Biological Approach in the Treatment of Crohn’s Disease

Introduction

Crohn’s Disease (CD) is a chronic inflammatory dis- ease of the gastrointestinal tract, with a low percent- age of patients remaining in clinical remission despite the long-term use of many drugs (steroids, immunosuppressants, aminosalicylates, antibiotics).

As a consequence of the limited efficacy and signifi- cant toxicity of current therapy, there is widespread interest in the development of novel drugs for the clinical management of CD.

In the early 1990s, the pathogenetic mechanisms of inflammatory bowel diseases (IBDs) were clarified.

Greater knowledge of systemic and local inflammato- ry cascade of CD (T1-helper-driven inflammation) [1] allowed identification of key mediators of inflam- mation, and pharmacologic research was addressed towards specific biologic drugs interfering with this cascade. Proinflammatory cytokines play a central role in amplification of the inflammatory process, inducing release of other cytokines and increasing the expression of adhesion molecules [2]. Tumour necrosis factor (TNF)- α and interleukin (IL)-1 are released at the beginning of the inflammatory cas- cade and contribute to recruitment and activation of inflammatory cells (macrophages, neutrophils, monocytes) by increasing mucosal intestinal perme- ability and stimulating the release of adhesion mole- cules. Anti-inflammatory cytokines play an impor- tant role in avoidance of an abnormal immune response. In particular, IL-10 controls TNF produc- tion, major histocompatibility complex (MHC)-II expression and release of other inflammatory media- tors whereas IL-12 induces the release of interferon gamma (INF- γ) by macrophages and natural-killer lymphocytes [3].

The term “biological agent” means any therapeu- tic agent derived from a living organism. Therefore, biological therapy may include [4]: (1) modified bio- logical compounds (i.e. vaccine), hormones, etc.; (2) recombinant peptides or proteins (i.e. growing fac- tors, growth hormones, etc.); (3) monoclonal anti- bodies or fusion proteins; (4) antisense oligonu-

cleotides. Many molecules have been used in con- trolled or open trials, mainly in fistulising or steroid- dependent CD (Table 1). Monoclonal antibodies and antisense nucleotides interfering with messenger ribonucleic acid (mRNA) are the drugs most fre- quently tested in clinical trials.

The Present: Indications, Efficacy, Problems and Open Fields Using Infliximab in Crohn’s Disease

The first drug approved by the Food and Drug Administration for the treatment of CD was inflix- imab (Remicade) [5], a monoclonal chimeric anti- body (human 75%, murine 25%). Infliximab neu- tralises biological activity of TNF- α by binding with high affinity to both soluble and transmembrane forms of TNF- α, inhibiting binding of TNF-α with its receptors. Therefore, infliximab heavy reduces TNF- α plasmatic levels and induces inflammatory cell apoptosis in the gut wall [6–9], thus promoting mucosal healing at colonoscopy [10, 11].

Efficacy on Remission and Maintenance Therapy

Infliximab, administrated intravenously at a dosage of 5 mg/kg of body weight at weeks 0, 2 and 6 (induc- tion therapy) and, later, every 8 weeks (maintenance therapy) is effective in the therapy of patients with active CD [12]. Infliximab is also a corticosteroid- sparing drug in patients with active CD [13–15]. Def- inite indications for induction therapy with inflix- imab (eligible patients) are [13]: (1) Fibrostenosing (inflammatory) disease nonrespondent or refractory to steroid and conventional therapy; (2) fistulising disease, draining enterocutaneous or perianal fistu- lae.

The use of infliximab as first-line treatment of patients with moderate to severe CD is still contro- versial. There are no studies demonstrating the effi- cacy of this scheduled therapy even though this approach has been suggested [16].

Biological Approach in the Treatment of Crohn’s Disease

Luca Frulloni, Laura Bernardoni, Chiara Scattolini, Italo Vantini

Induction therapy is effective in up to 70% of cases [10, 12, 17–30]. N ot responders to induction therapy do not respond to a maintenance therapy. In patients who respond to induction therapy, mainte- nance therapy may prolong complete clinical remis- sion [Crohn’s Disease Activity Index (CDAI) <150]

after 1 year in about 40% of cases, with clinical response in 60% of cases. This implies that, after 1 year of prolonged infliximab therapy, only 25% of patients attain complete clinical remission and 35%

clinical response [10, 12, 17–29]. There is evidence that an increase of infliximab dosage and/or a reduc- tion of interval time between infusions may main-

tain a higher number of patients in clinical remis- sion [21, 31–33].

Disease Type and Stage

The reported incidence of fistulae in patients with CD ranges from 17% to 50% [34]. Fistulae may be ente- rocutaneous (more commonly perianal), easily recognisable; or internal, enteroenteric or between gut and other organs. Internal fistulae may be asymp- tomatic but may be more aggressive and require urgent surgery (temporary colostomy allows the

Table 1.Biological therapy in Crohn’s disease

Proper name Trade name Action

Inhibitors of pro-inflammatory cytokines

Infliximab Remicade Chimeric (human/mouse) anti-TNF-

α

monoclonal antibody (IgG1)

CDP571 Humicade Humanized anti-TNF

α

monoclonal antibody (IgG4)

Certolizumab - Humanized anti-TNF-

α

monoclonal antibody (polyethylene glycolated Fab’ fragment)

Adalimumab Humira Humanized anti-TNF-

α

monoclonal antibody (IgG1) Thalidomide Thalomid Derivative of glutamic acid with anti-inflammatory and

anti-TNF-

α

^action

Semapimod - Interferes with the phosphorylation of both p38 and JNK Etanercept Enbrel Genetically engineered fusion protein consisting of two

identical chains of the recombinant human p75 TNF receptor linked to the Fc portion of human IgG1

Onercept - Recombinant form of the human soluble p55 TNF receptor

MRA - Humanized monoclonal antibody to IL-6 receptor

ABT-874/J695 - Humanized anti-IL-12 p40 (IgG1)

Anti-inflammatory cytokines

RhIL-10 Tenovil Humanized monoclonal antibody to IL-10

RhIL-11 - Humanized monoclonal antibody to IL-11

Inhibitors of adhesion molecules

Alicaforsen - Antisense phosphorothioate oligonucleotide targeting human ICAM-1 mRNA

N

atalizumab Antegren Humanized monoclonal antibody against

α

^{4 integrin} Immune modulators

Tacrolimus Prograf Inhibits production of IL- 2 by T-helper cells

Mycophenolate mofetil CellCept Antimetabolite strongly suppressing lymphocyte proliferation Miscellaneous

Sargramostim Leukine Yeast-derived recombinant human GM-CSF

Trichuris suis - Worm

Probiotics VSL#3 Bacteria

TNF, tumor necrosis factor; IgG, immunoglobulin G; JNK, Jun N-terminal kinase; ICAM, intercellular adhesion molecule; mRNA, mes- senger ribonucleic acid; IL, interleukin; GM-CSF, granulocyte macrophage colony stimulating factor

patient to heal by bypassing the inflammatory tract).

In perianal disease, induction therapy with inflix- imab (5 mg/kg at 0, 2 and 6 weeks) is effective on fis- tula healing in more than 60% of patients [28, 35].

Concomitant conventional therapy (antibiotics and immunosuppressants) is useful since 3 months’ treat- ment with antibiotic monotherapy (metronidazole and ciprofloxacin) showed 60–70% complete remis- sion, improved quality of life and reduced disease recurrences [20, 36, 37]. Azathioprine monotherapy is effective as well, but its full action is evident after 2–3 months of therapy. Therefore, its use in fistulising (perforating) CD is limited, at least as initial treat- ment. In patients intolerant to azathioprine, methotrexate at dose of 25 mg intramuscularly week- ly may be a possible alternative treatment [38].

In patients with perianal fistulae, first-line treat- ment with antibiotics is suggested. In case of relapse, antibiotics and immunosuppressant therapy (aza- thioprine or methotrexate) is effective. If fistulae per- sist, induction therapy with infliximab is recom- mended, followed by maintenance therapy with immunosuppressant or infliximab (every 8 weeks) [36, 39].

In our opinion, a more appropriate first-line treat- ment may include induction therapy with infliximab, followed in responders by a maintenance regimen with infliximab and introduction of immunosup- pressant drugs. In nonresponders or those intolerant to infliximab i.v., antibiotics, immunosuppressants and local injection of infliximab may be an alterna- tive treatment [40]. Internal fistulae are usually asso- ciated to a more aggressive subtype of CD and more difficult to treat conservatively [41]. However, treat- ment with azathioprine and antibiotics or infliximab may be tried [42, 43].

In some patients, clinical onset of CD occurs with subocclusive symptoms secondary to inflammatory strictures. In naïve patients, infliximab probably induces rapid anti-inflammatory effects with symp- tomatic improvement, reducing the need for resec- tive surgery. There are no studies confirming this hypothesis but a top-down therapy has been recently proposed [16] and, probably, in the near future, this may be an appropriate initial treatment [44]. Howev- er, concomitant therapy with immunomodulators increases the rate of clinical response and clinical remission, and smoking seems to be a negative pre- dictor of response to infliximab therapy [45, 46].

Therefore, an initial therapeutic approach with steroids and immunosuppressants followed by inflix- imab is suggested, as well as smoking cessation.

On the contrary, infliximab is not indicated in the presence of a fibrotic stenosis [44, 47]. Clinical (long- standing disease), laboratory data [inflammatory indexes, mainly C-reactive protein [48, 49] and instru-

mental findings by magnetic resonance imaging (MRI) or fluorodeoxyglucose positron emission tomography (FDG-PET)] may help in discriminating an inflammatory or fibrotic process in the intestinal wall [47].

Finally, infliximab significantly improved quality of life in patients with active CD, increasing their ability to work and significantly reducing hospitali- sations, surgeries and procedures compared with placebo [50–55].

Adverse Events and Side Effects

Infliximab is a drug with low side effects. Up to now, more than 300,000 patients have been treated with infliximab for rheumatoid arthritis and CD [56]. The more frequent side effects, generally mild, are headache, nausea, upper respiratory infections, skin rash and diarrhoea. However, the incidence of these side effects is similar to that observed in the placebo group, except for upper respiratory infections, which are more common in patients treated with infliximab [23, 56–58].

Adverse events include acute and delayed hyper- sensitivity reactions. Acute reactions (1–20%) appear during or within 2 h of infusion and are not immunoglobulin E (IgE) mediated. They include headache, nausea, dyspnea, urticaria and coercion chest perception. Treatment of acute reactions con- sists of temporarily stopping the infusion, adminis- trating antihistamine drugs and/or steroids and then restart infusion at a low rate [13, 22, 23, 57–59]. If acute reactions recur, retreatment is not recom- mended [13]. Delayed hypersensitivity reactions occur after 3–12 days after infusion in 2–3% of patients. Generally, these reactions are serious and consist in itch, headache, swelling of hands or face, rash, myalgia, polyarteritis, fever and leucocytosis.

Steroids induce complete and immediate resolution of symptoms without recurrences. When delayed hypersensitivity reaction occurs, retreatment is not recommended.

Adverse events are caused by the development of antibodies to infliximab (ATI) related to its chimeric nature as it consists of 25% murine proteins. The incidence of ATI is low (10–20%) and at low titre [12, 60, 61]. The presence of ATI reduces drug efficacy in term of percentage of active response (clinical remis- sion) and duration of effectiveness (lower in patients who develop ATI) [12, 62]. Strategies to decrease ATI production are:

– scheduled therapy (not “on demand”): single dose or sporadic infusion causes higher incidence of ATI [30, 63, 64].

– Steroids pretreatment before infliximab infusion

(particularly in the induction phase): hydrocorti- sone 100 mg i.v. [60, 65].

– Two- to four-month immunosuppressant pre- treatment before first infliximab infusion: azathio- prine 2 mg/kg per day [57, 65, 66].

Risks: Infections, Tumours, Heart and Neurologic Diseases

TNF- α plays an essential role in the immune-mediat- ed response against infection, especially towards intracellular pathogens [67–70]. Many data support the association between TNF- α blockers and infec- tions [71–74]. Susceptibility to all types of infections is higher, particularly upper respiratory tract infec- tions and bacterial skin infections [74, 75]. TNF- α antagonists should not be given to patients with active infections. Severe pulmonary and abdominal infections (pneumonia, abdominal abscess) are more frequently observed in patients treated with inflix- imab than placebo.

Many reactivation cases of pulmonary and extra- pulmonary tuberculosis have been reported during the first 3 months of infliximab therapy [72, 76–78].

Screening for tuberculosis (skin test and chest X-ray) before anti-TNF therapy is strongly recommended [67, 76]. Some authors suggested that if the skin test is negative, treatment with infliximab may be given without prophylactic therapy. If the skin test is posi- tive, an accurate clinical history, physical examina- tion and chest X-ray are required to rule out active disease [67]. However, it should be considered that:

(1) a concomitant use of steroids or immunosup- pressant drugs may interfere with the skin test; (2) a large number of patients with IBD are anergic, with a delayed hypersensitivity reaction; (3) more than 15%

of patients with active tuberculosis (TBC) infection are anergic; (4) more than 90% of patients with posi- tive skin test have a negative chest X-ray. Therefore, an accurate clinical history, skin test and chest X-ray are mandatory before infliximab administration in all cases. In the absence of tuberculosis lesions at chest X-ray but with a clinical history suggestive for TBC and/or in patients at strong risk for TBC infec- tion (immigrant, injection-drug users, HIV patients, diabetes mellitus, etc.) empirical isoniazid for 6–9 months is a reasonable prophylactic treatment, start- ing infliximab after 2 months if clinically required. In the presence of TBC lesions at chest X-ray, active therapy is mandatory for at least 2 months before infliximab therapy.

Very rare cases (less than 0.01%) of fungal infec- tion (cryptococcosis, coccidioidomycosis, listeriosis and histoplasmosis) during infliximab therapy are reported [71, 73, 79–94]. Screening and primary pro- phylaxis are not recommended, but patients with a

history of these infections can receive secondary pro- phylaxis with oral fluconazole.

Patients eligible for infliximab therapy should be tested for hepatitis B and C serological markers before treatment. It has been documented that TNF- α levels are increased in patients with viral hepatitis [95, 96]. Inhibition of TNF-α may alter host antiviral defence mechanisms and promote virological repli- cation [97]. The safety of infliximab in hepatitis C has been demonstrated in a retrospective study on patients with rheumatoid arthritis, as well as in case reports [98–102]. In hepatitis B, reactivation of infec- tion has been documented, as has a case of fulminat- ing hepatitis B after one infusion of infliximab [97, 103–107]. However, definitive conclusions cannot be made regarding the use of infliximab in patients suf- fering from viral hepatitis. If infliximab therapy is necessary in patients with hepatitis B and C, we sug- gest monitoring viral replication during therapy and treating hepatitis B patients with lamivudine if serum HBV-DNA levels increase [104]. Prophylaxis with lamivudine should be considered for every carrier patient (HbsAg-positive) [104, 106].

Newly diagnosed neoplasia has occasionally been reported in trials using infliximab in CD although the possible role of infliximab in the pathogenesis of can- cer is unclear [108]. However, in a recent multicentre matched-pair study, the frequency of a new diagnosis of neoplasia in CD patients treated with infliximab was comparable with CD patients never receiving infliximab [109]. In particular, the development of lymphoma seemed not to be associated with inflix- imab therapy but with immunosuppressive drugs, particularly azathioprine, as previously reported [110–115].

Infliximab is not indicated for patients with mod- erate to severe congestive heart diseases [New York Heart Association (NYHA) classification III–IV]

[116, 117]. Serum levels of TNF- α are elevated in patients with heart failure and correlates with its severity [118–121], but the use of infliximab (at a dosage of 10 mg/kg) significantly increases mortality and morbidity in patients with congestive heart fail- ure [122]. Possible pathogenic mechanisms worsen- ing congestive heart failure in infliximab-treated patients are quite complicated and are reported in a recent review [123].

Multiple sclerosis, demyelination and optic neuri- tis associated with anti-TNF- α therapy are reported [124–127], but a recent study shows that demyelinat- ing disease occurs more commonly among patients with IBD than among non-IBD patients [128]. Future studies should clarify whether treatment with inflix- imab results in further increased incidence of neuro- logic diseases among IBD patients.

In pregnant women, drug safety has not been

investigated, but some authors [129] found no differ- ence in live birth and spontaneous abortion in an infliximab-treated group compared with healthy women. It is no clear if the drug is present in moth- er’s milk. Patients who undergo infliximab therapy should use a contraceptive method and, in the pres- ence of a new pregnancy, biological therapy should be stopped until the end of lactation.

Postsurgical Patients

Prophylactic therapy after resection for CD may pre- vent recurrence of the disease. However, no clear prophylactic drug regimens have been identified. In the last decade, endoscopic recurrence has been con- sidered a relevant outcome in patients suffering from CD since they are strictly related to the clinical out- come. After surgery, more than 70% of patients at 1 year and more than 85% at 3 years will have an endo- scopic recurrence in the neoterminal ileum [130–134]. Consequently, more than 20% of patients will already have a clinical recurrence within the first year after surgery, with a 10% increase in each subse- quent year [135]. The pattern of CD remains unchanged after surgery. Fistulising disease before surgery will have the same complications after resec- tion and early recurrent symptoms [131].

Standard therapy with steroids or 5-ASA formula- tions are unable to prevent development of new lesions at 1 year after resection whereas nitroimida- zole antibiotics (metronidazole and ornidazole) seem to be effective in preventing endoscopic and clinical recurrences [136, 137]. However, some patients are intolerant to nitroimidazole antibiotics. The benefit of immunosuppression for preventing endoscopic and clinical recurrence is lower than expected yet still high (70% and more than 50%, respectively, at 1 year).

The role of biologic therapy in postsurgical patients is still controversial, and no controlled trials are present in literature. However, aggressive disease, young age and intolerance to standard therapies (steroid dependence; steroid-refractory or immuno- suppressant side effects) may represent an indication for infliximab therapy in the postsurgery period to prevent disease recurrence, as recently suggested [131].

New Drugs: What We Expect in the Near Future

Other Anti-TNF-Blocking Strategies

Adalimumab is a humanised immunoglobulin G (IgG)1 monoclonal antibody directed against TNF- α

containing only human peptide sequences. It is indis- tinguishable in structure and function from natural- ly occurring human IgG1 antibodies. This molecule binds soluble and membrane-bound TNF, fixes com- plement and induces macrophage and T-cell apopto- sis. The recently published CLinical assessment of Adalimumab Safety and efficacy Studied as Induc- tion therapy in Crohn’s (CLASSIC-I) study seems to be superior to placebo for induction of remission in patients with moderate to severe CD naïve to anti- TNF therapy [138], confirming results of previous open-labelled studies [139–143]. TNF- α exerts proin- flammatory effects by binding to two specific trans- membrane receptors, p55 and p75.

Etanercept is a human recombinant p75 recep- tor/IgG fusion protein highly effective in rheumatoid arthritis. Etanercept was shown to be ineffective in patients with moderate to severe CD [144, 145] and at the moment does not have marketing approval for any indication in IBD. In vitro examination of T lym- phocytes the lamina propria of patients with CD seems to demonstrate that etanercept can neutralise TNF-α but does not induce apoptosis of inflammato- ry cells, differently from infliximab [146]. This exper- imental evidence probably explains the differences in efficacy between the two TNF-α-neutralising drugs.

However, efficacy of etanercept administration on CD activity was assessed at 8 weeks [145]. Since the biological effects of the drug begin after 4–8 weeks, further clinical trials with a longer treatment period are necessary to reach definitive results.

Onercept is a recombinant form of the human sol- uble p55 TNF receptor, which neutralises the effect of TNF- α. An open-labelled pilot study with onercept suggests that this agent may be effective and well tol- erated in patients with active CD [147]. However, other randomised double-blind trials are necessary to make definitive conclusions.

CDP571 is a humanised monoclonal IgG4 anti-

body anti-TNF- α (95% human, 5% murine). The first

study using a single dose of 5 mg/kg i.v. in a limited

number of patients reduced disease activity in CD at

2 weeks [148]. A successive study using a higher dose

of CDP571 (10 or 20 mg/kg) demonstrated a signifi-

cant clinical response in patients with moderate-to-

severe CD compared with placebo. In the same study,

retreatment with 10 mg/kg CDP571 at dose intervals

of 8 or 12 weeks seemed to be beneficial although not

statistically significant, probably due to the low num-

ber of patients with long-term treatment [149]. In a

later randomised double-blind placebo-controlled

study, patients with steroid refractory CD in clinical

remission were enrolled to verify corticosteroid spar-

ing at weeks 8 and 16. CD patients were treated with

a loading dose infusion (20 mg/kg) at week 0 fol-

lowed by a maintenance dose infusion (10 mg/kg) at

week 8 to ensure adequate plasma concentrations of CDP571 throughout each 8-week dosing interval.

Corticosteroid sparing was achieved in a higher number of patients treated with CDP571 than in those treated with placebo at week 16 [150]. Repeat- ed treatment (every 8 weeks up to 24 weeks) did not seem to increase drug efficacy [151]. In conclusion, CDP571 may provide an alternative option in patients with previous hypersensitivity to infliximab considering the very low risk for TBC or opportunis- tic infections during CDP571 therapy.

Certolizumab pegol (CDP870) is a polyethylene glycolated Fab’ fragment of a humanised anti-TNF- α monoclonal antibody intended for subcutaneous administration. A phase II dose-ranging trial, enrolling patients with moderate to severe CD, was recently published. Certolizumab at 4-week intervals and dosage of 400 mg induces a rapid treatment effect (2 weeks) and is clinically effective compared with placebo [152]. Certolizumab was well tolerated and, similarly to CDP571, gives potentially more advantages in clinical practice compared with inflix- imab. However, a phase III trial is needed to confirm these results and to better understand the precise role of this drug in the management of CD.

Thalidomide, an old drug recalled for the terato- genic effects in pregnant women, has complex and incompletely understood immunomodulatory prop- erties. Multiple mechanisms of action have been reported, particularly its ability to inhibit production of TNF- α, inhibiting neoangiogenesis and shifting immune response Th1 to Th2. Some recently pub- lished papers seem to demonstrate that thalidomide administered for 12 weeks is effective in patients with steroid-resistant or steroid-refractory CD [153–157].

However, about 30% of patients withdrew from the clinical studies due to the development of severe adverse events. Preliminary reports also indicate that thalidomide is effective as maintenance treatment after induction of remission with infliximab [157].

Despite side effects (teratogenicity and peripheral neuropathy) and adverse events, thalidomide may be an alternative medical treatment in a select subgroup of patients.

Anti-Inflammatory Cytokines

IL-10, discovered in 1989, has shown potent anti- inflammatory properties in in vitro and in vivo ani- mal studies. IL-10 exerts its anti-inflammatory effects by down-regulating production of proinflammatory cytokines by dendritic cells, macrophages and mono- cytes. In clinical trials, a modest benefit was observed in patients treated with subcutaneous injection of humanised IL-10 [158-161]. In postsurgical CD

patients, IL-10 administration did not show any effect [162]. The efficacy of IL-10 is lower than expected, probably as a consequence of the stimula- tion of INF-γ production [158]. Furthermore, serum levels and tissue concentrations of IL-10 are general- ly elevated in CD patients [5].

Recent experimental studies demonstrated that the delivery of tissutal immunoregulatory proteins, including IL-10, by gene therapy may be a potential- ly exciting approach to treatment of IBD [163].

Future challenges for using gene therapy in CD will be to establish safety, localisation and duration of the expression of the transduced gene, as well as to ensure that delivery of immunoregulatory proteins is therapeutic.

IL-11 has been shown to be expressed and have activity in multiple other tissues, including gut, but to date, the physiologic role of this protein remains unknown. Preliminary data on short-term treatment with humanised IL-11 demonstrated that given sub- cutaneously for 3 weeks (16 mg/kg per week), the drug is well tolerated in patients with active CD [164].

A recent randomised trial reported that weekly sub- cutaneous injection with rhIL-11 (15 mmg/kg per week) compared with placebo is effective in inducing remission in patients with active CD [165]. Potential- ly, IL-11 may be an alternative or sparing agent for corticosteroids in the management of CD, but its role remains unclear.

Selective Inhibition of Adhesion Molecules

Adhesion molecules, up-regulated in inflamed CD mucosa, play a role in the trafficking of leukocytes and are involved in local lymphocyte stimulation and antigen presentation. Integrins are heterodimeric receptors consisting of an α4 subunit and either a β1 or β7 subunit (α4 β1 and α4 β7 integrins). Integrins bind leukocytes in the vascular endothelium and have a role in the migration of leukocytes across the vascular endothelium. Therefore, they contribute to recruitment, activation and survival of leukocytes within the parenchyma. There is evidence that in IBDs, integrins are overexpressed on the vascular endothelium at the site of inflammation. In animal models of colitis, antibodies against the a4 integrin are able to significantly reduce gut inflammation [166].

Recent double-blind placebo-controlled studies

using natalizumab, a humanised mouse monoclonal

antibody anti-a4 integrin (95% human, 5% murine),

in patients suffering from CD administrated at

dosage of 3 mg/kg per month twice improve clinical

remission and clinical response rates, as well as qual-

ity of life [167–169]. However, three cases of progres-

sive multifocal leukoencephalopathy (PML), proba- bly secondary to reactivation of latent JC poly- omavirus infection, have been reported [170–172].

Retrospective analysis of serum samples showed that JC virus became detectable only after three injections of natalizumab monotherapy, and the serum viral load increased by a factor of ten after two additional injections. Although the large number of patients enrolled in this clinical trial (over 3,000) and, conse- quently, the low incidence of neurologic disease in natalizumab-treated patients, the lack of diagnosis of latent infection before natalizumab treatment limits, due to ethical concerns, its clinical use [173]. In fact, there is no specific treatment for PML at the moment.

A better understanding of the risk of PML in natal- izumab-treated patients and the diagnostic possibili- ties for a latent JC polyomavirus infection will define the role of the drug in the treatment of CD.

Immunomodulators

Tacrolimus (FK506) is a macrolide antibiotic isolated from Streptomyces tsukubaensis with greater immunomodulatory properties than cyclosporine and good oral absorption. Consequently, it has been approved for the prophylaxis of organ rejection in transplant patients. The proposed dosage in CD patients is 0.1 mg/kg administered orally, adjusted for each patient to reach a blood concentration range between 5 and 15 ng/ml. Clinical open-labelled stud- ies suggest the effectiveness and safety of short-term and long-term oral tacrolimus therapy in patients with perianal and enterocutaneous fistulising CD [174–178]. However, side effects are numerous and frequently reported by the patients. They include gastrointestinal symptoms (abdominal pain, diar- rhea, nausea), headache, insomnia, paresthesias, tremor, temporary rise of creatinine, hyperkaliemia, hypertension and opportunistic infections. There- fore, tacrolimus may be an alternative drug in fis- tulising CD if no success is achieved with conven- tional therapy, including infliximab.

Mycophenolate mofetil (MMF) is an antimetabo- lite that undergoes ester hydrolysis in mycophenolic acid, its active form. Mycophenolic acid inhibits an enzyme required for de novo guanosine nucleotide biosynthesis and consequently blocks production of necessary precursors for synthesis of DNA and RNA.

The action of MMF is considered to be directed in particular against T and B lymphocytes, interfering with the immune system. MMF has been used as an immunosuppressant in the posttransplant patients but also in chronic inflammatory diseases, particu- larly in patients suffering from rheumatoid arthritis and psoriasis, as with other drugs presented in this

review. Earlier studies showed a potential role of MMF in CD patients intolerant to azathioprine that was not confirmed in other studies [179–183]. How- ever, a potential steroid-sparing effect has been sug- gested. A recent open-labelled long-term-treatment study in patients with complicated CD shows that MMF at dosage of 1 g/b.i.d. induces an initial response (at 6 months) in more than one half of the patients, with a significant reduction in steroid dosage. However, a large number of patients relapsed during the long-term follow-up (median 18 months).

Furthermore, adverse events determined discontinu- ation of treatment in 20% of long-term-treated patients [184]. Therefore, we strongly believe that MMF, similarly to other immunosuppressant, has a possible therapeutic role in short-term treatment of steroid-resistant or steroid-refractory CD patients intolerant to azathioprine.

Helminths

Recent experimental and clinical evidence suggest that helminthic parasites may be considered as a therapeutic option in IBDs. CD is a result of an inap- propriate immune response towards normal gut flora and, probably, helminths down-regulate the host immune response towards bacterial antigens. The high prevalence of CD in industrialised Western regions and the low incidence in developing coun- tries may be related to the different incidence of helminthic infestation. ‘‘IBD hygiene hypothesis’’ has been formulated based on induction of the disease by extremely hygienic environments in genetically pre- disposed subjects. Data from experimental colitis seems to show that helminths reduce inflammation, probably counteracting the Th1-driven immune response [185]. In fact, experimental colitis induced in mice and rats with di- or trinitrobenzene sulfonic acid (DNBS, TNBS), develop a Th1-cytokine-driven colitis that shares features with CD [186, 187]. Expo- sure to Schistosoma mansoni or Trichinella spiralis attenuates DNBS-induced colitis and indicates a pro- tective role of nematode infection in Th1-cell-driven inflammation [188]. The authors suggested the possi- bility of an immunological distraction with helminths as a novel therapeutic strategy in CD.

Recently, therapy with Trichuris suis eggs in

patients suffering from CD has been postulated. The

life cycle of T. suis begins with the ingestion of

embryonated eggs. Embryonated eggs hatch in the

proximal small bowel, delivering larvae that migrate

aborally and attach to the mucosa of the distal small

bowel and proximal colon. After several weeks, they

mature and begin to spread eggs. T. suis ova are

capable of colonising a human host for several weeks

and are eliminated from the body without any specif- ic therapy (self-limiting infection). In an preliminary study, a single dose of 2,500 live T. suis eggs given orally in patients suffering from active CD induced a clinical response in all patients treated and a clinical remission in the large part of cases [189]. Later, a repeated dose of 2,500 live ova given orally every 3 weeks for 24 weeks induced a clinical response in 80% of patients and a clinical remission in 70% [190].

The positive results of these preliminary open- labelled studies showed that helminth therapy is tol- erated and effective in the treatment of CD.

Helminths inhibit intestinal inflammation by mecha- nisms different from current medications, and the efficacy of T. suis therapy supports the hypothesis that helminthic exposure provides protection against CD.

Probiotics

A possible pathogenesis of IBD includes mucosal inflammation secondary to abnormal immune response against resident bacteria. The intestinal environment is a persisting stimulus in normal sub- jects. In CD patients, normal intestinal bacteria in genetically predisposed subjects or altered intestinal flora may induce and amplify gut inflammation.

Therefore, manipulation of intestinal bacterial flora is the rationale for the use of antibiotics. In the last decade, probiotics (living micro-organisms with a beneficial effect on the host), prebiotics (dietary components that induce the growth of beneficial bac- teria) and synbiotics (both the previous) have been used in the treatment of CD. Despite the lack of ran- domised clinical trials demonstrating efficacy of this therapeutic approach to CD patients, a number of review articles have been published during the last years outlining its efficacy in the treatment of IBD [191–193]. Based on experimental evidence, many strains are tested for maintenance of remission in CD patients.

Single-strain probiotics (Lactobacillus GG) at dif- ferent dosages is not effective to maintain remission in patients suffering from CD [194–196]. Probiotics containing miscellaneous strains at high dosage may be theoretically more effective than a single strain in the treatment of CD, probably due to a synergic effect. Recently, VSL#3, containing three strains of Bifidobacterium (B. longum, B. infantis, B. breve), four strains of Lactobacillus (L. acidophilus, L. casei, L. delbrueckii, L. plantarum) and one strain of Strep- tococcus salivarius subsp. thermophilus has been proposed in the treatment of IBD. The postulated pathogenic mechanism of VSL#3 is induction of tis- sutal levels of the anti-inflammatory cytokine IL-10

and a greater expression of intestinal mucin, pre- venting adhesion of pathological Escherichia coli strains. Efficacy of VSL#3 is being tested in a double- blind randomised trial for maintenance of remission in recurrent chronic pouchitis.

Results of randomised double-blind clinical trials are being awaited in order to make definitive conclu- sions on the use of probiotics in CD patients.

Conclusions

The advent of biological therapy provides the oppor- tunity to advance the care of patients with IBD because we have opportunities to interrupt the dis- ease pathophysiology in a more specific way. The infliximab experience teaches that interfering with cytokine-mediated inflammation improves the clini- cal outcome of severe CD.

New biological drugs are coming in the near future. Can we treat severe CD by blocking more than one inflammatory mediator? The mechanism of inflammation in cytokine-mediated CD is quite com- plex. Cytokines are numerous, act at very low titre (potent biological activity), have many target cells and the cytokine response is mediated by specific cel- lular receptors. Furthermore, different cytokines produce the same effects, and large interactions among cytokines are documented. We can postulate that interfering with two or more cytokines may improve the clinical course of CD. However, we do not know yet if this approach may alter immune defence towards pathogens and immune surveillance on carcinogenesis, and we must consider that biolog- ics are expensive drugs.

Biological drugs present a major challenge: the more effective they are, the more side effects they can produce. Therefore, biological therapy should care- fully managed, with close observance for possible adverse events and provision for any complication resulting from treatment. Inappropriate manage- ment of these potent drugs will compromise their use in clinical practice.

Future clinical studies should address efficacy, safety and cost effectiveness of biologic therapies for CD and should provide the necessary information to take maximum advantage of these new therapies.

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