Allergen-specific subcutaneous immunotherapy in allergic asthma: immunologic mechanisms and improvement

Abstract

Allergic asthma is a disease characterized by persistent allergen-driven airway inflammation, remodeling, and airway hyperresponsiveness. CD4⁺ T-cells, especially T-helper type 2 cells, play a critical role in orchestrating the disease process through the release of the cytokines IL-4, IL-5, and IL-13. Allergen-specific immunotherapy (SIT) is currently the only treatment with a long-term effect via modifying the natural course of allergy by interfering with the underlying immunological mechanisms. However, although SIT is effective in allergic rhinitis and insect venom allergy, in allergic asthma it seldom results in complete alleviation of the symptoms. Improvement of SIT is needed to enhance its efficacy in asthmatic patients. Herein, the immunoregulatory mechanisms underlying the beneficial effects of SIT are discussed with the ultimate aim to improve its treatment efficacy.

• allergic asthma
• immunotherapy
• dendritic cell
• regulatory T cells
• Th2 lymphocytes
• hyperresponsiveness
• eosinophilia
• IgE
• IL-10

Allergic asthma is recognized as a chronic inflammatory disease of the airways in which many types of cells play a role, in particular mast cells, eosinophils, B- and T-lymphocytes, and epithelial cells. It is characterized by the production of allergen-specific immunoglobulin (Ig)E antibodies, reversible airflow obstruction, airway hyperresponsiveness (AHR) to a wide variety of specific or non-specific stimuli, chronic airway inflammation, and airway remodeling 1. During the past several decades, the prevalence of allergic asthma is increasing dramatically (> 20% in every 10 years), especially in children 2. Approximately, it affects 10–15% of the population in most developed industrial countries 2. Both genetic and environmental factors are involved in allergic asthma. Genetic studies indicate that multiple genes are linked to the onset of this disease 345. However, the epidemic increase of asthma cannot be explained by genetic changes in the population. Changes in environment and lifestyle including isolation of houses which increases numbers of house dust mite, and childhood immunizations have contributed to the increase 6789. In addition, epidemiological and clinical studies have suggested a link between the relative decreased exposure to bacteria or other pathogenic agents (such as helminthes) during the first years of life, and the increase in allergic asthma 101112; this is referred to as the ‘hygiene hypothesis.’

Pathogenesis of asthma

The concepts of the pathogenesis of allergic asthma are illustrated in Fig. 1. The development of allergic asthma requires sensitization to an environmental allergen, which can occurs years before the onset of clinical symptoms 613. For allergic sensitization allergens are taken up and processed by professional antigen-presenting cells (APCs), such as dendritic cells (DCs) and presented to allergen-specific T-cells that subsequently differentiate into T-helper (Th) 2-type cells. Activation of Th2 cells leads to the production of inflammatory cytokines, such as interleukin 4 (IL-4) and IL-13, thereby stimulating allergen-specific B-cells to proliferate and switch to production of IgE 1415. Then IgE binds to high-affinity IgE receptor FcɛRI on mast cells. Upon re-exposure, the allergen will bind to allergen-specific IgE on mast cells thereby cross-linking the IgE–FcɛRI complexes which subsequently cause the release of preformed mediators like histamine and several other inflammatory mediators 1617. These mast cell-derived mediators cause the early asthmatic reaction which is characterized by airway smooth muscle constriction, extensive vascular leakage, mucus hypersecretion, enhanced airway responsiveness, and recruitment of inflammatory cells 171819.

Fig. 1.  Schematic representation of the pathophysiology of allergic asthma. In genetically predisposed individuals, primary exposure to an allergen leads to activation of Th2 lymphocytes and stimulation of IgE synthesis. Later on, exposure causes immediate release of the biologically active mediators (histamine, leukotrienes) via mast cell degranulation and further activation of Th2 cells with resulting eosinophil inflammation and AHR.

In approximately 50% of the asthmatic patients this early phase reaction is followed by the late phase reaction. This phase is characterized by significant involvement of infiltrated inflammatory cells, such as eosinophils, T lymphocytes and macrophages, and resident epithelial, endothelial and smooth muscle cells in promoting the chronic symptoms of airway inflammation. In addition, these cells may also be an important source of inflammatory mediators like chemokines, cytokines, and leukotrienes in asthma.

Asthma: a CD4⁺ T-helper type 2 (Th2)-mediated disease

CD4⁺ T-cells play a crucial role in controlling inflammation in allergic asthma. They are the predominant lymphocyte population that infiltrates the airways in asthmatics and are activated in these sites, expressing the surface activation markers class II histocompatibility antigen [HLA-DR], CD25 (IL-2R), and very late activation antigen-1 (VLA-1) 2021. In addition to CD4⁺ T-cells, CD8⁺ T-cells, and γ/δ T-cells have been identified in the airways of allergic asthmatics 21. About 60% of the CD4⁺ T-cells in the airway of persons with asthma are invariant natural killer T (iNKT) cells 22232425. However, there is still controversy whether iNKT-cells can induce allergic asthma in the absence of CD4⁺ T-cells 262728.

Human and murine CD4⁺ T-cells are divided into two broad functional subsets according to their profiles of cytokine secretion 2930. The conditions under which CD4⁺ Th cells become activated during an immune response determine the development toward the Th1- or the Th2-phenotype 31. CD4⁺ Th1-type cells secrete predominantly IL-2 and interferon gamma (IFN-γ) and are particularly implicated in cell-mediated immune responses against invading intracellular pathogens, such as viruses 3233. CD4⁺ Th2-type cells produce a different panel of cytokines including IL-4, IL-13, and IL-5 34. IL-4 promotes the development of Th2 cells and together with other cytokines, promotes the growth of mast cells, basophils, and eosinophils 3536. Furthermore, IL-4 and IL-13 are required for the induction of proliferation and isotype switching to IgE by B-cells that recognize the allergen 1437. IL-5 secretion by Th2 cells is critical for eosinophil differentiation and maturation 3839.

Over the years it became clear that the allergen-induced airway inflammation is orchestrated by activated Th2 cells. Th2-type lymphocytes play a critical role in the initiation, progression, and persistence of allergic asthma. Initially, a disturbed balance between Th1- and Th2-mediated immune responses has been postulated to underlie aberrant Th2 reactions to ‘innocuous’ environmental antigens. Indeed, allergen-specific T-cell clones isolated from the blood of allergic individuals express a Th2 cytokine profile secreting IL-4, IL-5, and minimal IFN-γ and IL-2, whereas those clones from non-atopic individuals displayed a Th1 profile 40. Furthermore, allergic asthma is associated with the expression of IL-3, IL-4, and IL-5 in bronchoalveolar cells, strongly supporting Th2 activation 41. Moreover, it has been shown that when antigen-specific Th2 cells activated with inhaled antigen, Th2 cells-induced airway eosinophilia, mucus hypersecretion, and AHR after short-exposure to antigen, while Th1 cells resulted in a neutrophil-predominant inflammatory response without mucus production or AHR 4243, indicating that Th1 cells alone do not produce any of the characteristic features of asthma. In human studies, CD4⁺ T-cells producing IL-4, IL-5, and IL-13 have been identified in bronchoalveolar lavage fluid and airway biopsies from asthmatics patients. These cytokines are secreted in the airways of patients with mild or asymptomatic disease 41. Furthermore, it has been shown that CD4⁺ Th2 lymphocytes are increased in the airways of asthmatic patients after antigen challenge 214445.

Allergen-specific immunotherapy (SIT)

Current pharmacologic therapies for allergic asthma, such as bronchodilators and inhaled corticosteroids, are effective in reducing and preventing symptom development, but do not reverse the progression of or cure this disease. SIT is unique in that it not only reduces symptoms, but also induces long-lasting disease remission. SIT has been shown to improve allergic dysfunction and to re-direct the immune system away from the allergic response.

SIT was first introduced at St. Mary's Hospital London, at the end of the nineteenth century and many of the basic principles described remain valid today. In 1911, Noon carried out the first study of active immunization to prevent allergy against grass pollen using s.c. injection of a distilled water extract of the pollen of timothy grass, Phleum pretense 46. In 1918, it was generally accepted that hay fever, asthma, and anaphylaxis were the result from antibodies produced after exposure to sensitizing antigen 47.

By 1961, a new technique was developed to allow in vitro measurements of histamine release from cells in whole blood in the presence of specific allergen before and after treatment with ragweed extract 48. The findings demonstrated that after SIT, histamine release was completely abolished in a few patients and was reduced in others. Five years later, a double-blind study performed by Lichtenstein and Osler 49 demonstrated that the treatment with crude ragweed extract or the major allergen of ragweed ‘Amb a 1’ resulted in reduction of cellular sensitivity in some patients. Treated patients showed little correlation between cellular sensitivity and symptom scores. This finding was accompanied with a rise in blocking antibodies. After these results, the term immunotherapy has been used to describe the process because it greatly deals with complex immunologic changes.

Specific immunotherapy (SIT) for allergic asthma

SIT for the management of allergic asthma continues to be a matter for discussion 50515253. Abramson and colleagues 515455 performed a meta-analysis of all trials published over the past 50 years of the twentieth century examining the impact of SIT in patients with allergic asthma. Overall, SIT was efficacious of decreasing asthma medication use, reducing bronchial hyperresponsiveness, and improving asthma symptom scores. While there was no consistent effect on lung function, SIT significantly reduced the airways response to inhalation of specific allergen, with some reduction in non-specific AHR as well.

Mechanisms involved in specific immunotherapy (SIT)

The potential mechanisms that have been proposed to explain the beneficial effects of SIT are illustrated in Fig. 2.

Fig. 2.  Schematic representation of the potential immune deviation leading to the beneficial effects of SIT. Allergen SIT results in both a shift in allergen-specific T-cells from Th2 to Th0/Th1 responses and the generation of IL-10 and TGF-β producing T regulatory (Treg) cells. Allergen-specific Th1 immune responses protect against the development of allergic disorders by inducing the production of IFN-γ, which inhibits the development of Th2 cells. The regulatory cytokines IL-10 and TGF-β induce switching of B cell responses in favor of IgG4 antibodies and IgA antibodies, respectively, and suppress IgE production. IL-10 and TGF-β directly or indirectly suppress effector cells of allergic inflammation such as mast cells and eosinophils thereby preventing release of mediators and late-phase inflammation. Sold gray arrows represent immune response pathway to natural exposure; dotted arrows represent immune response pathway to IT; blocked lines represent inhibition.

To explain the immunological mechanisms underlying the clinical improvement, intensive research has concentrated upon the specific antibody response in serum with respect to class and subclass distribution 56. Studies showed that the allergen-specific IgE levels rise temporarily during initial phase of SIT, but fall back to pre-treatment levels during maintenance therapy 57. Subsequently, it was demonstrated that SIT also induces allergen-specific IgG (mostly IgG1 and IgG4) and in few reported cases IgA levels 585960. These findings led to the hypotheses that these IgG antibodies contribute to the immunosuppressive effects of SIT by blocking IgE-facilitated antigen presentation 6162 and that such antibodies act as blocking antibodies by engaging low-affinity Fc receptors for Ig (e.g. FcγRII) expressed by B lymphocytes, basophils, and mast cells 60. In fact, a substantial number of studies demonstrated increased specific IgG4 levels together with clinical improvement 6364. However, the role of IgG (and IgA) in SIT has been questioned and it is not clear whether the significant increase in IgG levels has a causal role in alleviating symptoms or simply represents a bystander effect, occurring as a consequence of high allergen exposure. In Fcγ knockout mouse model of allergic asthma we found that bronchoalveolar lavage (BAL) eosinophilia, Th2 cytokines, and serum ovalbumin (OVA)-specific IgE levels were strongly suppressed by SIT, demonstrating that IgG Fcγ receptors have no major role in the beneficial effects of SIT (unpublished data). Moreover, results demonstrated that the suppression of allergen-induced airway inflammation by SIT is not B-cell dependent, ruling out a critical role for B-cells, IgGs, and IgA in SIT in the mouse model of allergic asthma.

Thus, in the context of allergic diseases, including asthma, most research indicates that during SIT the increase of IgG is an epiphenomenon, however, the role of allergen-specific antibodies, as putative ‘blocking factors(s),’ in SIT providing protection against allergic disease appears unlikely.

T-cell tolerance

The generation of an effective immune response involves antigen-specific T-cell expansion and differentiation of effector function. T-cell activation requires at least two distinct signals, including signaling via the antigen-specific T-cell receptor and a co-stimulatory pathway 31. Antigen stimulation of T-cells can lead either to a positive immune response, characterized by proliferation, differentiation, clonal expansion, and effector function, or in the absence of an appropriate co-stimulatory signal to a state of long-lasting unresponsiveness, termed anergy 65. The most important co-stimulatory molecules involved are CD28/CD152 (CTLA-4)–CD80/CD86 (B7-1/-2) and CD40-CD154 (CD40 ligand) 666768. Anergy induction can down-regulate both cellular and humoral immune responses. The induction of T-cell non-responsiveness or anergy as the mechanism of SIT is supported by the observed diminution of allergen-specific T-cell proliferation and cytokine production 69, although in some instances IL-2 was able to restore these functions 70. However, the usually increased IgG production during SIT cannot be explained by the induction of T-cell anergy 7172. Various methods for anergy induction have been described. Stimulation of T-cells with high doses of antigen in the absence of appropriate co-stimulation or in the presence of IL-10 has been found to induce a profound form of anergy 7374. Under these conditions anergic T-cells showed limited peripheral expansion and a significant amount of death, with a residual subset of cells remaining that were unresponsive to antigen restimulation both in vivo and in vitro. It has been suggested that T-cells rendered anergic in vivo and in vitro become regulatory T-cells that can regulate other T-cell responses 7576.

Immune deviation toward T-helper type 1 (Th1) responses

In the late 1980s, a favorable explanation was based on the Th1/Th2 dichotomy in specific immune reactions 29. Mechanistic studies demonstrated that SIT alters the balance of cytokines released from Th-lymphocytes with a shift from Th2-cells in association with allergic inflammation toward Th1-cells that release IFN-γ, which inhibits Th2-cells 777879. Early studies showed that allergen-specific T-cell clones shift from IL-4 to IFN-γ production after SIT 7780. However, the effects of SIT on Th1 cytokine secretion are less consistent 81. In humans, several clinical trials demonstrated a reduction of the allergen-specific Th2 response, whereas the Th1 response remained undetectable 7882 and in a separate study, SIT diminished both allergen-specific Th2 and Th1 responses 83. In experimental allergic asthma models some studies have confirmed that the relation between presence of Th1-associated cytokines and amelioration of disease, but others have not found such effects 84. In recent years, the concept of immune deviation toward Th1 response by SIT lost favor with the alternative observation on regulatory T-cells.

Adaptive regulatory T (Treg) cells are taking the center stage as crucial immunoregulatory cells that may offer an explanation for many of the observations that occur during SIT that cannot be adequately explained by the Th2 to Th1 shift 858687.

Regulatory T-cells

Several subsets of regulatory T-cells with distinct phenotypes and mechanism of actions have been identified. These include: 1 naturally occurring CD4⁺CD25⁺Foxp3⁺ T-cells (nTregs) that inhibit immune responses through cell-to-cell contact, 2 CD4⁺Foxp3⁺ iTregs induced in the periphery, in contrast to nTregs not generated in the thymus; the capacity of these cells to suppress the proliferation of naïve T-cells requires cell–cell contact, and 3 cells induced in the periphery following antigen exposure (aTreg cells). aTreg cells are subdivided into type 1 regulatory T (Tr1) cells which secrete high levels of IL-10 and low to moderate levels of transforming growth factor (TGF)-β and type 3 regulatory T (Th3) cells which secrete TGF-β.

Natural CD4⁺CD25⁺Foxp3⁺ T regulatory cells

The natural CD4⁺CD25⁺Foxp3⁺ regulatory T (nTreg) cells are generated in the thymus and represent 5–10% of the CD4⁺ T lymphocytes both in mice and humans. These cells are thought to perform a specialized role in controlling both the innate and the adaptive immune response 8889. Depletion of nTreg cells leads to the spontaneous development of various autoimmune diseases 90919293949596. Although the mechanism of immune regulation mediated by nTreg cells is not well understood, these cells can both directly suppress responding T-cells and down-modulate APC function in vitro 9798. nTreg cells may also convert CD4⁺ Th cell into regulatory cells expressing IL-10 and/or TGF-β in culture systems 99100. It has been shown that nTreg cells induce infectious tolerance in vivo by catalyzing the formation of IL-10 producing regulatory T-cells 101.

Adaptive T regulatory cells

In recent years, it has been demonstrated that SIT is associated with the induction of aTreg cells. The aTreg cells are generated in the periphery from naïve T-cells after encountering antigens presented by tolerogenic DCs. In studies of SIT for bee venom anaphylaxis, Akdis et al. 85 were the first to provide evidence for a role of IL-10 and Tr1 cells in the beneficial effects of SIT. They observed that the therapy-induced increase in secretion of IL-10 in T-cell cultures along with decreased allergen-driven proliferation and decreased production of Th2 and Th1 cytokines. Moreover, in clinical trials, SIT has been shown to increase the production of IL-10 by APCs, including B cells, monocytes, and macrophages 6187102. Findings were extended by SIT studies with airborne allergens that showed increases in IL-10 and TGF-β producing antigen-specific Treg cells in the blood and airway tissue 61858687. Induction of IL-10 producing Tr1 cells has been shown to be associated with successful SIT 8586. Animal studies confirmed the suppressive role of IL-10 during allergic inflammatory diseases 84103104105106. In human studies, the regulatory cytokine IL-10 has been shown to increase the production of IgG4 while preventing IL-4-mediated class switching to IgE 107. Furthermore, IL-10 down-regulate IgE-dependent activation of basophils and mast cells, and decrease survival and activation of eosinophils 108. In addition, IL-10 (and TGF-β) might act on APCs. IL-10 down-regulates Major Histocompatability Complex (MHC) class II expression on monocytes and reduces their antigen presentation capacity 109. Moreover, IL-10 down-regulates the CD80 expression on DCs and macrophages 110111. IL-10 may therefore block the APC dependent CD28–CD80 interaction and subsequent co-stimulatory signaling in T-cells 112.

The other immunoregulatory cytokine associated with regulatory T-cells is TGF-β. Studies have provided evidence for increases in the amount of TGF-β-driven allergen-specific IgA following SIT, indicating other antibody classes than IgG might contribute to clinical efficacy 87. Moreover, TGF-β has been shown to induce the expression of IL-10 by T-cells 113. Literature data suggest that early increase in IL-10 85 and sustained TGF-β production may be required for successful SIT to induce stable tolerance to non-pathogenic environmental antigens 114.

Role of dendritic cells (DCs) in generation of adaptive regulatory T (aTreg) cells

Although there is suggestive evidence that SIT achieves beneficial effectiveness by the induction of aTreg cells, the mechanism by which SIT induces aTreg cells is not completely understood. Over the past decade, it has become clear that DCs play a critical role in the generation of all aTreg cell subsets 115 and regulation of immune responses to a variety of antigens 116. The ability of DCs to induce immunity or tolerance depends on their maturation state. Tolerogenic DCs are semi-mature cells with increased expression of MHC II and CD86 but low levels of expression of CD40 and absence of the pro-inflammatory cytokines IL-6 and TNF-α 117. It has been shown that aTreg cells induced by immature DCs are characterized by high levels of IL-10 cytokine secretion 118119. The nuclear factor-κB (NF-κB) protein Re1B activity is critically required for DCs maturation 120. Re1B-deficient bone marrow-derived DCs (BMDCs) or BMDCs in which Re1B activity is inhibited have the potential to induce antigen-specific immune tolerance in vivo 121. Inhibition of NF-κB signaling by various drugs 122123124 induces DCs to acquire tolerogenic properties that favor the induction of Tr1-like cells in vitro and tolerance in mouse models of transplantation and autoimmune diseases 125126127.

Besides this, recent findings demonstrate that DCs induce development of aTreg cells by several mechanisms including high expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) 128129. IDO expressing DCs inhibit T-cell proliferation in vitro and promote tolerance in vivo 130131, including maternal tolerance during pregnancy 132, control of allograft rejection 133, and protection against autoimmunity 134. A number of studies have also demonstrated that IDO is implicated in tolerance induction by regulatory T-cells expressing cytotoxic T-lymphocyte antigen-4 (CTLA-4) 135136. Moreover, IDO may directly mediate inhibition of T-cells proliferation by tryptophan depletion or downstream tryptophan metabolites 137138. Recently, we showed that inhibition of IDO throughout SIT abrogated the efficacy of SIT on the reduction of BAL eosinophil numbers and AHR to methacholine, but no changes in serum OVA-specific IgE levels were observed 139. In addition, we demonstrated that inhibition of IDO throughout the aerosol challenge period did not interfere with tolerance induction by SIT. These findings reveal that IDO plays a role only during induction of tolerance by SIT and that activity of IDO is irrelevant for the effects of SIT thereafter. Moreover, considering the conceptual possibilities that either tryptophan depletion or downstream tryptophan metabolites mediated-IDO immunoregulation we showed that formation of tryptophan metabolites, rather than tryptophan depletion, is the mechanism by which SIT induces tolerance to the induction of airway manifestations of asthma.

Improvement of specific immunotherapy (SIT)

The goal of SIT is the transformation of an allergic individual into one who can tolerate allergen exposure by converting deleterious allergic immune response into protective responses. Despite the impressive efficacy of allergen (injection) SIT for treatment of allergic rhinitis and insect venom allergy, its efficacy in allergic asthma remains controversial 51. Furthermore, it has been reported that s.c. administration of allergens can induce severe systemic reactions due to cross-linking of allergen-specific IgE on mast cells 140141. Thus, for development of a more effective and safe form of SIT, more insight into the underlying immunological mechanisms of allergen IT is considered necessary to improve efficacy, in particular in asthmatic patients. There is growing evidence that the positive effects of SIT are associated with aTreg cells (Tr1 and Th3) and the immunosuppressive cytokines that they produce. The mechanisms by which these cells are induced by SIT are still not fully understood. Well-described pathways to induce aTreg cells are antigen presentation to T-cell by immature DCs or the presence of IL-10 or TGF-β in the local microenvironment. In this regard, combination of SIT with administration of compounds that inhibit DCs maturation, such as the biologically active form of vitamin D₃ (1α,25-dihydroxyvitamin D₃) or glucocorticoids, might be novel immunotherapeutic strategies to improve SIT for better treatment of allergic diseases, including allergic asthma. Interestingly, in a mouse model of allergen immunotherapy, we recently demonstrated that co-administration of 1α,25-dihydroxyvitamin D₃ effectively suppressed AHR to methacholine and potentiated the reduction of serum allergen-specific IgE levels and BAL eosinophilia in the suboptimal SIT regime matched by a reduction in Th2 cytokines, IL-5, and IL-13 142. We further observed that in vitro antigen-induced productions of Th2-cytokines are decreased in lung-draining lymph node cells after this combination SIT. Moreover, data demonstrates that the immunoregulatory cytokines IL-10 and TGF-β are involved in tolerance to allergen-induced airway manifestation of asthma since the suppressive effects of combined SIT are completely abrogated by blocking of the IL-10R and neutralizing of TGF-β by using mAbs at the time of antigen inhalation challenge.

Conclusion

Peripheral (naturally occurring and adaptive) T- cell tolerance is the key immunological mechanism in healthy immune response to self- and non-infectious non-self-antigen. Changes in the balance between allergen-specific Treg and Th2, and/or Th1 cells are very crucial in the development and also treatment of allergic diseases. Understanding of the immunological mechanisms that lead to tolerance induction by SIT, Fig. 3, in particular the role of regulatory T-cells in allergen-specific peripheral tolerance may lead to more rational and safer approaches that could result in prevention and cure of allergic diseases, including asthma.

Fig. 3.  Hypothetical scheme of the proposed mechanisms contributing to the induction of tolerance by SIT. (A) Natural exposure to an allergen leads to activation of Th2 cells. (B) nTreg cells are generated in thymus, but can also develop from conventional CD4⁺ T-cells by specific condition or signals. nTreg cells induce IDO expression on dendritic cell (IDO⁺DC). This is partially mediated via the interaction of CTLA-4 expressed on nTreg and CD80 (B7) expressed on DC. (C) IDO catalyzes the initial and rate-limiting step of tryptophan degradation. (D) IDO expressing DC induces development of Treg after allergen IT by a mechanism at present not completely defined. (E) 1,25(OH)₂D₃ inhibits transcription factor NF-κB thereby inhibiting maturation of DC and resulting in tolerogenic DC, which direct the induction of Treg cells. Treg cells suppressed Th2 responses and effector cells by the release of immunoregulatory cytokines IL-10 and TGF-β. (F) The role of allergen-specific IgA is unclear. Dotted arrows represent immune response pathway to SIT; blocked lines represent inhibition.

Conflict of interest and funding

Y.A.T was supported by a grant 965.02 from the Libyan Ministry of Higher Education

Acknowledgements

This study was partly financially supported by the Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.