thickness of the epidermis. The atypical keratinocytes exhibit eosinophilic, sometimes pale or vacuolated cytoplasm, a sign of faulty cornification, as well as whorls of parakeratosis within aggregates of neoplastic cells (vacuoles) (Fig. 15.12A). The nuclei of the atypical keratinocytes are crowded, pleomorphic, and often large and hyperchromatic. By definition, the atypical keratinocytes throughout the epidermis do not penetrate into the dermis. SCC in situ may develop into invasive SCC. Histologically, the different types of SSC exhibit the same morphology; however, their architectural patterns are different. BCC is an epithelial neoplasm that is believed to derive from the basal layer of the epidermis or follicular epithelium. The classic histologic presentation of BCC is that of nodules and/or strands of atypical basaloid cells that show nuclear palisading, cellular apoptosis, and scattered mitotic activity (Fig. 15.12B). Artifactual cleft formation may be seen between the tumor lobules and its surrounding stroma, which may be mucinous. Tumor calcification may be seen, especially in long standing tumors, although this phenomenon has been reported to be more commonly associated with more aggressive BCC subtypes (Suarez et al., 2007). Multiple growth patterns of BCC have been described, and these act as prognosticators of biologic behavior (O'Driscoll et al., 2006). The approach to diagnosis of many skin conditions today usually depends on the histopathological microscopic analysis of excised and processed tissue. The latter is a well-established technique that provides high-resolution cellular and subcellular tissue detail. It requires, however, that a biopsy be performed, which can be painful, time consuming, costly, only provides information on the excised tissue examined and carries a potential risk of scarring and infection. It also typically requires processing and staining of the specimen, which may induce artifacts.

FIGURE 15.12 Nonmelanoma skin cancer (SCC and BCC of skin). Whorls of malignant squamous cells contain keratin pearls in their center (A), basal carcinoma appears below the epidermis with typical nuclear palisading at the peripheral layer of the tumor (B) (Harsh, 1998).

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Recent advances in imaging techniques provide the potential for noninvasive high-resolution skin imaging in vivo. These can overcome some of the disadvantages of the biopsy and histologic analysis. Such advances include optical coherence tomography (Gambichler et al., 2015), high-frequency ultrasound (US) (Mlosek et al., 2013), magnetic resonance imaging (MRI), and reflectance confocal microscopy (RCM) (Barral et al., 2010; Calzavara-Pinton et al., 2008). Of these, RCM proposed the topmost resolution imaging in comparison to regular histology.

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15.8.7.1 Use of confocal reflectance microscopy for evaluating response to treatment

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To assess histologic changes that occur after new treatments, a biopsy would normally be required. If a treated lesion was small, this might only be possible once. Unlike surgical tissue sampling such as a biopsy, it is possible to repeat RCM on the same skin lesion or site repeatedly. This permits evaluation of dynamic processes such as response to therapy. As an example of this, RCM monitoring of the response to laser treatment of cherry angiomata has been reported (Gonzalez et al., 2001). This showed that several minutes after treatment with pulsed dye laser, blood flow within the lesions ceased and was replaced by amorphous cords of brightly refractile material. There followed an early inflammatory cell infiltrate, which gradually disappeared, and the development of new small vessels, so that by 3 weeks after treatment, normal epidermis and dermis replaced the lesion. It was also shown that the effect of a different laser, the krypton laser, was slightly different, with no amorphous cord development but only dark spaces, epidermal necrosis as an early event, and healing and normalization of architecture seen by 4 weeks after treatment. There are ongoing RCM studies evaluating the histopathological responded of actinic keratoses treated with 5-aminolevulanic acid photodynamic therapy, as well as those of BCCs treated with imiquimod. To date, RCM has proven to be accurate in establishing the presence of BCC before treatment and responsiveness of the BCC to the treatment regimen with imiquimod (Torres et al., 2004).

15.8.7.2 Reflectance confocal microscopy of neoplastic skin lesions

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RCM characterization of neoplastic lesions is an important area for research, with the potential to aid in the noninvasive diagnosis and management of a variety of skin cancers. With the advent of newer, less invasive, or topical therapies, it is desirable to use a noninvasive diagnostic tool that can allow high resolution, accurate identification of tumor subtypes and tumor margins and response to treatment.

15.8.8 HISTOLOGICAL ASSESSMENT OF SKIN SENSITIZATION The degree of irritation was rated using a scoring system discriminating between “nonirritant,” “less irritant,” “intermediate irritant,” and “extreme irritant.” To the best of our knowledge, a full-fledged model of allergic contact dermatitis has not

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been described as yet, which is easy to understand considering the complexity required. Yet, there is an approach based on Langerhans cells integrated into the suprabasal layers of reconstructed human epidermis. Established sensitizers, namely dinitrofluorobenzene and NiSO4, induced a reduction in the number of Langerhans cells. Moreover, dendritic cells discriminate between haptens and irritants, which can be judged from the adaptation of their morphology and number (Ahaghotu et al., 2005). There was also an increase in mast cells and plasma cells at the epidermal separation from the basement membrane. According to previous reported research the skin excised at 104 h after dermal exposures are shown in Fig. 15.13. The control skin showed intact SC and epidermal layers without any disruption. Dermal exposures of benzene showed slight swelling and disruption of SC, but there was not much change in the epidermal and dermal structure. Xylene demonstrated SC swelling and disruption, granulocyte infiltration in the epidermis and epidermal separation from the basement membrane and vacuolation. At areas of epidermal–dermal separation, there was accumulation of homogeneous eosinophilic material, suggesting xylene induced local skin damage and/or inflammation and dermal exposure of trimethylbenzene (TMB) showed severe histopathological changes in

FIGURE 15.13 Effect of unocclusive dermal exposure of various aromatic chemicals on the histological changes in the skin of hairless rats: (A) control (untreated), (B) benzene, (C) xylene, (D) TMB. The skin of the exposed areas was excised from the animals at 104 h for histological studies (Ahaghotu et al., 2005).SC, stratum corneum; E, epidermis; D, dermis.

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cluding severe disruption of SC, granulocyte infiltration, and swelling of the epidermis. In the dermis area, the collagen fibers bundles were found thicker, less coarse and there were greater number of fibroblasts. The number of mast cells in the dermis increased tremendously in areas that showed severe inflammation and this suggests immune reaction induced by TMB (Souris et al., 2014; Theoharides et al., 2012). Hydrocarbon chemical exposures at periods of 1 h to several weeks in hairless rats.

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15.9.1 SKIN TUMOR DEVELOPMENT IN ANIMAL MODEL 15.9.1.1 Chemically induced skin carcinogenesis

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Chemically induced mouse skin carcinogenesis was the main animal model of cutaneous tumorigenesis (Marinescu et al., 2010; Schwarz et al., 2013; Prasad and Katiyar, 2014). This model was used for evaluating antitumor drugs, but also for understanding the nature of epithelial cancers as a multistage process (Bhatia et al., 2012). Several protocols have been developed for “two-stage” carcinogenesis in which tumor incidence, tumoral latency, multistaging and the progression of the skin cancer are studied. In the model of two-stage skin carcinogenesis, the tumor is initiated after a single sub-carcinogenic dose of 7,12-dimethylbenz[a]-anthracene (DMBA). This irreversible event leads to visible tumors only after the repeated application of a promoter agent, such as the phorbol ester, 12-O-tetra decanoylphorbol-13-acetate. Therefore, unlike one-step carcinogenesis, in two-stage carcinogenesis, the initiation and promotion phases can be noticeably separated, both functionally and temporally (Neagu et al., 2016). This distinction of phases offers a tremendous advantage when studying the effects of environmental factors and/or drugs in the different stages of tumorigenesis. Further in various literature skin tumor was induced by topical treatment with the skin carcinogen DMBA followed by croton oil. The treatment was applied topically on a shaved area. Within 6–8 weeks of treatment, a small papilloma can be observed after the treatment in mice (Gupta et al., 2011). The histopathological report of skin tumor induced by two-stage carcinogenesis suggests invasive moderately differentiated keratinizing SCC in one animal group and in other animals early invasive cell carcinoma, which comprised vacuoles and bag-like structures containing malignant squamous cells.

15.9.1.2 Xenograft model Xenograft models of human malignancy play a crucial role in the screening and characterization of candidates for new anticancer molecules. The models, which are originated from human tumor cell lines and are categorized according to the transplant site, include ectopic xenograft and orthotopic xenograft, are still utilized to characterize therapeutic efficacy and toxicity. The malignant model is modified for

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the evaluation and prediction of cancer progression (Jung et al., 2012).

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• Ectopic tumor xenografts model In general, human malignant cells are subcutaneously injected into the hind leg or back of mice (Fig. 15.14A). In an ectopic tumor xenografts model, the transplanted site is varied from the origin of the cultured cells. The ectopic model is the optimal model of cancer used for validation and measurement in oncology investigations (Choi et al., 2012; Ho et al., 2012). • Orthotopic tumor xenograft model The orthotopic model is another model for assessment of tumor sensitivity. The orthotopic tumor xenograft model is an advanced tool (Banyard et al., 2013), but is based on an immunosuppressive murine microenvironment. In the orthotopic model, the human cancer cells are transplanted into the same origin site of the tumor (Fig. 15.14B). • Metastatic cancer model Cancers that form locally by exposure to ultraviolet (UV), ionizing radiation, and carcinogens proliferate within vessels and lymph nodes by invasion, causing

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FIGURE 15.14

Various xenograft models. (A) Ectopic xenograft model. The cancer cells were subcutaneously injected into Balb/c nude mice. After approximately 2 weeks, the tumor was observed; (B) orthotopic xenograft model. Human nonsmall cell lung cancer cells (A549 cells) were injected into the thoracic cavity of Balb/c nude mice. Tumor was observed by in vivo optical imaging. Isolated lung tissue was stained and observed by microscopy; (C) metastasis model. Luciferase-expressing cancer cells were injected into the tail vein. Tumor was observed by in vivo optical imaging; (D) patient-derived tumor xenograft model. Patient-derived tumor tissues were transplanted into the SCID mouse (Jung, 2014).

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metastasis (malignant cancer) at sites that are responsive to invasion. For setting of a metastasis model, various techniques have been established and there are two categories of human xenograft models. First, in orthotopic transplantation, transplanted tumor cells give rise to the primary tumor, the tumor is isolated, and then metastasis is observed. For example, WM239 melanoma cells were transplanted into severe combined immune deficiency (SCID) mice and the primary tumor was removed after 4 weeks. Then, lung metastasis was found (Madan et al., 2010). The orthotopic model was made from prostate cancer cells (DU145), and the removed lymph node was cultured and isolated tumor cells were reinjected into mice to obtain a metastasis model (Yano et al., 2005). Another one, cancer cells were intravenously injected into nude (Fig. 15.14C) or SCID mice, where they migrated such as cancer stem cells and triggered metastasis (Cook et al., 2012). This model is produced faster than the earlier model. • Patient-derived tumor xenograft model

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Xenograft models are limited in their ability to demonstrate how a cancer patient would respond to a definite treatment. The reliable prediction of drug response in a clinical trial is required, and these models are not sufficient. In a practice to report the shortcomings of these models, a patient-derived tumor xenograft (PDTX) was developed and applied (Tentler et al., 2012; Moro et al., 2012). Because PDTX associated with transplanting cancer patient tissue directly into immunecompromised mice (Fig. 15.14D) genetic information and immunohistological markers are correlative to the patient and can be used to evaluate new anticancer drugs (Maekawa et al., 2002) and personalized chemotherapies.

15.9.2 ANTITUMOR ACTIVITY

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An evaluation of the antitumor effect was reported by various researchers according to following method (Manchun et al., 2012). In an assessment of the antitumor effect using skin tumor bearing mice after drug application through topical nanocarriers in various groups, the mean tumor volume (V) of the treated group (T) and the control group (C) is estimated by using the following equation. (15.1)

where d1=longest tumor diameter (length), d2=the diameters crossing the longest diameters at right angles (widths), and d3=height of the tumor. These are measured with a Vernier Caliper.

15.9.2.1 Assessment of tumor growth inhibition rate Evaluation of the antitumor effect is carried out according to the well-established procedure (Jun et al., 2015). In assessment of the antitumor effect (Yokoyama et al., 1990), tumor growth inhibition rate (IR) is calculated using tumor bearing mice. The mean estimated tumor volume in the treated group (T) and that in the control

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group (C) is measured.

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(15.2)

15.9.2.2 Assessment of mean survival time or increase in life span of animal Antitumor activity is also determined by comparing the mean survival time (MST) of the treated group with that of the control group. Antitumor activity (Yokoyama et al., 1991; Paudel et al., 2010) is expressed as an increase in life span (ILS).

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15.9.3 SKIN IRRITATION AND SENSITIZATION STUDY 15.9.3.1 Recommendations for a cumulative skin irritation study

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Recommended designs for skin irritation and skin sensitization studies for the comparative evaluation of topical/transdermal drug delivery systems (Chudasama et al., 2011) are as follows: A randomized, controlled, repeated skin irritation study in subjects is used to find out the irritation upon application of topical/transdermal drug delivery systems. For comparison a reference formulation may be used. Placebo (topical formulation without active drug substance) and/or high and low irritancy controls (e.g., sodium lauryl sulfate 0.1% w/v and 0.9% w/v sodium chloride) can be included as additional test arms. The study is conducted in 30 subjects for 22 days. Formulations should be applied for 23 h (±1 h) daily for 21 days to the same skin site and should be evaluated for reaction. Application of formulation should be discontinued at a site if predefined serious reactions occur at the site of repeated applications. Application at a different site may subsequently be initiated. Scoring of skin reactions should be performed. Dermal reactions should be scored on a scale that describes the amount of erythema, edema and other features indicative of irritations. The mean cumulative irritation score, the total cumulative irritation score and the number of days until sufficient irritation occurred to preclude formulation application for all the study subjects should be calculated for each test product and a statistical analysis of the comparative results should be performed.

15.9.3.2 Recommendations for a skin sensitization study (modified Draize test) A randomized, controlled study should be done for skin sensitization study (Nair et al., 2013). Test sites should be randomized among subjects. Two hundred subjects are required for the study and the duration is 6 weeks. The study is divided into

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three sequential periods:

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• Induction phase Applications of the test drug carrier should be made to the same skin sites three times weekly for 3 weeks, for a total of nine applications. Dermal reactions should be scored on a scale that describes the amount of erythema, edema and other features indicative of irritation. • Rest phase The induction phase is followed by a rest phase of 2 weeks, during which no applications are made. • Challenge phase

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The drug carrier should be applied to new skin sites for 48 h. Evaluation of skin reactions should be made at 30 min and at 24, 48, and 72 h after application. The individual daily observations should be provided, as well as a tabulation of the percentage of subjects with each grade of skin reaction. The mean cumulative irritation score and the total cumulative irritation score for all the study subjects should be calculated for each test product, and a statistical analysis of the comparative result should be performed.

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15.9.4 BIODISTRIBUTION AND PHARMACOKINETIC STUDIES

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Bioavailability is defined as the rate and extent at which a drug reaches the general circulation from an administered dosage form. While this definition is adequate for systemically acting drugs given by, for example, the oral or transdermal routes, its relevance to dermatological actives applied and targeted to local skin sites is less clear. In particular, the correlation between the availability of a topically applied drug in the skin (and its therapeutic activity at the site of action) with the resulting blood levels, has not been established, primarily owing to analytical problems. It follows that, until the relevance of systemic concentrations to those of the drug within, for example, the viable epidermis, can be shown, the evaluation of topical bioavailability must involve quantification of the target tissue itself, that is, one or more components of the skin adjacent to the application site. Although skin biopsy is a logical solution to this challenge (Ding and Wu, 2012), the approach is invasive and unacceptable for routine use, such as the need to perform repeated biopsies to characterize a DPK drug concentration versus time profile. The properties of biodistribution and pharmacokinetics play an important role in affecting and determining the efficacy and safety for the treatment with the therapeutics. Now days, several image supervised modalities have been used in biomedicine and even in clinic, including MRI, X-ray computed tomography (CT), positron emission tomography (PET), single-photon emission computed tomography (SPECT), electron microscopy, autoradiography, optical imaging and US, etc. They are all noninvasive imaging techniques and demonstrated clinical applications, and some of them are only expanded to in vivo research models as small as mice

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(Kunjachan et al., 2015). Among them, PET and optical imaging are considered as quantitative or semiquantitative imaging tools that employ radiotracers or optical tracers to image biodistribution of the labeled drugs or marker loaded delivery systems in the body, along with that CT and MRI are generally used for anatomical imaging purposes (Alhareth et al., 2012). In these imaging tools, noninvasive technique drawn more attentiveness because of its characteristics of no breaking in the skin and no contact with the mucosa, internal body cavity beyond a natural or artificial body orifice. Noninvasive in vivo molecular imaging can be obtained from PET, MR, CT, and visible infrared in vivo optical imaging systems. The ability to quantitatively measure the biodistribution of therapeutics or drug delivery systems in a noninvasive manner can facilitate in the development of new theranostic, dose establishment, and treatment monitoring. Generally, the information of biodistribution can often be acquire by dissecting the animal, collecting plasma or tissue specimens and being analyzed by HPLC, enzyme-linked immunosorbent assay, etc. (Kamath et al., 2012; Bao et al., 2004), the key advantage of noninvasive imaging is less time consuming and more cost effective of animals and analysis reagents (Wang et al., 2007). There is a certain need to establish an effective noninvasive tool to clearly diagnose diseases along with the treatment. If a high influencing noninvasive technique exists, a cancer patient could be more potentially received effective treatment at an primary stage with concise targeted drug delivery (Iyer et al., 2013).

15.9.5 TOXICITY ASSESSMENT

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Exposure to drugs and chemicals can result in toxicity manifest in specific organs, known as target organs of toxicity. Factors contributing to the susceptibility of an organ to toxicity include pharmacokinetics, metabolism of the drug or chemical, and the ability of the organ to respond to toxic insult (Pappinen et al., 2012). Typically, mechanistic toxicity testing is conducted retrospectively to understand mechanisms of action and relevance to human safety. These studies provide information applicable to early diagnosis or prevention of target toxicity in humans and may contribute to development of metabolites or analogs of the drug or chemical with the same or better efficacy but with reduced side effects.

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15.9.5.1 Skin toxicity Substances when applied to human skin might exert a sensitizing potential on the skin and need, therefore, to be evaluated and classified for their possible toxicity. Every substance that provokes immunologically mediated cutaneous reactions (i.e., skin sensitization or allergic contact dermatitis) is referred to as skin sensitizer. Several tests are recommended, but no single method is able to identify all potential substances capable of inducing sensitization of human skin (Moore et al., 2013). Widely used test methods for the investigation of skin sensitization, the so-called adjuvant and nonadjuvant tests, are described below.

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• Guinea pig maximization test The guinea pig maximization test (GPMT) is a preferred method for the detection of skin sensitizers. It belongs to the class of adjuvant-tests, where the substance will be applied in Freund’s complete adjuvant. The test is based on the possible induction of an immune response of the skin during an induction period (at least 1 week). This pretreatment of the subject will eventually result in a hypersensitive reaction during a further exposure, the so-called challenging phase. The dose used for the induction exposure is chosen in such a way that it is systemically well tolerated and causes only mild-to-moderate skin irritations. The dose during the challenging period should be the highest nonirritating dose. Both doses need to be determined in preliminary tests, in case no other information on the test substances is available. The test is started with an intradermal and/or epidermal application of the test substance, using the induction dose on young adult guinea pigs of either gender. • Buehler test The Buehler test is the preferred nonadjuvant test method, even though it might be not as accurate as other tests. Like the GPMT, the Buehler test consists of two successive phases, the induction treatment followed by the challenging exposure. The induction dose needs to be chosen in such a way that it is high enough to cause mild irritation on the skin, while the challenging dose should equal the highest nonirritating concentration of the investigated substance. Both doses have to be determined in a pilot study. Guinea pigs of either sex can be used. If female animals are used, these have to be nonpregnant and nulliparous. • Local lymph node assay The murine local lymph node assay (LLNA) was developed as an alternative to the GPMT. Even though it cannot fully replace the GPMT, fewer animals are necessary to perform the test. The LLNA enables the hazard classification of substances that induce allergic contact dermatitis, while offering animal welfare advantages, compared to the GPMT (elimination of pain and reduction in animal numbers required). Furthermore, the LLNA allows assigning substances into specific potency categories (classes 1–3). The LLNA has recently been accepted by the SCCNFP and is published as OECD test guideline (Jones and Grainger, 2009) (TG) 429 updated (2002). The test method is based on the fact that sensitizers induce a proliferation of lymphocytes in the lymph node draining the site of substance administration. The increased proliferation is proportional to the applied dose of the chemical and the potency of the allergen. Consequently, the murine LLNA assesses proliferation in a dose–response manner, comparing it to the proliferation in a control group. The ratio of the proliferation after sensitizer application to the control group defines the Stimulation Index. • Rat skin transcutaneous electrical resistance (TER) The rat skin TER assay enables to reliably discriminate between skin corrosives and noncorrosive substances. The assay relies on the change in the bioelectrical properties of the skin in response to the application of test chemicals. For the

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15.9 In Vivo Characterization

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measurements, small discs of rat skin are necessary onto which the substances are applied to the epidermal surface for up to 24 h. • Human skin model assay The human skin model assay involves measuring the effects of corrosives on viable cells in a reconstituted human skin equivalent. To be accepted as a valid human skin model, several criteria must be met. The artificial skin must comprise a functional SC with an underlying layer of viable cells. Furthermore, the barrier function of the SC, as well as the viability of the epidermis, must be verified with appropriate experimental set-ups. The chemicals to be tested are applied up to 4 h as a liquid or a wet powder onto the skin model. Afterward, careful washing has to be performed, followed by investigation of the cell viability (e.g., with a MTT reduction assay). The human skin model assay can provide further data on the degree of corrosiveness and allows ranking corrosives among each other. It is, therefore, accepted as a replacement method of animal tests for skin corrosion in the EU. • 3T3-Neutral red uptake

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This test enables to identify compounds which show a phototoxic effect in vivo [285]. It allows inter alia assessing the photo irritancy potential of UV filter candidates after topical application and distribution to the skin (Maryam et al., 2010). The 3T3-neutral red uptake test, however, is unable to predict other adverse effects that might result from the combined interaction of chemicals with light. The test is based on an in vitro assay of the uptake of the dye, neutral red (NR), in Balb/c 3T3 fibroblasts. It was developed to detect the phototoxicity induced by the combined interaction of the test substance and light of the wavelength range from 315 to 400 nm, the so-called UVA. The cytotoxicity is evaluated in the presence (+UVA) or absence (−UVA) of UVA light exposure, after application of a nontoxic dose of the compound. The cytotoxicological impact is accessed via the inhibition of the fibroblasts to take up the vital dye NR (NR is a weak cationic dye, penetrating easily into the cell membrane by a nonionic diffusion and accumulates in the lysosomes) 1 day after the initial treatment.

15.9.5.2 Hepatotoxicity

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The hepatotoxins induce a broad variety of clinical and histopathological indicators of hepatic injury. Hepatic injury can be observed by specific biochemical markers such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and bilirubin. Elevations in serum enzyme levels are consider as the relevant indicators of hepatic toxicity whereas increases in both total and conjugated bilirubin levels are indications of overall liver function. An elevation in transaminase biochemical levels in conjunction with an increase in bilirubin level to more than double its normal upper level is considered as an inauspicious marker for liver toxicity (Kalra et al., 2007). Macroscopic and in particular histopathological observations and determination of additional clinical biochemistry parameters allows assurance of hepatotoxicity. Hepatotoxicity can be charac

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terized into two main categories, each with a different mechanism of injury: hepatocellular and cholestatic (ICH 2010). Hepatocellular or cytolytic injury associated with predominantly initial increase in serum aminotransferase level, usually preceding increases in total bilirubin levels and modest elevation in ALP levels. Such injury is associated with drugs such as acetaminophen, allopurinol, amiodarone, diclofenac, isoniazid, ketoconazole, methotrexate, nevirapine, nonsteroidal antiinflammatory drugs, pyrazinamide, rifampicin, retonavir, statins, tetracyclines, trazodone, troglitazone, and valproic acid (Bjornsson and Hoofnagle, 2016). Cholestatic injury is characterized by predominantly initial ALP level elevations that precede or are relatively more dominant than increases in the levels of serum aminotransferases. Such injury is related with amoxicillin clavulanic acid, anabolic steroids, chlorpromazine, erythromycins, estrogens, phenothiazines, or tricyclics (Agarwal et al., 2010). Generally mixed type of injuries, involving both hepatocellular and cholestatic mechanisms (Karie et al., 2010). Azathioprine, captopril, clindamycin, ibuprofen, nitrofurantoin, phenobarbital, phenytoin, sulfonamides, and verapamil are associated with causing mixed pattern liver injury (Agarwal et al., 2010; Karie et al., 2010). The ratio ALT:ALP plays a crucial role in deciding the category of liver damage by hepatotoxins. The ratio is more than or equal to 5 during hepatocellular damage whereas the ratio is less than or equal to 2 during cholestatic liver damage. During mixed type of liver damage, the ratio ranges between 2 and 5. ALT and AST or in combination with total bilirubin are primarily considered for the determination of hepatocellular injury in rodents and nonrodents in nonclinical investigations. ALT is reported a more specific and sensitive biochemical parameter of hepatocellular injury than AST.

15.9.5.3 Nephrotoxicity

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Nephrotoxicity defined as a renal disease or dysfunction, is often caused by drugs, chemicals, industrial, or environmental toxic agents. Drug-induced renal dysfunction is frequent in clinical practice. Kidneys are particularly vulnerable to drug toxicity because they are highly vascularized and play a major role in metabolism and elimination of toxicants (Karie et al., 2010). There are various methods in assessing drug renal toxicity after drug application. However, utilization of classical markers of kidney injury (proteinuria, plasma creatinine, and blood urea nitrogen (BUN)) in nephrotoxicological studies is limited. Indeed, because of the great ability of the kidneys to compensate renal mass loss and to recover after acute insult, the sensitivity of serum creatinine and BUN is very poor. It has been observed that a reduction of renal functionality occurs only after approximately two-thirds of renal biomass has been lost. In addition, these markers lack specificity. Indeed, the serum level of creatinine, a breakdown product of muscle tissue, depends on age, gender, muscle mass, and weight. It has also been observed that gastrointestinal bleeding or enhanced protein catabolism and other pathologic situations can lead to a rise in serum BUN without any negative effect on the kidneys.

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Short-duration studies measuring interactions with genetic material (DNA and chromosomes) are widely applicable to scrutinized molecules for the potential to cause cancer or heritable mutations. Most of these investigations include the use of in vitro assays for mutation in bacteria or isolated mammalian cells that have been shown to predict the potential for a component to be carcinogenic or mutagenic through interaction with DNA. Animal studies, usually in the mice, are used only when one or more of these in vitro tests have given a positive response, and with the aim of demonstrating that the chemical can or cannot target a sensitive tissue and cause genetic changes in the intact animal. In practice, very few molecules that have been considered to be mutagenic in vitro are tested any further in animals. However, in the case of therapeutics, regulatory requirements demand that an in vivo test be completed before the start of Phase II clinical studies in humans. In vivo tests include the rodent bone marrow micronucleus test (Hendriks et al., 2011), which is an early indicator of carcinogenic response. A single topical application of molecule can be applied to rats or mice which are sacrificed either 24 or 48 h later for examination of chromosomal changes in bone marrow cells. It is predicted that the highest dose level used will show evidence of adverse events if the molecule is genotoxic, and the mean tolerated dose is generally used to set this dose level.

15.10 CONCLUSION

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The permeation of components into the skin through the dried SC is a major obstacle to the delivery of high concentrations of anticancer drugs into tumor cells. Nanocarriers appear to be promising systems because they offer several advantages, such as low skin irritation and increased protection of encapsulated drug. An especially important advantage of these formulations is that they often increase anticancer drug penetration through the skin. Various strategies have been employed over the last few decades to optimize the topical nanocarriers for the skin cancer treatment. Approaches based on lipid-based system, polymeric system, prodrugs, and ion pairing have been used with success to improve the topical and site-specific delivery of anticancer drugs. Further, topical drug delivery reduces the burden of systemic therapies to patients and optimizes localization of drugs to deep skin strata for the treatment of skin cited malignancies in future.

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