Macrolide immunosuppressants

ARTICLE

Macrolides have been known for decades to physicians as antibiotics of preference for use with children and for a number of special indications such as legionellosis and mycoplasma infections.

During a screening program for naturally occuring immunosuppressants, in 1984, Fujisawa Pharmaceutical Company isolated a 822 kDa macrolide compound with potent T cell inhibitory activity from the fermentation broth of Streptomyces tsukubaensis, which was subsequently termed FK506 [1]. Later on the name of the substance was changed to the INN tacrolimus, an acronym for Tsukuba macrolide immunosuppressant. The structural formulas of tacrolimus as the first member of the group of macrolide immunosuppressants and rapamycin are displayed in Figure 1.

The mechanism of action of tacrolimus is similar to that of the first immunosuppressant of fungal origin, cyclosporin A [2]. Both cyclosporin A and tacrolimus are used worldwide for the prevention of transplant rejection. In dermatology, cyclosporin A was shown to be highly effective in the treatment of severe psoriasis and atopic dermatitis as well as in a number of other indications such as pyoderma gangrenosum and generalised lichen planus.

A major disadvantage in the clinical use of cyclosporin A for the treatment of dermatological disorders is its lack of topical effectiveness [3]. With the exception of the ulcerative phase of pyoderma gangrenosum and erosive lichen planus of the mucosa, topical treatment with cyclosporin A fails to improve dermatoses such as psoriasis and atopic dermatitis where the systemic delivery is highly effective [4, 5].

Tacrolimus, being a macrolide compound, was found however, to be efficacious by topical application in animal and human models of contact dermatitis as well as for atopic dermatitis.

Recently new macrolide immunosuppressants for topical use have been developed which share the T cell-inhibitory activity of tacrolimus and cyclosporin A.

These compounds are produced by different strains of Streptomyces (Table I). For future dermatotherapy the ascomycins seem to be the most interesting group of new macrolide immunosuppressants [6, 7].

This review aims to summarize the mechanism of action of the new class of immunosuppressive macrolides and to describe the clinical and experimental experience obtained so far.

Mechanism of action

The mechanisms of action of macrolide immunosuppressants have been thoroughly investigated and characterized. The pharmacological pathways leading to an inhibition of T cell activation has been elucidated in great detail (Fig. 2).

a: cyclosporin A, tacrolimus and ascomycin

After cellular uptake macrolide immunosuppressants are bound to cytosolic proteins called immunophilins. A number of different immunophilins have been identified including cyclophilin binding cyclosporin A and the group of FK-binding proteins for the macrolide compounds. FK-binding protein 12 (FKBP-12), also called macrophilin 12, is most important for binding tacrolimus as well as ascomycins [8].

The major effects of macrolide immunosuppressants are mediated through the inhibition of the cytosolic phosphatase calcineurin [9]. The binding of the macrolide/immunophilin-complex to calcineurin has been attributed to as "molecular glue" and leads to the inactivation of phosphatase activity [10].

Calcineurin is a key enzyme regulating the translocation of cytosolic components of nuclear factors which regulate the promotor activities of a number of mediators during mRNA transcription [11]. Studies have shown that cyclosporin A -cyclophilin and macrophilin 12-tacrolimus have distinct binding targets which are highly conserved regions of calcineurin A-isoform that overlap the binding domain for the calcineurin B regulatory subunit [9, 12].

After translocation into the nucleus the cytosolic dephosphorylated component of the nuclear factors assembles with a nuclear component to form the active molecule. For macrolide immunosuppressants as well as for cyclosporin A the transcription factor "nuclear factor of activated T cells (NF-AT)" is a prominent target. NF-AT regulates mRNA transcription of a variety of mediators of which IL-2 is of primary importance as a T cell-growth and -activation factor [13, 14]. As compared to cyclosporin A, tacrolimus and the ascomycins are more potent in inhibiting IL-2 production [2].

Beside IL-2, the transcription of genes and the production of a variety of other mediators is influenced by macrolide immunosuppressants as recently reviewed [8].

NF-AT not only mediates cytokine transcription in T-lymphocytes but also in a number of non-lymphoid cells. It has recently been demonstrated that histamine could induce mRNA-expression for IL-8 and monocyte chemotactic protein 1 (MCP-1) in human umbilical vein endothelial cells (HUVEC) [15]. The cellular response was mediated via the H₁ histamine-receptor and NF-AT dependent. Tacrolimus as well as cyclosporin A potently inhibit histamine-induced HUVEC-stimulation in a dose-dependent fashion through an inhibition of NF-AT.

Another important target for the action of macrolide immunosuppressants are mast cells. Mast cells play a prominent role in allergic diseases such as atopic dermatitis as well as for psoriasis where these cells are increased in number and found to be activated in the early development of psoriatic lesions [16, 17].

The mechanism of mast cell inhibition seems to be dependent on the cytosolic receptor protein, as it has been shown for tacrolimus, using FKBP-12 transfection experiments in a mouse model [18]. In human tissue mast cells, tacrolimus inhibits stem cell factor (SCF)- or anti-IgE-induced histamine release with the same potency as observed for cyclosporin A [19]. Tacrolimus decreased TNFalpha and IL-6 transcripts in mouse bone-marrow-derived mast cells dose-dependently [18]. Recently, Hultsch and co-workers showed inhibition of IgE-induced serotonin and ß-hexosaminidase release by SDZ ASM 981 in RBL 2H3 mast cells [20]. It was also demonstrated that this ascomycin-derivative dose-dependently decreased TNFalpha-release with an IC₅₀ of about 100 nM. The effect of SDZ ASM 981 was mediated by an inhibition of macrophilin 12, not, however, by cyclophilin.

Beside their strong inhibitory effect on IL-2 gene transcription, macrolide immunosuppressants decrease the production of a number of other cytokines. In human T cells SDZ ASM 981 decreased production of IL-5, IL-10 and TNFalpha in a dose-dependent fashion with IL-10 secretion being similarly suppressed as IL-2 [21]. For comparison, 3-10 fold higher doses of cyclosporin A were needed to achieve similar effects.

Recently it was demonstrated that ascomycin was able to potently inhibit interleukin-8 (IL-8) production by human neutrophils [27]. It was shown that thapsigargin, a compound releasing calcium from intracellular stores and opening calcium influx pathways, induced IL-8 mRNA-transcription, production and secretion of IL-8 in these cells. Ascomycin was able to inhibit IL-8 production with a ten-fold higher potency as compared to cyclosporin A, whereas rapamycin was without effect. This effect seems to be mediated by the inhibition of calcineurin phosphatase.

b: rapamycin

In contrast to tacrolimus, the ascomycins and cyclosporin A, the macrolide immunosuppressant rapamycin acts differently on a molecular level. Rapamycin also binds to macrophilin 12, however, this complex does not bind to calcineurin [22]. Therefore rapamycin does not inhibit early T cell activation or directly reduce the synthesis of cytokines. A mammalian target protein for rapamycin (TOR, FRAP, RAFT, SEP) has been identified. Binding to the target protein seems to influence cell cycle pathways. It has been shown that rapamycin inhibits cellular proliferation by affecting G1- to S-phase transition [23]. It has also been reported that rapamycin prevents CD28-dependent down-regulation of IkappaBalpha resulting in the inhibition of the nuclear translocation of c-rel, thereby inhibiting the up-regulation of IL-2 gene transcription [24].

While the anti-proliferative capacity of cyclosporin A, tacrolimus and the ascomycins is limited not only in T cells but also in cells like keratinocytes, rapamycin reduced the proliferation of mouse bone marrow mononuclear cells about 4 fold, whereas tacrolimus and cyclosporin A were without effect [25]. In human keratinocytes, rapamycin inhibited the synthesis of proliferating cell nuclear antigen (PCNA), a cell cycle regulatory protein necessary for cells to traverse from G1- into S-phase [26].

Taken together, macrolide immunosuppressants are able to inhibit a number of pro-inflammatory cells and mediator systems with major importance for cutaneous inflammatory disorders. Therefore this group of compounds has been used for the treatment of dermatological diseases from the beginning of drug development.

Use of macrolide immunosuppressants in dermatology

Systemic application of macrolide immunosuppressants

In 1992, Jegasothy et al. [28] first reported the clinical use of a macrolide immunosuppressant in dermatology. Treating psoriasis patients systemically with tacrolimus a rapid clearing of lesions was observed, similar to the therapeutic results obtained with cyclosporin A, in several studies [29, 30].

These first data were proven by a placebo-controlled, double-blind study of the European FK 506 Multicenter Study Group in 50 patients with severe psoriasis vulgaris [31]. Patients receiving 0.05 to 0.15 mg/kg/day tacrolimus showed a 70% reduction of the psoriasis area and severety index (PASI) after 9 weeks of treatment. Diarrhea, paresthesia, insomnia, pharyngitis and headache were the most frequently noted adverse events.

Topical application of macrolide immunosuppressants

Great efforts have been made to create a galenical formulation of cyclosporin A for the topical treatment of cutaneous disorders. With the exception of disorders of the mucous membranes (i.e. lichen planus) or the ulcerative phase of pyoderma gangrenosum there was no clinical response to topically applied cyclosporin A [3].

The chemical structure of the macrolide immunosuppressants, however, allows the development of topical formulations. Recently it has been shown that tacrolimus penetrates into human cadaveric skin to a much greater extent than cyclosporin A which may be due to its higher molecular weight (cyclosporin A: 1.202 kD; tacrolimus: 0.822 kD) and its lipophilic nature [32].

In 1992 Lauerma and co-workers first reported the inhibition of allergic contact eczema by topical tacrolimus in man [33]. Pre-treatment of the skin with creams containing 0.01 to 1% tacrolimus resulted in an inhibition of subsequent dinitrobenzene (DNCB)-induced contact allergic reaction as compared to the vehicle-treated site.

These results could be confirmed in a guinea-pig model of allergic and irritant contact dermatitis for topical tacrolimus by the same group [34]. In this study topical tacrolimus showed the most suppressive effects when skin sites were pre-treated with the drug before allergen challenge and suppressed local lymph node cell accumulation during contact allergy induction.

The latter observation was investigated in great detail in a recent study using a mouse model of allergic contact dermatitis (35). The authors found that topical tacrolimus (0.01 to 1%) dose-dependently suppressed oxazolone-induced lymph node cell proliferation and expression of both Th1 (IL-2, interferon gamma) and Th2 (IL-4) cytokines. On a cellular level, expression of T cell activation markers such as CD25 and CD69 induced by oxazolone was down-regulated by topical tacrolimus.

In another guinea-pig model of delayed-type hypersensitivity to dinitrofluorobenzene (DNFB), cyclosporin A and rapamycin (25 mg/kg/day), or tacrolimus (2.5 mg/kg/day) were given systemically at the time of DNFB challenge or several hours after [36]. Tacrolimus was also given topically (0.02 and 2%) in the same experimental setting. The results of the study showed a significant inhibition of T cell infiltration and skin reddening when given by both routes, whereas cyclosporin A only suppressed the erythema response. Rapamycin proved to be ineffective in this system. The study further investigated the effect of the three compounds on keratinocyte proliferation and found that cyclosporin A and rapamycin inhibited keratinocyte growth, however, tacrolimus did not.

Since the lack of influence of tacrolimus on keratinocyte proliferation may point towards the lack of atrophogenic potential, Reitamo and co-workers investigated the effect of tacrolimus ointment on collagen synthesis in man [37]. In a combined group of atopic dermatitis patients and healthy volunteers, 0.3% and 0.1% tacrolimus ointment, betamethasone-valerate (0.1%) and a vehicle control were randomly applied to abdominal skin under occlusion for 7 days. Peptides necessary for collagen synthesis (amino-terminal propeptides of procollagen) were quantified in suction blister fluid after ultrasound measurement of skin thickness. The data clearly indicate that only betamethasone-valerate, not, however, tacrolimus or the vehicle alone, significantly reduced collagen synthesis and reduced skin thickness.

The same results were obtained in another study using the ascomycin-derivative SDZ ASM 981 in a pig model of skin atrophy [38].

These experimental data provide evidence that topical treatment with macrolide immunosuppressants does not carry the risk of skin atrophy as is well known with corticosteroids.

Topical use of macrolide immunosuppressants in skin disorders

a: atopic dermatitis

Nakagawa et al. [39] showed for the first time that tacrolimus ointment (0.03 to 1%) induced substantial improvement of atopic dermatitis lesions with and without lichenification in an open trial involving 50 patients. Mild skin irritation was noted in one third of the patients but did not lead to drug withdrawal.

In a randomized, double-blind, placebo-controlled multicenter study Ruzicka and co-workers [40] confirmed the beneficial effect of topical tacrolimus for the short-term treatment of atopic dermatitis. It was shown that an ointment containing 0.1% tacrolimus was significantly superior to a placebo (vehicle control), and slightly more effective than a 0.03% or 0.3% tacrolimus formulation. A sensation of burning at the sites of application was the only adverse event clearly linked to the three groups receiving tacrolimus-containing ointments. In this study blood levels were found to be low with values below the detection limit of the assay in a large number of patients. Among all patients who were treated an area of approx. 800 cm², the highest blood level measured was 4.9 ng/ml in the group receiving the 0.3% tacrolimus ointment. This is clearly within the range used in transplant recipients where tacrolimus is given to achieve blood levels up to 20 ng/ml [41].

Van Leent et al. [42] conducted a randomized, double-blind, placebo-controlled study to investigate the efficacy and safety of topical SDZ ASM 981 in patients with atopic dermatitis. A twice daily application of an ointment containing 1% SDZ ASM 981 was found to be significantly superior to the vehicle control. A once daily application of the same ointment also improved the eczematous skin condition, however, the twice daily regimen was clearly more effective. In contrast to the experience with tacrolimus ointment for atopic dermatitis, patients' skin irritation such as burning sensations were not observed under SDZ ASM 981 therapy.

b: psoriasis

Although tacrolimus proved to be effective after systemic application, there are limited data about topical treatment of psoriasis with macrolide immunosuppressants. Rappersberger et al. [43] first reported the effectiveness of the experimental macrolide immunosuppressant SDZ 281-240 in psoriasis when applied under Finn-chamber occlusion. Using the same clinical approach, an ointment containing 1% SDZ ASM 981 was shown to completely resolve a psoriatic lesion after two weeks and to be comparable to clobetasol-propionate with regard to clinical efficacy [44]. In this investigation SDZ ASM 981 used in a 0.6% concentration was still significantly more effective as compared to the vehicle control.

However, an unoccluded treatment with a tacrolimus-containing ointment (0.3%) for 6 weeks twice daily did not improve psoriatic lesions as compared to the vehicle control [45]. Calcipotriol ointment was shown to be significantly superior to placebo and tacrolimus in this study.

c: pyoderma gangrenosum

Beside the main dermatological indications, severe psoriasis and atopic dermatitis, cyclosporin A proved to be very effective in the treatment of pyoderma gangrenosum either by systemic, topical or intralesional application [4, 46, 47]. A recent report demonstrated for the first time that tacrolimus ointment produced in the hospital pharmacy of the authors rapidly cleared pyoderma gangrenosum lesions. A combination of systemic cyclosporin A therapy and topical tacrolimus proved to be an effective regimen in one patient, whereas in the other complete resolution was achieved within three weeks giving tacrolimus ointment twice daily alone [48].

d: alopecia areata

Alopecia areata seems to be another interesting application of macrolide immunosuppressants in dermatology. It has been shown years ago that cyclosporin A treatment in transplant recipients and in patients with autoimmune disorders led to hypertrichosis as an adverse side effect [49]. This observation prompted investigations about the use of this drug in alopecia areata and male pattern baldness where systemic treatment with cyclosporin A was effective [50, 51]. A recent study using a rat bald model showed that topical tacrolimus was as effective as systemic cyclosporin A in inducing hair re-growth and decreasing the inflammatory infiltrate around the hair follicles [52]. Further studies in humans are needed to elucidate fully the therapeutic potential of topical macrolide immunosuppressants for diseases such as alopecia areata.

CONCLUSION

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