**10. Novel therapies and strategies**

#### **10.1. Biological agents**

#### *10.1.1. Rituximab*

Rituximab is a chimeric (human/murine) monoclonal antibody directed against CD20, a cell surface molecule specific to B cells. Although it was initially approved for use in B-cell non-Hodgkin's lymphoma, a growing number of reports have described the efficacy of rituximab for B-cell depletion in the treatment of autoimmune diseases [101]. The mode of action of rituximab in autoimmune diseases includes the removal of precursors of autoantibodyproducing plasma cells, and impairment of autoantigen presentation to CD4 cells [102]. In PV, Eming et al, show that rituximab not also causes marked reduction of anti-desmoglein 3 antibodies but also depletion of desmoglein-specific CD4 cells. In contrast, tetanus toxoidspecific CD4 cells were not affected nor were the overall number of CD4 cells [103]. Tetaus toxoid has been used in as an antigen in the assessment of memory CD4 cell responses [104]. Eming et al, speculate that this specific effect of rituximab on autoreactive rather than patho‐ gen-specific T cells is that the latter do not require CD20 B cells as antigen-presenting cells. A number of case reports and series have demonstrated the benefit on the use of rituximab in over 40 patients with either PV or PF [40],[105]-[115]. The largest case series to date comprising 42 patients, 37 with PV and 5 with PF, utilized the rheumatoid arthritis protocol where two 1g infusions of rituximab are administered 15 days apart [116]. Patients were followed for up to 5 years and 36 of the 42 patients achieved a complete response and were able to cease corti‐ costeroids within 6 months from induction therapy. Twenty patients experienced relapses with the time to relapse ranging from 8 to 64 months. Relapses were treated with rituximab (500 mg) without corticosteroids resulting in a new complete response. Importantly, no serious adverse events were observed.

PF) who were treated with two infusions of rituximab (500 mg each) at an interval of 2 weeks [121]. All 15 patients responded to therapy. Eight patients achieved complete remission in a median period of 5 weeks. Seven patients achieved partial remission in a median period of 34

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In the majority of these studies, abrogation of peripheral B cells and concomitant reduction in the level of circulating antipemphigus autoantibodies for 6 to 12 months occurs with only two to four infusions of rituximab. Interestingly, clinical remission in both PV and PF is often sustained beyond B cell recovery. The reason as provided Mouquet et al, is that the phenotype of restored B cells following rituximab treatment is that of a naïve B cell with a diverse

Accumulation of the data from case reports and series reveals that 16% of patients with PV developed the serious complications of bacterial sepsis, fatal *Pneumocystis jirovecii* pneumonia, persistent hypogammaglobulinemia or pulmonary embolism [123]. Concerns have also been raised regarding the role of rituximab and other biological agents used in patients with other immune-mediated diseases such as rheumatoid arthritis developing Progressive Multifocal Leukoencephalopathy (PML) [124]. PML is an inevitably fatal demyelinating disease of the central nervous system, that occurs almost exclusively in immunosuppressed individuals due to reactivation of the polyomavirus JC (JCV) [125]. This is in contrast to rarity of adverse events observe in patients treated for non-Hodgkin's lymphoma [126]. Although, the question has been raised of the use of rituximab as a first line agent due to the impressive rates of remission [127], the incidence of serious side effects may at present preclude its role in initial therapy. Ongoing surveillance of patients treated with rituximab for pemphigus and other autoimmune

There is enough evidence to suggest that rituximab should be the therapy of choice for patients with pemphigus who have refractory disease or contraindications to first-line immunosup‐ pressive therapy although randomized controlled trials have not been performed and as such, there is no uniform protocol on its administration. The value of adjunctive therapies such as IVIg and IA in patients treated with rituximab for pemphigus needs to be also further elucidated. However, one of the most important questions to address is whether it is cost

Studies have demonstrated that TNF released by keratinocytes plays a role in acantholysis in PV. Human keratinocytes pretreated with anti-TNF antibodies, are resistant to the acantholytic effect of anti-desmoglein 3 antibodies [128],[129]. Furthermore, TNF-deficient mice are more susceptible to blister formation after injection with anti-desmoglein 3 antibodies [128]. The role of TNF in PF has not been as extensively studied. Etanercept [130]-[133], infliximab [134],[135], and adalimumab [136], have all demonstrated benefit in a small number of patients with refractory PV. Randomized controlled trials of infliximab and etanercept in refractory PV are currently in progress. However, the effect of these agents in refractory PF has not been reported

effective and safe to administer rituximab for PV and severe PF as a first line agent.

5 weeks. Relapses (40%) were seen between 53 and 103 weeks after start of therapy.

repertoire rather than a primed autoreactive B cell [122].

diseases is required to monitor for long-term complications.

*10.1.2. TNF-antagonists*

to date.

The largest case series to date using the lymphoma protocol where rituximab is administered weekly (375 mg/m2 ) for 4 weeks induced remission in 12 of 14 patients with PV and 6 of 7 patients with PF within 3 months [117]. These patients had previously not responded to firstline immunosuppressive agents or had contraindications to corticosteroid therapy. The treatment was generally well tolerated; however, two cases were complicated by severe infection, one resulting in death. Similarly, Canchini et al, showed that the administration of rituximab, 375 mg/m2 once weekly for 4 weeks, induced remission in all 10 patients with PV and both patients with PF [118]. Another study comprising 11 patients with refractory PV, evaluated the effect of combination therapy consisting of 10 infusions of rituximab (375 mg/m2 ) and 6 infusions of intravenous immunoglobulin (IVIg) (2 g/kg body weight) admin‐ istered over a 6-month period [119]. Remission was induced in 9 patients for a period of 22 to 37 months following treatment and there were no reports of serious adverse events.

More recently, Kasperkiewicz et al reported use of rituximab in combination with immunoad‐ sorption, pulsed dexamethasone and azathioprine or mycophenolate mofetil in 23 patients. [120] IA was performed at initially 3- and later 4-week intervals until lesions healed by 90%; 1 g rituximab was given at weeks 1 and 3, and intravenous dexamethasone pulses were administered at first every 3 weeks and then at increasing intervals in addition to daily azathioprine or mycophenolate mofetil. All patients demonstrated clinical improvement within the first few weeks of therapy accompanies by a concomitant rapid fall in anti-desmo‐ glein antibody levels. However, two patients had non -atal severe adverse events; one developed *Staphylococcus aureus* sepsis from a central intravenous line followed by spinal haemorrhage and transient paraplegia, and another developed extensive herpes simplex infection

The high frequency of treatment failure with corticosteroids and first line immunosuppressive therapies has raised the issue of whether rituximab should be implemented earlier in the treatment of PV. Horvath et al conducted a study comprising 15 patients (12 with PV, 3 with PF) who were treated with two infusions of rituximab (500 mg each) at an interval of 2 weeks [121]. All 15 patients responded to therapy. Eight patients achieved complete remission in a median period of 5 weeks. Seven patients achieved partial remission in a median period of 34 5 weeks. Relapses (40%) were seen between 53 and 103 weeks after start of therapy.

In the majority of these studies, abrogation of peripheral B cells and concomitant reduction in the level of circulating antipemphigus autoantibodies for 6 to 12 months occurs with only two to four infusions of rituximab. Interestingly, clinical remission in both PV and PF is often sustained beyond B cell recovery. The reason as provided Mouquet et al, is that the phenotype of restored B cells following rituximab treatment is that of a naïve B cell with a diverse repertoire rather than a primed autoreactive B cell [122].

Accumulation of the data from case reports and series reveals that 16% of patients with PV developed the serious complications of bacterial sepsis, fatal *Pneumocystis jirovecii* pneumonia, persistent hypogammaglobulinemia or pulmonary embolism [123]. Concerns have also been raised regarding the role of rituximab and other biological agents used in patients with other immune-mediated diseases such as rheumatoid arthritis developing Progressive Multifocal Leukoencephalopathy (PML) [124]. PML is an inevitably fatal demyelinating disease of the central nervous system, that occurs almost exclusively in immunosuppressed individuals due to reactivation of the polyomavirus JC (JCV) [125]. This is in contrast to rarity of adverse events observe in patients treated for non-Hodgkin's lymphoma [126]. Although, the question has been raised of the use of rituximab as a first line agent due to the impressive rates of remission [127], the incidence of serious side effects may at present preclude its role in initial therapy. Ongoing surveillance of patients treated with rituximab for pemphigus and other autoimmune diseases is required to monitor for long-term complications.

There is enough evidence to suggest that rituximab should be the therapy of choice for patients with pemphigus who have refractory disease or contraindications to first-line immunosup‐ pressive therapy although randomized controlled trials have not been performed and as such, there is no uniform protocol on its administration. The value of adjunctive therapies such as IVIg and IA in patients treated with rituximab for pemphigus needs to be also further elucidated. However, one of the most important questions to address is whether it is cost effective and safe to administer rituximab for PV and severe PF as a first line agent.

#### *10.1.2. TNF-antagonists*

producing plasma cells, and impairment of autoantigen presentation to CD4 cells [102]. In PV, Eming et al, show that rituximab not also causes marked reduction of anti-desmoglein 3 antibodies but also depletion of desmoglein-specific CD4 cells. In contrast, tetanus toxoidspecific CD4 cells were not affected nor were the overall number of CD4 cells [103]. Tetaus toxoid has been used in as an antigen in the assessment of memory CD4 cell responses [104]. Eming et al, speculate that this specific effect of rituximab on autoreactive rather than patho‐ gen-specific T cells is that the latter do not require CD20 B cells as antigen-presenting cells. A number of case reports and series have demonstrated the benefit on the use of rituximab in over 40 patients with either PV or PF [40],[105]-[115]. The largest case series to date comprising 42 patients, 37 with PV and 5 with PF, utilized the rheumatoid arthritis protocol where two 1g infusions of rituximab are administered 15 days apart [116]. Patients were followed for up to 5 years and 36 of the 42 patients achieved a complete response and were able to cease corti‐ costeroids within 6 months from induction therapy. Twenty patients experienced relapses with the time to relapse ranging from 8 to 64 months. Relapses were treated with rituximab (500 mg) without corticosteroids resulting in a new complete response. Importantly, no serious

The largest case series to date using the lymphoma protocol where rituximab is administered

patients with PF within 3 months [117]. These patients had previously not responded to firstline immunosuppressive agents or had contraindications to corticosteroid therapy. The treatment was generally well tolerated; however, two cases were complicated by severe infection, one resulting in death. Similarly, Canchini et al, showed that the administration of rituximab, 375 mg/m2 once weekly for 4 weeks, induced remission in all 10 patients with PV and both patients with PF [118]. Another study comprising 11 patients with refractory PV, evaluated the effect of combination therapy consisting of 10 infusions of rituximab (375

) and 6 infusions of intravenous immunoglobulin (IVIg) (2 g/kg body weight) admin‐ istered over a 6-month period [119]. Remission was induced in 9 patients for a period of 22 to

37 months following treatment and there were no reports of serious adverse events.

More recently, Kasperkiewicz et al reported use of rituximab in combination with immunoad‐ sorption, pulsed dexamethasone and azathioprine or mycophenolate mofetil in 23 patients. [120] IA was performed at initially 3- and later 4-week intervals until lesions healed by 90%; 1 g rituximab was given at weeks 1 and 3, and intravenous dexamethasone pulses were administered at first every 3 weeks and then at increasing intervals in addition to daily azathioprine or mycophenolate mofetil. All patients demonstrated clinical improvement within the first few weeks of therapy accompanies by a concomitant rapid fall in anti-desmo‐ glein antibody levels. However, two patients had non -atal severe adverse events; one developed *Staphylococcus aureus* sepsis from a central intravenous line followed by spinal haemorrhage and transient paraplegia, and another developed extensive herpes simplex

The high frequency of treatment failure with corticosteroids and first line immunosuppressive therapies has raised the issue of whether rituximab should be implemented earlier in the treatment of PV. Horvath et al conducted a study comprising 15 patients (12 with PV, 3 with

) for 4 weeks induced remission in 12 of 14 patients with PV and 6 of 7

adverse events were observed.

126 Skin Biopsy - Diagnosis and Treatment

weekly (375 mg/m2

mg/m2

infection

Studies have demonstrated that TNF released by keratinocytes plays a role in acantholysis in PV. Human keratinocytes pretreated with anti-TNF antibodies, are resistant to the acantholytic effect of anti-desmoglein 3 antibodies [128],[129]. Furthermore, TNF-deficient mice are more susceptible to blister formation after injection with anti-desmoglein 3 antibodies [128]. The role of TNF in PF has not been as extensively studied. Etanercept [130]-[133], infliximab [134],[135], and adalimumab [136], have all demonstrated benefit in a small number of patients with refractory PV. Randomized controlled trials of infliximab and etanercept in refractory PV are currently in progress. However, the effect of these agents in refractory PF has not been reported to date.

#### **10.2. Intravenous immunoglobulin**

IVIg is a fractionated and purified blood product derived from the pooled plasma of up to 15, 000 healthy donors. Hence it has a high concentration of IgG with a broad range of specificities against various antigens [137]. The mode of action of IVIg in autoimmune disease has not been clearly defined but is probably multifactorial and includes provision of anti-idiotypic anti‐ bodies, modulation of expression and function of Fc receptors thereby neutralizing the effect of pathogenic antibodies, blocking of complement activation, reduced secretion of proinflammatory cytokines through modulation of dendritic, T and B-cell activation [138],[139]. IVIg upregulates endogenous caspase inhibitors protecting keratinocytes from proapoptotic molecules and thereby inhibits acantholysis [140]. In PV, IVIg causes a selective and rapid decline in serum levels of pathogenic antibodies, specifically IgG1 and IgG4 anti-desmoglein-1 and anti-desmoglein-3 antibodies without affecting total serum IgG levels [141],[142]. A reduction in anti-desmoglein-1 antibodies also occurs in PF [143]. FcRn receptors, which normally function to protect serum IgG from degredation, are saturated following IVIg resulting in catabolism of all IgG molecules including autoreactive antibodies [144]. However, pathogenic autoantibodies are selectively reduced because catabolized normal antibodies are replaced by those present in the IVIg preparation [141].

of IVIg may result in new synthesis of autoantibodies exceeding that initially present [154] and

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Comparative trials in refractory disease with other modalities such as biological agents and extracorporeal treatments still need to be performed. The considerable expense of IVIg warrants clarification of the optimal dose, frequency and duration of therapy. Further studies are required to determine whether IVIg can be ceased once remission is achieved, and the patient maintained on conventional first line immunosuppressive agents to minimize rebound

Plasmapheresis results in the potential removal of pathogenic antibodies from the pa‐ tient's plasma. Forty patients with pemphigus were recruited in a multicentre study and randomized to receive prednisone alone or prednisone and plasma exchange, which consisted of 10 treatments over 4 weeks [155]. No adjuvant immunosupressive therapy was used for any of the patients. No difference was observed between the 2 groups. Four patients in each group did not achieve disease control. Four patients in the plasmaphere‐ sis group died from either sepsis or thromboembolism. The lack of response is surprising as Nagasaka et al, demonstrated in 15 patients with PV and 1 patient with PF that one centrifugal plasmapheresis treatment eliminates 15% of the IgG autoantibodies as meas‐ ured in the effluents and this is reflected in serum measurements performed one day later [156]. Numerous case series have demonstrated benefit in severe or recalcitrant pemphi‐ gus when plasmapheresis is combined not only with corticosteroids but other immunosup‐ pressive agents as well [157]-[161]. Many of these patients experienced side effects including thrombocytopenia, hypocalcemia, urticaria, fever, hypotension, acute hepatitis, nausea, dizziness and leg cramps. The beneficial response observed in these case series studies in contrast to the randomized controlled study of Guillaume et al, may therefore be ex‐ plained by the concurrent use of immunosuppressive agents in order to prevent the rebound production of autoantibodies. Hence further randomized control studies are needed to clarify the value of plasmapheresis combined with immunosuppressive therapy.

Immunoadsorption (IA) is an extracorporeal treatment for the selective removal of antibodies and circulating immune complexes from plasma. This differs from plasmapheresis, which nonspecifically removes plasma proteins including clotting factors, hormones and albumin, thus requiring substitution of fresh frozen plasma or albumin. The Food and Drug Adminis‐ tration approved IA for the treatment of rheumatoid arthritis, idiopathic thrombocytopenic purpure and hemophilia with inhibitors. It has also been used off label for the treatment of various autoimmune mediated conditions including dilated cardiomyopathy, systemic lupus

erythematosus, myasthenia gravis and autoimmune bullous disorders [162].

this rebound in antibody levels may minimized by concurrent cytotoxic therapy.

synthesis of pathogenic antibodies.

**10.3. Extracorporeal treatments**

*10.3.1. Plasmapheresis*

*10.3.2. Immunoadsorption*

Three case series and 1 retrospective analysis comprising 54 patients with refractory PV documented the induction of clinical remission following IVIg in all but 2 patients [145]-[148]. Two case series involving a total of 15 patients with refractory PF all responded to IVIg [149], [150]. One of these studies featuring 7 patients revealed a prolonged mean remission time of 18.6 months following discontinuation of IVIg [150]. Two retrospective analyses that included 17 patients with refractory PV and 2 patients with refractory PF, however, demonstrated a much less favourable response with the majority of patients harbouring active disease following IVIg [151],[152]. Recently, a double blind randomized study investigated the effect of a 5 day course of IVIg at varying doses (400, 200 or 0 mg/kg/day) in 40 patients with PV and 21 patients with PF resistant to doses of steroids greater that 20 mg daily [153]. The study did not specify whether these patients had previously been treated with or were currently receiving corticosteroid-sparing immunosuppressive agents. The patients were maintained on their study entry dose of corticosteroids for the duration of the trial. The primary end point was the time to escape from the protocol, which was defined as the length of period that the patient remained on the protocol, without any additional treatment. Patients that showed no improvement after 2 weeks or developed fresh lesions necessitating an increase in corticoste‐ roids or additional immunosuppression were considered as having escaped from the protocol. The study demonstrated a significantly longer time to escape the protocol, and a lower disease activity index accompanied by a fall in anti-desmoglein antibody levels in the 400 mg/kg/day group at days 43 and 85 for both PV and PF patients. There were no significant differences in the side effects observed between the groups. Adverse events that were reported in a minority of patients included fever, headache, palpitations, hypertension, gastrointestinal bleeding, increased creatinine, abnormal liver function tests, and anemia. One patient in the 200 mg/kg/ day died as a result of liver failure from exacerbation of pre-existing chronic hepatitis C. Hence, this study does provide useful evidence for the efficacy of IVIg in both PV and PF. Cessation of IVIg may result in new synthesis of autoantibodies exceeding that initially present [154] and this rebound in antibody levels may minimized by concurrent cytotoxic therapy.

Comparative trials in refractory disease with other modalities such as biological agents and extracorporeal treatments still need to be performed. The considerable expense of IVIg warrants clarification of the optimal dose, frequency and duration of therapy. Further studies are required to determine whether IVIg can be ceased once remission is achieved, and the patient maintained on conventional first line immunosuppressive agents to minimize rebound synthesis of pathogenic antibodies.

#### **10.3. Extracorporeal treatments**

#### *10.3.1. Plasmapheresis*

**10.2. Intravenous immunoglobulin**

128 Skin Biopsy - Diagnosis and Treatment

replaced by those present in the IVIg preparation [141].

IVIg is a fractionated and purified blood product derived from the pooled plasma of up to 15, 000 healthy donors. Hence it has a high concentration of IgG with a broad range of specificities against various antigens [137]. The mode of action of IVIg in autoimmune disease has not been clearly defined but is probably multifactorial and includes provision of anti-idiotypic anti‐ bodies, modulation of expression and function of Fc receptors thereby neutralizing the effect of pathogenic antibodies, blocking of complement activation, reduced secretion of proinflammatory cytokines through modulation of dendritic, T and B-cell activation [138],[139]. IVIg upregulates endogenous caspase inhibitors protecting keratinocytes from proapoptotic molecules and thereby inhibits acantholysis [140]. In PV, IVIg causes a selective and rapid decline in serum levels of pathogenic antibodies, specifically IgG1 and IgG4 anti-desmoglein-1 and anti-desmoglein-3 antibodies without affecting total serum IgG levels [141],[142]. A reduction in anti-desmoglein-1 antibodies also occurs in PF [143]. FcRn receptors, which normally function to protect serum IgG from degredation, are saturated following IVIg resulting in catabolism of all IgG molecules including autoreactive antibodies [144]. However, pathogenic autoantibodies are selectively reduced because catabolized normal antibodies are

Three case series and 1 retrospective analysis comprising 54 patients with refractory PV documented the induction of clinical remission following IVIg in all but 2 patients [145]-[148]. Two case series involving a total of 15 patients with refractory PF all responded to IVIg [149], [150]. One of these studies featuring 7 patients revealed a prolonged mean remission time of 18.6 months following discontinuation of IVIg [150]. Two retrospective analyses that included 17 patients with refractory PV and 2 patients with refractory PF, however, demonstrated a much less favourable response with the majority of patients harbouring active disease following IVIg [151],[152]. Recently, a double blind randomized study investigated the effect of a 5 day course of IVIg at varying doses (400, 200 or 0 mg/kg/day) in 40 patients with PV and 21 patients with PF resistant to doses of steroids greater that 20 mg daily [153]. The study did not specify whether these patients had previously been treated with or were currently receiving corticosteroid-sparing immunosuppressive agents. The patients were maintained on their study entry dose of corticosteroids for the duration of the trial. The primary end point was the time to escape from the protocol, which was defined as the length of period that the patient remained on the protocol, without any additional treatment. Patients that showed no improvement after 2 weeks or developed fresh lesions necessitating an increase in corticoste‐ roids or additional immunosuppression were considered as having escaped from the protocol. The study demonstrated a significantly longer time to escape the protocol, and a lower disease activity index accompanied by a fall in anti-desmoglein antibody levels in the 400 mg/kg/day group at days 43 and 85 for both PV and PF patients. There were no significant differences in the side effects observed between the groups. Adverse events that were reported in a minority of patients included fever, headache, palpitations, hypertension, gastrointestinal bleeding, increased creatinine, abnormal liver function tests, and anemia. One patient in the 200 mg/kg/ day died as a result of liver failure from exacerbation of pre-existing chronic hepatitis C. Hence, this study does provide useful evidence for the efficacy of IVIg in both PV and PF. Cessation

Plasmapheresis results in the potential removal of pathogenic antibodies from the pa‐ tient's plasma. Forty patients with pemphigus were recruited in a multicentre study and randomized to receive prednisone alone or prednisone and plasma exchange, which consisted of 10 treatments over 4 weeks [155]. No adjuvant immunosupressive therapy was used for any of the patients. No difference was observed between the 2 groups. Four patients in each group did not achieve disease control. Four patients in the plasmaphere‐ sis group died from either sepsis or thromboembolism. The lack of response is surprising as Nagasaka et al, demonstrated in 15 patients with PV and 1 patient with PF that one centrifugal plasmapheresis treatment eliminates 15% of the IgG autoantibodies as meas‐ ured in the effluents and this is reflected in serum measurements performed one day later [156]. Numerous case series have demonstrated benefit in severe or recalcitrant pemphi‐ gus when plasmapheresis is combined not only with corticosteroids but other immunosup‐ pressive agents as well [157]-[161]. Many of these patients experienced side effects including thrombocytopenia, hypocalcemia, urticaria, fever, hypotension, acute hepatitis, nausea, dizziness and leg cramps. The beneficial response observed in these case series studies in contrast to the randomized controlled study of Guillaume et al, may therefore be ex‐ plained by the concurrent use of immunosuppressive agents in order to prevent the rebound production of autoantibodies. Hence further randomized control studies are needed to clarify the value of plasmapheresis combined with immunosuppressive therapy.

#### *10.3.2. Immunoadsorption*

Immunoadsorption (IA) is an extracorporeal treatment for the selective removal of antibodies and circulating immune complexes from plasma. This differs from plasmapheresis, which nonspecifically removes plasma proteins including clotting factors, hormones and albumin, thus requiring substitution of fresh frozen plasma or albumin. The Food and Drug Adminis‐ tration approved IA for the treatment of rheumatoid arthritis, idiopathic thrombocytopenic purpure and hemophilia with inhibitors. It has also been used off label for the treatment of various autoimmune mediated conditions including dilated cardiomyopathy, systemic lupus erythematosus, myasthenia gravis and autoimmune bullous disorders [162].

Initially, 4 case series and 2 case reports totalling 31 patients with PV and 5 patients with PF reported efficacy for IA in combination with immunosuppressive therapies in the treatment of recalcitrant disease [163]-[167]. The treatment schedule generally consisted of 3 initial cycles on consecutive days, a fourth cycle on day 8, followed by 19 cycles in incrementally prolonged intervals of 1 to 4 weeks. However, relapses are common once IA is discontinued and concur‐ rent immunosuppressive therapy tapered [167]. More recently, a small case series comprising 7 patients with refractory PV demonstrated that 23 cycles of IA administered 40 weeks, as described above, in combination with rituximab (375 mg/m2 weekly for 4 weeks) and con‐ comitant conventional therapy resulted in complete remission in 3 patients for a period of between 13 and 30 months with minimal or no maintenance immunosupresion [168]. One patient attained partial remission but required significantly less dose of corticosteroids than prior to IA and rituximab. The remaining 3 patients relapsed following the completion of treatment. Two of these patients achieved remission when IVIg (2 g/kg body weight every 4 weeks) was administered and the other only partially responded to IVIg. A retrospective study on refractory PV compared the efficacy of IA in 6 patients with rituximab in 5 patients and showed remission in all patients at 6 months [169]. This remission was sustained in all patients who had received rituximab compared to half of those that received IA. Randomized trials are required to compare the efficacy of rituximab or IVIg with IA and determine any additional benefit from a combination of these modalities.

**10.4. Cholinergic agonists**

process in vitro [179].

**10.5. Peptide immunotherapy**

a beneficial effect in PF.

*10.5.1. Inhibitors of intracellular signalling and apoptosis*

p38MAPK inhibitor, KC706 (Kémia, Inc), in refractory PV.

reported.

Studies have suggested that acetylcholine and its receptors are involved in the acantholysis of pemphigus. Approximately 85% of patients with pemphigus have antibodies against acetyl‐ choline receptors on keratinocytes [178]. Cholinergic antagonists mediate similar acantholytic effects on keratinocytes as PV IgG [179]. Acantholytic antibodies can recognize the alpha-9 acetylcholine receptor [180] and pemphaxin [181], which can function as an acetylcholine receptor. Finally, cholinergic agonists can prevent acantholysis *in vivo* [182] and reverse the

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Grando demonstrated a response in 3 of 6 patients with PV treated with pyridostigmine bromide and conventional immunosuppression with 2 responders ultimately able to control their disease with pyridostigmine bromide alone [183]. A recent double blind placebo con‐ trolled study comprising 3 PV patients showed a superior epithelialisation effect from 4% pilocarpine gel compared with placebo [184]. The use of these agents in PF has not been

Immunization with intravenous desmoglein-3 peptides was developed to suppress production of anti-desmoglein-3 antibodies through inactivation of disease specific CD4 cells. A phase I clinical trial in PV patients found no significant change in anti-desmo‐ glein-3 antibodies following administration of intravenous desmoglein-3 peptides [185]. Additional studies utilizing higher doses and longer treatment are in progress. It remains to be determined whether peptide immunotherapy with desmoglein-1 peptides will have

Studies performed by Berkowitz's group have demonstrated the role of p38 mitogen-activated protein kinase (p38MAPK) in the pathogenesis of pemphigus. Human keratinocytes treated with PV IgG show a time and dose dependent increase in levels of p38MAPK and heat shock protein 27 (HSP27) proteins involved in regulating cytoskeletal components such as keratin intermediate filaments [186]. Inhibitors of MAPK signalling blocked phosphorylation of HSP27 following PV IgG stimulation of human keratinocytes and importantly prevented keratin filament retraction, an early change evident in acantholysis [186]. Inhibition of p38MAPK in murine and cell culture models of pemphigus vulgaris also prevented blister formation [187],[188]. The same group also recently demonstrated p38MAPK inhibition and prevention of blister formation in a murine model of PF [189]. An open labelled uncontrolled study is currently in progress to determine the safety and efficacy of the oral allosteric

Activation of protein kinase C (PKC) followed by plakoglobin dislocation and subsequent dissociation of desmogleins from desmosomes also appear important in the pathogenesis of pemphigus [190]-[194]. Inhibitors of PKC and plakoglobin/c-myc proto-oncogene axis have been shown to inhibit PV IgG induced blister formation in the neonatal passive

The treatment is generally well tolerated. Rare adverse events that have been reported include catheter related sepsis, *Pneumocystis jerovecii* pneumonia, mild hypotension, bradycardia and in relation to anticoagulant use, hypocalcemia and paraesthesia. The trials by Schmidt et al, and Shiminovich et al, showed that the combination of IA and immunosuppression resulted in anemia in 30% of patients [164],[167].

#### *10.3.3. Extracorporeal Photochemotherapy (ECP)*

In ECP, a patient's leukocytes are collected, exposed to 8-methoxypsoralen, irradiated with ultraviolet-A light and reinfused into the patient. The principle of ECP is to induce apoptosis of leukocytes with ultraviolet-A radiation after their presentation by psoralens [170]. Early apoptotic cells produce anti-inflammatory cytokines such as IL-10 and TGF-beta, which stimulates their engulfment by macrophages and immature dendritic cells. The further production of IL-10 and TGF-beta by these antigen presenting cells with subsequent down regulation of proinflammatory cytokines such as TNF, IL-1 and IL-12 results in immunosu‐ pression and absence of co-stimulation of effector T cells [171]. A deficiency of apoptotic cell clearance may contribute to the pathogenesis of autoimmune diseases including pemphigus and therefore ECP may enhance clearance of autoreactive cells and the reduce formation of pathogenic autoantibodies by B cells [172],[173]. Collectively, 9 patients, 8 with PV and 1 with PF, originating from a small number of case studies and series, received ECP for refractory disease in conjunction with their baseline immunosuprressive agents. [174]-[177]. In contrast to the patients with PV, the lone patient with PF achieved only partial remission and long term immunosuppression was unable to be weaned successfully [177]. ECP was well tolerated in these patients with no adverse effects reported.

#### **10.4. Cholinergic agonists**

Initially, 4 case series and 2 case reports totalling 31 patients with PV and 5 patients with PF reported efficacy for IA in combination with immunosuppressive therapies in the treatment of recalcitrant disease [163]-[167]. The treatment schedule generally consisted of 3 initial cycles on consecutive days, a fourth cycle on day 8, followed by 19 cycles in incrementally prolonged intervals of 1 to 4 weeks. However, relapses are common once IA is discontinued and concur‐ rent immunosuppressive therapy tapered [167]. More recently, a small case series comprising 7 patients with refractory PV demonstrated that 23 cycles of IA administered 40 weeks, as

comitant conventional therapy resulted in complete remission in 3 patients for a period of between 13 and 30 months with minimal or no maintenance immunosupresion [168]. One patient attained partial remission but required significantly less dose of corticosteroids than prior to IA and rituximab. The remaining 3 patients relapsed following the completion of treatment. Two of these patients achieved remission when IVIg (2 g/kg body weight every 4 weeks) was administered and the other only partially responded to IVIg. A retrospective study on refractory PV compared the efficacy of IA in 6 patients with rituximab in 5 patients and showed remission in all patients at 6 months [169]. This remission was sustained in all patients who had received rituximab compared to half of those that received IA. Randomized trials are required to compare the efficacy of rituximab or IVIg with IA and determine any additional

The treatment is generally well tolerated. Rare adverse events that have been reported include catheter related sepsis, *Pneumocystis jerovecii* pneumonia, mild hypotension, bradycardia and in relation to anticoagulant use, hypocalcemia and paraesthesia. The trials by Schmidt et al, and Shiminovich et al, showed that the combination of IA and immunosuppression resulted

In ECP, a patient's leukocytes are collected, exposed to 8-methoxypsoralen, irradiated with ultraviolet-A light and reinfused into the patient. The principle of ECP is to induce apoptosis of leukocytes with ultraviolet-A radiation after their presentation by psoralens [170]. Early apoptotic cells produce anti-inflammatory cytokines such as IL-10 and TGF-beta, which stimulates their engulfment by macrophages and immature dendritic cells. The further production of IL-10 and TGF-beta by these antigen presenting cells with subsequent down regulation of proinflammatory cytokines such as TNF, IL-1 and IL-12 results in immunosu‐ pression and absence of co-stimulation of effector T cells [171]. A deficiency of apoptotic cell clearance may contribute to the pathogenesis of autoimmune diseases including pemphigus and therefore ECP may enhance clearance of autoreactive cells and the reduce formation of pathogenic autoantibodies by B cells [172],[173]. Collectively, 9 patients, 8 with PV and 1 with PF, originating from a small number of case studies and series, received ECP for refractory disease in conjunction with their baseline immunosuprressive agents. [174]-[177]. In contrast to the patients with PV, the lone patient with PF achieved only partial remission and long term immunosuppression was unable to be weaned successfully [177]. ECP was well tolerated in

weekly for 4 weeks) and con‐

described above, in combination with rituximab (375 mg/m2

benefit from a combination of these modalities.

in anemia in 30% of patients [164],[167].

130 Skin Biopsy - Diagnosis and Treatment

*10.3.3. Extracorporeal Photochemotherapy (ECP)*

these patients with no adverse effects reported.

Studies have suggested that acetylcholine and its receptors are involved in the acantholysis of pemphigus. Approximately 85% of patients with pemphigus have antibodies against acetyl‐ choline receptors on keratinocytes [178]. Cholinergic antagonists mediate similar acantholytic effects on keratinocytes as PV IgG [179]. Acantholytic antibodies can recognize the alpha-9 acetylcholine receptor [180] and pemphaxin [181], which can function as an acetylcholine receptor. Finally, cholinergic agonists can prevent acantholysis *in vivo* [182] and reverse the process in vitro [179].

Grando demonstrated a response in 3 of 6 patients with PV treated with pyridostigmine bromide and conventional immunosuppression with 2 responders ultimately able to control their disease with pyridostigmine bromide alone [183]. A recent double blind placebo con‐ trolled study comprising 3 PV patients showed a superior epithelialisation effect from 4% pilocarpine gel compared with placebo [184]. The use of these agents in PF has not been reported.

#### **10.5. Peptide immunotherapy**

Immunization with intravenous desmoglein-3 peptides was developed to suppress production of anti-desmoglein-3 antibodies through inactivation of disease specific CD4 cells. A phase I clinical trial in PV patients found no significant change in anti-desmo‐ glein-3 antibodies following administration of intravenous desmoglein-3 peptides [185]. Additional studies utilizing higher doses and longer treatment are in progress. It remains to be determined whether peptide immunotherapy with desmoglein-1 peptides will have a beneficial effect in PF.

#### *10.5.1. Inhibitors of intracellular signalling and apoptosis*

Studies performed by Berkowitz's group have demonstrated the role of p38 mitogen-activated protein kinase (p38MAPK) in the pathogenesis of pemphigus. Human keratinocytes treated with PV IgG show a time and dose dependent increase in levels of p38MAPK and heat shock protein 27 (HSP27) proteins involved in regulating cytoskeletal components such as keratin intermediate filaments [186]. Inhibitors of MAPK signalling blocked phosphorylation of HSP27 following PV IgG stimulation of human keratinocytes and importantly prevented keratin filament retraction, an early change evident in acantholysis [186]. Inhibition of p38MAPK in murine and cell culture models of pemphigus vulgaris also prevented blister formation [187],[188]. The same group also recently demonstrated p38MAPK inhibition and prevention of blister formation in a murine model of PF [189]. An open labelled uncontrolled study is currently in progress to determine the safety and efficacy of the oral allosteric p38MAPK inhibitor, KC706 (Kémia, Inc), in refractory PV.

Activation of protein kinase C (PKC) followed by plakoglobin dislocation and subsequent dissociation of desmogleins from desmosomes also appear important in the pathogenesis of pemphigus [190]-[194]. Inhibitors of PKC and plakoglobin/c-myc proto-oncogene axis have been shown to inhibit PV IgG induced blister formation in the neonatal passive transfer murine model of pemphigus vulgaris [195]. It has been demonstrated that p53 knockout mice are protected from PV IgG induced disease [196]. Neonatal mice pretreated with p53 inhibitor pifithrin-alpha were resistant to both PV and PF IgG mediated blister formation [197].

maintaining remission and having a comparatively favourable side effect profile. An enhanced understanding of the pathogenesis of pemphigus has resulted in the implementation of a number of novel agents. These therapies have also been mainly studies through case series reports, are expensive and difficult to access in some centres, and are associated with a number of deleterious side effects. Rituximab, has emerged as the therapy of choice in severe refractory disease and is now being explored as a first line agent. We eagerly await further studies on the effects and safety profiles of more specific agents, especially those targeting signalling

Pemphigus Vulgaris and Pemphigus Foliaceus

http://dx.doi.org/10.5772/56423

133

We have formulated guidelines on the treatment of pemphigus as suggested by the current level of evidence [204]. Corticosteroids remain the mainstay of treatment and should be initiated at a dose of 0.5 mg /kg of oral prednisone per day and continued at this dose until disease control is obtained. This is defined as the time at which new lesions cease to form and existing lesions begin to heal and in responsive patients, this usually occurs within weeks. At the end of the consolidation phase defined as the point in time where no new lesions have occurred after 2 weeks and 80% of established lesions have healed, the corticosteroid dose is tapered. Unless disease is mild we would recommend adding an adjuvant agent. Either mycophenolate mofetil or azathioprine could be utilized at this stage. For severe or recalcitrant disease rituximab is recommended given the current level of evidence. Other biologic agents, extracorporeal therapies or cytotoxic agents can be considered if rituximab is unavailable or

and Mark Schifter3,4

2 Sydney Medical School – Northern, Sydney University, Sydney, Australia

4 Faculty of Dentistry, University of Sydney, Sydney, Australia

1209 cases. Int J Dermatol 2005;44:470-6.

1 Department of Clinical Immunology and Allergy, Royal North Shore Hospital, Sydney,

3 Department of Oral Medicine, Oral Pathology and Special Care Dentistry, Westmead Hos‐

[1] Chams-Davatchi C, Valikhani M, Daneshpazhooh M, et al. Pemphigus: analysis of

molecules involved in the pathogenesis of pemphigus.

contraindicated.

**Author details**

Australia

**References**

Suran L. Fernando1,2, Jamma Li1

pital, (Western) Sydney, Australia

As shown by the studies of Waschke et al, PV autoantibodies directly block desmoglein-3 transinteraction in contrast to anti-desmoglein antibodies found in PF, which disrupt desmoglein-1 transinteraction via cellular signalling events rather than by direct inhibi‐ tion [198]-[200]. Hence targeting these signalling proteins in PF may provide a more specific target of therapy as compared to immunosuppressive or biological agents. It remains to be determined whether targeting these signalling proteins results in mediating disease remission in humans. Although they target specific areas of pemphigus pathogenesis, these proteins mediate numerous cellular functions and hence the outcome of early safety studies is eagerly awaited.
