You are on page 1of 9

Biology of Blood and Marrow Transplantation 9:417-425 (2003) 2003 American Society for Blood and Marrow

Transplantation 1083-8791/03/0907-0001$30.00/0 doi:10.1016/S1083-8791(03)00151-4

Halofuginone to Treat Fibrosis in Chronic Graftversus-Host Disease and Scleroderma
Mark Pines,1 David Snyder,2 Shai Yarkoni,2 Arnon Nagler3
Institute of Animal Science, Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel, 2Collgard Biopharmaceutical Ltd, Tel Aviv, Israel; and 3Department of Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer, Israel Correspondence and reprint requests: Dr. Mark Pines, Institute of Animal Science, ARO, the Volcani Center, Bet Dagan 50250, Israel (e-mail: Received February 28, 2003; accepted May 1, 2003

Chronic graft-versus-host disease (cGvHD) and systemic sclerosis (scleroderma [SSc]) share clinical characteristics, including skin and internal organ fibrosis. Fibrosis, regardless of the cause, is characterized by extracellular matrix deposition, of which collagen type I is the major constituent. The progressive accumulation of connective tissue results in destruction of normal tissue architecture and internal organ failure. In both SSc and cGvHD, the severity of skin and internal organ fibrosis correlates with the clinical course of the disease. Thus, there is an unmet need for well-tolerated antifibrotic therapy. Halofuginone is an inhibitor of collagen type I synthesis in cells derived from various tissues and species and in animal models of fibrosis in which excess collagen is the hallmark of the disease. Halofuginone decreased collagen synthesis in the tight skin mouse (Tsk) and murine cGvHD, the 2 experimental systems that show many features resembling those of human GvHD. Inhibition of collagen synthesis by halofuginone is achieved by inhibiting transforming growth factor – dependent Smad3 phosphorylation. Dermal application of halofuginone caused a decrease in collagen content at the treated site of a cGvHD patient, and reduction in skin scores was observed in a pilot study with SSc patients. The results of the human studies provide basis for using halofuginone treatment for dermal fibrosis. As a first step toward future treatment of internal organ involvement, an oral administration study was performed in which halofuginone was well tolerated and plasma levels surpassed the predicted therapeutic exposure. © 2003 American Society for Blood and Marrow Transplantation




INTRODUCTION Tissue fibrosis, including pulmonary, renal, hepatic, and cardiac fibrosis, is manifested as excessive production of connective tissues that results in destruction of normal tissue architecture and function. This review focuses mainly on the antifibrotic potential of a novel and promising agent, halofuginone [1-3], with emphasis on chronic graft-versus-host disease (cGvHD) and systemic sclerosis (SSc) that share clinical characteristics including skin and internal organ fibrosis. FIBROSIS Fibrosis represents the response of the organ to diverse acute and chronic insults. Fibrosis, regardless

of the cause, is characterised by an increase in extracellular matrix (ECM) constituents, of which collagen type I is the major one. Although the synthesis and systemic accumulation of collagen and noncollagen matrix proteins are essential for normal tissue development and wound repair, excessive matrix accumulation leads to pathologic fibrosis. The pathogenesis of the fibrotic disorders is similar, regardless of the tissue, and the cellular mechanism of fibrosis is shared among the various insults. This mechanism in many aspects mirrors the scarring and wound-healing processes, but the persistent activation of the genes encoding for the ECM proteins distinguish controlled wound repair from the uncontrolled connective tissue deposition that results in fibrosis. In addition to the


M. Pines et al.

ECM synthesis and deposition, and not less important to the progression of fibrosis, there are inadequate degradation, removal, and clearance of the ECM. In the extracellular space, matrix degradation occurs predominantly as a consequence of the actions of the matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) that closely regulates MMP activity. The MMPs and TIMPs play a crucial role in the fine regulation of the ECM turnover that is altered in most pathologic states associated with fibrosis [4,5]. The fibrotic reaction is thought to involve the stimulative response of tissue cells that results in increased cell proliferation as well as ECM deposition. In some cases, such as skin, pulmonary, and kidney fibrosis, fibroblasts are thought to play a pivotal role in collagen synthesis [6,7]. In the liver and pancreas, other resident cells, the stellate cells, were found to be the cellular source of collagen and other ECM molecules [8,9]. Regardless of their origin, and in response to the fibrogenic stimulus, the cells that are usually quiescent with low proliferation rates differentiate into myofibroblast-like cells, with high proliferative capacity and an ability to synthesize large numbers of ECM molecules, especially collagen type I [10,11].

CGVHD AND SSC cGvHD is a major late complication of allogeneic stem cell transplantation (alloSCT) and is a principal cause of morbidity and nonrelapse mortality. As a result of increasing numbers of unrelated and mismatched alloSCT and of the change from bone marrow to peripheral blood grafts, the number of patients with cGvHD has increased [12]. Chronic GvHD may develop in 25% to 45% of recipients of alloSCT from matched, related siblings depending upon age, and its frequency may increase to 40% to 70% in patients receiving alloSCT from matched, unrelated donors [13,14]. The incidence is significantly lower in patients receiving human umbilical cord blood grafts or haploidentical CD34 purified grafts [15,16]. Chronic GvHD may develop as an extension of acute GvHD (progressive onset), after resolution of acute GvHD (quiescent onset), or without proceeding acute GvHD (de novo onset). Chronic GvHD is categorized as either limited (localized skin and/or hepatic involvement) or extensive (diffuse skin and/or multiorgan involvement); the latter is associated with a more severe prognosis [17]. The introduction of low-intensity conditioning (LIC) and nonmyeloablative alloSCT has not resulted in a reduction in the frequency of cGvHD [18]. cGvHD usually develops more than 100 days after transplantations, with increased frequency in patients receiving peripheral blood stem cell grafts and in those undergoing LIC alloSCT [12,18].

Chronic GvHD typically resembles connective tissue autoimmune-like immunologic disorder such as SSc characterized by lichenoid or sclerodermoid lesions of the skin, along with joint contractors with eosinophilia, circulating autoantibodies, hypergammaglobulinemia, and plasmacytosis of viscera and lymph nodes [17]. The skin includes papulosquamous dermatitis, plaques, desquamation, depigmentation and vetiligo, and thickened reticular dermis because of increased synthesis of collagen [19]. Cutaneous appendages become encased in collagen and tend to disappear. There is occurrence of a variable perivascular and interstitial inflammatory cell infiltrate, composed predominantly of lymphocytes and occasional plasma cells [20]. Less-frequent skin findings include poikiloderma, reticulated hyperpigmentation, alopecia, dystrophic nails, leucoderma bullae, discoid lupus erythematosus, and photosensitivity. The pathophysiologic mechanism involves autoreactive lymphocytes and cytokine dysregulation [21,22]. Current therapeutic options are limited. Fewer than 20% of patients with untreated extensive cGvHD with Karnofsky performance scores 70% survive [17]. Currently, corticosteroids, alone or in combination with cyclosporine, remain the treatment of choice for established cGvHD. Other therapeutic options that have been tried include thalidomide, mycophenolate mofetil, tacrolimus, rapamycin, clofazimine, etretinate, hydroxychloroquine, ursodeoxycholic acid, penicillamine, cyclophenile, and nedocromil sodium, as well as medical procedures such as total lymphoid irradiation, phototherapy (PUVA), and extracorporeal phototherapy [23]. SSc is a connective tissue disease with an unknown cause. As an autoimmune disease involving cellular and humoral immunity, it affects multiple organ systems, including skin, gastrointestinal tract, lungs, kidneys, and heart. The severity of skin and internal organ involvement correlates with the clinical course of the disease [24]. According to the degree of skin involvement, SSc can be classifies as limited cutaneous SSc or diffuse cutaneous SSc. Limited cutaneous SSc, which is slowly progressive and frequently associated with anticentromere antibodies, is characterized by sclerodactyly or acrosclerosis, with distal involvement of the extremities with or without facial involvement, Raynaud’s phenomenon, dysphagia, calcinosis cutis, and telangiectasis [24]. The most severe systemic complications are pulmonary hypertension and biliary cirrhosis. Diffuse cutaneous SSc is more severe and shows proximal involvement of the extremities, trunk, or both, and is associated with pulmonary interstitial fibrosis, renal crises, and gastrointestinal involvement (dysphagia, hypomotility, and other disorders). Diffuse cutaneous SSc is frequently associated with Scl-70 (antitopoisomerase) and nucleolar autoantibodies

Halofuginone, cGvHD, and SSc

(polymerase I and III, fibrillarin). The mechanism of fibrosis in SSc is not fully understood, although it is known that soluble mediators (transforming growth factor [TGF- ], platelet-derived growth factor, interleukin-4 and -6, tumor necrosis factor [TNF- ] can be secreted by lymphocytes, monocytes, and endothelial cells and can affect the behavior of fibroblast growth, proliferation, collagen synthesis, and chemotaxis [25,26]. The excessive tissue fibrosis is caused by expansion of fibrogeneic clones of tissue fibroblasts, which behave relatively autonomously and overexpress genes encoding ECM components, leading to excessive deposition of collagen and other ECM proteins in skin and internal organs and blood vessels [27]. Present therapies are directed to improve peripheral blood circulation with vasodilators and antiplatelet aggregation drugs, to prevent the synthesis and release of harmful cytokines by means of immunosuppressants, and to inhibit or reduce fibrosis with agents that interfere with collagen synthesis or enhance collagenase production. Autologous stem cell transplantation was performed after immunoablative pretreatment in diffuse SSc patients with involvement of 1 or more internal organs [28]. The mortality rate was 17%, in part caused by interstitial pneumonitis, possibly as a result of an exaggerated response to total body irradiation. Seventy percent of patients had over 25% improvement in their skin score and stabilization of lung function. This is still an experimental therapy and currently limited to patients with severe, progressive, life-threatening disease [29]. The treatment of SSc remains a challenge to the clinician, and there is an unmet need for new therapies [30].

II [33] or III [34]. Halofuginone inhibited collagen synthesis in a variety of cells, including primary cultures of mouse skin fibroblasts, avian growth plate chondrocytes, a transformed rat embryo cell line, vascular smooth muscle cells, bovine aortic endothelial cells, and rat liver stellate cells [33-36], all of which produce and secrete collagen type I. In animal models of fibrosis in which excess collagen is the hallmark of the disease, administration of halofuginone prevented the increase in collagen synthesis and collagen 1(I) gene expression. These models include rat postoperational abdominal and uterine horn adhesions [37,38], rat urethral stricture formation [39], thioacetamideand dimethylnitrosamine-induced rat cirrhosis [36,40], rat pulmonary fibrosis after bleomycin treatment [41], and tight skin (Tsk) and cGvHD-afflicted mice [4244]. Inhibition was independent of the route of administration (intraperitoneally, administered locally, or given orally). The reduction in collagen synthesis by halofuginone appears not to be a direct effect of halofuginone on the collagen 1(I) promoter but rather dependent on new protein synthesis, because simultaneous treatment of fibroblasts with protein synthesis inhibitors such as cycloheximide or actinomycin D blocks the suppressive effect of halofuginone on collagen 1(I) mRNA gene expression [45]. The nature of the newly synthesized protein(s) and how they function to decrease procollagen mRNA levels is unknown.

HALOFUGINONE IN ANIMAL MODELS OF CGVHD AND SSC Although there is no animal model that exhibits all aspects of cGvHD or SSc [46], 2 experimental systems are available that show many features resembling those of the human disease [47]. The first is murine cGvHD, in which changes develop that are similar to human cGvHD and SSc, including cutaneous fibrosis, loss of dermal fat, atrophy of dermal appendages, deposition of mast cells, and mononuclear cell infiltration [48]. The second model is the Tsk mouse possessing a single gene mutation in the fibrillin-1 gene on chromosome 2 [49]. The mutation is transmitted as an autosomal dominant trait, whereas the heterozygous Tsk/ mice develop coetaneous hyperplasia and connective tissue abnormalities in skin, heart, and lungs [50,51] associated mainly with an increase in collagen type I [52,53]. These mice produce autoantibodies characteristic of SSc patients [54]. In vitro halofuginone inhibited collagen production by Tsk skin fibroblasts by inhibition of the collagen gene expression [44]. The reduction in collagen synthesis preceded any effect on cell proliferation or synthesis of other proteins, which suggests a specific effect on the collagen biosynthesis pathway. In vivo

HALOFUGINONE For centuries, the roots of Dichroa febrifuga, a saxifragaceous plant, have been employed in China in the treatment of malarial fever. Febrifugine and its stereoisomere, isofebrifugine, were identified as the active components against malaria. Febrifugine analogues bearing a modified or unmodified 4-quinazolinone ring are active, whereas those produced through the modification of the side chain attached to the N-3 position of the 4-quinazolinone are ineffective [31]. Halofuginone (7-bromo-6-chloro-3-[3-(3-hydroxy2-piperidinyl)-2-oxopropyl]-4(3H)-quinazolinone) is one of the febrifugine analogoues used worldwide in commercial poultry production for coccidiosis [32].

HALOFUGINONE AND THE COLLAGEN CONNECTION Serendipity was the driving force beyond the finding that halofuginone has an inhibitory effect on collagen synthesis [1-3]. In vitro halofuginone was found to inhibit collagen type I synthesis but not that of type


M. Pines et al.

halofuginone administered intraperitoneally eliminated the increase in skin collagen and prevented the thickening of the dermis and the loss of the subdermal fat in both the cGvHD and the Tsk models in a dose-dependent manner attributed to a decrease in collagen type I gene expression [42]. These effects of halofuginone were correlated with a decrease in the number of cells expressing the collagen 1(I) gene that reached the expression level of the matched controls. Moreover, reductions in the levels of autoantibodies specific for human target antigens (anti-topoisomerase I and antifibrillin antibodies) were observed [55]. These results raised an interesting question regarding the association of the ECM biosynthesis pathway and autoimmunity. It has previously been suggested that TGF- plays a critical role in fibrosis and is the pivotal immunoregulatory cytokine affecting the immune responses by regulating monocyte function, T-cell proliferation, and inflammation [56]. In an attempt to begin developing a local-therapy modality for SSc and cGvHD, the effect of dermal application of halofuginone on skin collagen and collagen 1(I) gene expression in the Tsk mice was determined. Dermal application of halofuginone (0.01% in cream) for 60 days was equal to the systemic administration (1 g per mouse per day) in reduction of skin collagen 1(I) gene expression and was almost as effective in reducing skin thickness. The effect of halofuginone was time-dependent; 40 days of treatment were needed for a significant reduction in the collagen 1(I) gene expression [43]. Physiologically, skin fibroblasts synthesize low levels of collagen. In the Tsk mouse, the onset of fibrosis coincides with the presence of a large number of fibroblasts expressing elevated levels of collagen type I gene [57], as was demonstrated in SSc [57,58]. The selective growth of the subpopulation of fibroblasts producing elevated levels of collagen has been suggested to be the causes of the fibrotic condition [59]. These fibroblasts are probably the target for halofuginone [43]. The effect of halofuginone was restricted to the dermis fibroblasts and did not affect other proliferating, but collagen-nonproducing cells, such as epidermal keratinocytes.

vated levels of TGF- were found in involved skin lesions of SSc patients [62,63], in murine models of sclerodermatous GvHD [64], and in the pathogenesis of graft vascular disease [65]. There was association between TGF- expression and severe GvHD in allogeneic bone marrow transplantation (BMT) [66]. TGF- is co-localized with collagen type I mRNA in involved lesions in the early stages of inflammation before the onset of fibrosis and was implicated in the initiation of fibrosis in SSc [26]. Following binding of TGF- to its receptor, signaling to the nucleus occurs predominantly by phosphorylation of cytoplasmatic mediators of the Smad family that are divided into 3 groups [60]. The regulation of matrix proteins in general [67] and of collagen type I gene expression in particular by TGF- involved the Smad3 signaling pathway [68,69], and increased presence of nuclear Smad3 has been observed in SSc fibroblasts [70].





TGF- is one of the leading candidates for eliciting overproduction of ECM proteins in wound healing and in various fibrotic conditions [60]. TGFmeets the criteria of a major participant in fibrosis: it is present in the affected tissues, and it induces profibrotic cellular and transcriptional responses such as induction of the synthesis of ECM proteins, especially collagen, as well as fibronectin and laminin, and it inhibits the matrix degradation enzymes [61]. Ele420

As a result of the extremely pleiotropic nature of the effects of TGF- , therapies targeting the expression of the genes encoding TGF- or its receptors are likely to be associated with an array of side effects caused by abnormal cell proliferation, inflammation, autoimmunity, and other potentially serious complications [71-73]. Drugs that selectively target individual signaling pathways downstream of the TGFreceptor are, therefore, likely to be more successful. Halofuginone was found to overcome the TGF- – induced collagen synthesis in human skin fibroblasts [45] and fibroblasts derived from control and Tsk mice [44]. A similar inhibitory effect was observed on promoter activity in fibroblasts transfected with the collagen 2(I) construct or with a construct with the Smad binding site, which suggests that the target of halofuginone is probably downstream in the TGFpathway. Although increased gene expression for TGF- receptors was observed in SSc fibroblasts [74], no effect of halofuginone was observed on the expression of the TGF- receptors gene in the Tsk fibroblasts, which further supports the hypothesis that downstream effects were involved. Halofuginone was found to inhibit phosphorylation of TGF-&beta– dependent Smad3, but not that of Smad2, which resulted in inhibition of Smad3 binding to DNA [44]. These results demonstrate the specificity of halofuginone activity, although the involvement of other members of the Smad family in halofuginone-dependent inhibition of collagen synthesis cannot be ruled out. In most animal models of fibrosis, regardless of the tissue, halofuginone had a minimal effect on collagen content in the control, nonfibrotic animals, whereas it exhibited a profound inhibitory effect in the fibrotic organs. These results suggest a different regulation of

Halofuginone, cGvHD, and SSc

the house-keeping and usually low level of expression of collagen type I genes on one hand, and the overexpression induced by the fibrogenic stimulus, which is usually an aggressive and a rapid process, on the other. Halofuginone mainly affects the stimulated collagen synthesis; therefore, when it was administered systemically, it was actually targeted to the desired fibrotic location without affecting collagen synthesis in other locations. In culture, halofuginone phosphorylated Smad3 and was more effective in reducing collagen synthesis by Tsk fibroblasts after they had been stimulated with a profibrotic agonist such as TGF- ; whereas no effect on Smad3 phosphorylation and reduced effect on collagen synthesis was observed in control cells [44].

HALOFUGINONE AFFECTS PREEXISTING FIBROSIS In cGVHD and SSc, the skin is not the only organ affected by fibrosis; survival studies in SSc and cGvHD patients have indicated a distinct dependency on the involvement of internal organs [17,24]. In cases of SSc, the prognosis was dependent on the involvement of internal organs, particularly the lungs, heart, and kidneys. Although the gastrointestinal tract is the most frequently affected organ, its involvement is less prognostic. As mentioned above, halofuginone has been found to be efficacious in inhibiting collagen synthesis in multiple organs. In most of these studies, halofuginone was used as a preventive agent; it was administered before— or together with—the fibrotic stimulus [37-41]. In the Tsk mouse, halofuginone treatment caused a decrease in the preexisting fibrotic condition as measured by changes in collagen gene expression, collagen content, and skin morphology [42-44]. In a liver fibrosis model using rats with established fibrosis, halofuginone administration resulted in complete resolution of the fibrosis. The levels of collagen, collagen 1(I) gene expression, and -smooth muscle–positive cells, all of which are characteristic of the fibrotic condition, were reduced almost to the control levels [36]. Furthermore, liver regeneration that was blocked in rats with established fibrosis occurred almost in a normal rate in halofuginone-treated rats [75].

HUMAN STUDIES FOR THE LOCAL TREATMENT OF SSC AND CGVHD A Phase I study with topical application of 0.1% halofuginone was conducted in 14 healthy volunteers. The results of the study showed safety and tolerability following repeated topical applications of halofuginone cream. There was no skin irritation or systemic absorption of halofuginone (Dr. S. Tamin, unpublished data, 1998). As a proof of principle, halofugi-

none cream was locally applied on the skin of a 22year-old male with acute promyelocytic leukemia who underwent successful BMT from his HLA-matched brother. Three-and-a-half months after BMT, he developed severe, extensive, de novo cGvHD with skin, mucosal, gastrointestinal tract, and liver involvement. The skin cGvHD was manifested by scattered lichenoid papules and mottled hyperpigmentation, mainly of the neck and upper chest and back, as well as of both axillae, lower abdomen, and flanks. Oral examination disclosed severe lichenoid lesions with hypertrophy of all the buccal and labial mucosa, atrophy of the papillae of the tongue with oral pain, xerostomia, and loss of taste. The skin cGvHD progressed, and within 8 months, the patient developed severe bilateral sclerosis and tightening of the skin of the neck resembling sclerodermoid changes that then progressed to contractures with pain and limitation of movements [76]. The protocol included once-daily topical application of 0.03% halofuginone ointment to the left side of the neck and the posterior aspect of the left shoulder for a period of 6 months, while the right and corresponding side served as control. After 1 month, softness of the left side of the neck and upper back was noted by clinical observation. There was no itching, rash, or erythema. Over the next few weeks, the softness intensified and was accompanied by gradual loosening of the sclerosis and tightness in the left side of the neck. In comparison, no change was observed in the untreated, control right side. No change in the skin pigmentation was noticed on either side. Within 3 months, there was an improvement in the pain and dysphagia, most probably related to the improvement in the severe sclerosis and fibrosis of the neck, and after 6 months of therapy rotation of the neck, only to the treated side, had improved from 30% to 70%. Skin biopsies were taken pretherapy from clinically involved and uninvolved areas at the left and right upper back below the scapulas, from the involvedtreated areas after 3 and 6 months of topical halofuginone therapy and 3 months following termination of halofuginone treatment [76]. The level of collagen 1(I) gene expression and the collagen content were much higher in the affected area compared with the macroscopically nonaffected area. Three months of halofuginone treatment caused a marked decrease in collagen 1(I) gene expression and collagen content, and a further decrease in both parameters was observed after an additional 3 months of treatment (Figure 1). However, 3 months after cessation of treatment, the levels of collagen 1(I) gene expression returned almost to baseline levels, indicating that the halofuginone-mediated inhibitory effect of collagen synthesis is reversible [76]. The transient effect of halofuginone was probably the result of the chronic process providing a continuing stimulus to fibrogenesis. Throughout the treatment period, there was no


M. Pines et al.

Figure 1. Collagen 1(I) gene expression. Skin biopsies were taken from the posterior upper back of the cGvHD patient before (A) and 6 months after (B) topical halofuginone therapy. The sections were hybridized with digoxigenin-labeled collagen 1(I) probe. Note the reduction in cells expressing the collagen 1(I) gene (arrows).

local or systemic toxicity, and no side effects were noticed. Repeated blood biochemistry tests of liver and kidney functions did not disclose any changes from the pretreatment values, blood counts were normal, and no halofuginone could be detected in the patient’s sera (high-performance liquid chromatography assay sensitivity for halofuginone is 1 ng/mL), all of which suggest that topical application of halofuginone is safe and will not cause any systemic effects in other locations. A phase II trial was performed to assess the safety and efficacy of halofuginone in diffuse SSc patients with changes in the upper extremities resulting in a modified Rodnan skin score of at least 10/21. The modified Rodnan skin thickness score measured on a 0-3 scale (0, normal; 1, mild thickness; 2, moderate thickness; 3, severe thickening) of 7 regions of the upper extremity was used [77]. Thirteen patients were treated once daily with topical application of 0.01% halofuginone on either left or right upper extremities (hand, forearm, upper arm). Eleven of these patients completed a 6-month treatment period. Safety results showed that there were no clinically relevant changes in vital signs (blood pressure, heart rate, body temperature, and body weight) and laboratory evaluations that included hematology (red blood cell, white blood cell, and platelet counts, hemoglobin, and hematocrit) and clinical chemistry (creatine phosphokinase, aspartate transaminase, -glutamyl transferase, lactose dehydrogenase, albumin, total and indirect bilirubin, urea, creatinine, glucose, sodium, and potassium). The most frequently reported adverse event that was at422

tributed to the treatment was dermatitis of varying degree and severity observed macroscopically that did not dictate cessation of the treatment. Efficacy evaluation results (changes in total skin score from baseline by 2 raters) showed a statistically significant reduction in the mean total skin score after 3 months of treatment (Table 1). Of 12 patients, 5 met responder criteria of at least 25% reduction in the total skin score in the treated arm, and the mean time for response was 2.6 months. The results of the human safety and efficacy studies provide a basis for the proof of concept of using halofuginone as the drug for treatment of dermal fibrosis and paved the way for a phase III placebo-controlled, double-blind trial.

Table 1. Total Skin Score of the Treated Arm of Responders and Nonresponders
Total Skin Score (Means Time of Treatment (months) Baseline Baseline 1 2 3 4 6 Efficacy-Eligible Patients (n 12) 12.5 11.8 10.0 10.8 9.6 10.4 10.1 2.4 2.0 4.5 4.0 3.6 2.9 3.7 Nonresponders (n 7) 13.1 12.0 12.1 12.4 10.9 12.7 11.3 2.2 1.7 1.9 2.3 2.0 1.2 1.8 STD) Responders (n 5) 11.7 11.5 7.2 8.7 8.2 8.6 8.6 2.6 2.6 5.7 5.2 4.4 3.4 5.0

Haloluginone caused a significant (P 0.03) reduction in the mean total skin score of the efficacy-eligible patients after 3 months of treatment compared with baseline.

Halofuginone, cGvHD, and SSc

PHARMACOKINETICS AND SAFETY: ORAL FORMULATION An ascending, single, oral-dose Phase I study in healthy volunteers was performed to assess the safety, tolerability, and pharmacokinetics of orally administered halofuginone. The study was double-blind in a single center and involved 26 healthy, male volunteers. The doses tested were between 0.07 to 2.5 mg/d, and the drug was given with food during dietcontrolled meals. Single, oral doses of 0.07 and 0.5 mg halofuginone were found to be safe and well tolerated. No clinically significant adverse events were recorded in these groups, whereas simulated steady state from the 0.5-mg group surpassed the predicted therapeutic exposure. At 1.5 to 2.5 mg, halofuginone was moderately tolerated, with incidence of gastrointestinal adverse events (nausea and vomiting) associated with dose increments. A daily dose of 1.5 mg halofuginone was designated as the maximal tolerated dose. To improve the tolerability profile of orally administered halofuginone, a 3-way, crossover, split-dose study in 8 male volunteers was initiated in which 3 dose regimens (0.25 mg 8; 0.5 mg 4 or 2.0 mg 1) were assessed. Dividing the dose into several daily portions, thereby reducing the slope increment of plasma levels of halofuginone, allowed the exposure to be increased without increasing gastrointestinal adverse events. CONCLUSION Any perturbation in the cellular function of any tissue that causes excessive collagen deposition and results in fibrosis may be a target for halofuginone therapy. Because halofuginone inhibited collagen type I synthesis on the transcriptional level and an increase in the ECM degradation capabilities, it is a promising candidate for treatment of diseases caused by, or associated with, excessive ECM, such as SSc and cGvHD. The local application of halofuginone is safe, without indications of systemic absorption. Systemically, therapeutically effective plasma levels can be reached at a dosage that is tolerated. Thus, halofuginone meets the criteria as a potential antifibrotic drug for further evaluation in the treatment of cGvHD. ACKNOWLEDGMENTS This paper is a contribution from the Agriculture Research Organization, Volcani Center, Bet Dagan, Israel. The authors would like to thank Dr. CM Black and Dr. AL Herrick for the SSc trial.

1. Pines M, Vlodavsky I, Nagler A. Halofuginone: a novel antifibrotic therapy. Drug of the Future. 1996;21:569-599.

2. Pines M, Vlodavsky I, Nagler A. Halofuginone: a novel antifibrotic therapy. Gen Pharmacol. 1997;30:445-450. 3. Pines M, Vlodavsky I, Nagler A. Halofuginone: from veterinary use to human therapy. Drug Develop Res. 2000;50:371-378. 4. Benyon RC, Arthur MJ. Extracellular matrix degradation and the role of hepatic stellate cells. Semin Liver Dis. 2001;21:373384. 5. Ravanti L, Kahari VM. Matrix metalloproteinases in wound repair. Int J Mol Med. 2000;6:391-407. 6. Rodemann HP, Bamberg M. Cellular basis of radiation-induced fibrosis. Radiother Oncol. 1995;35:83-90. 7. Rodemann HP, Binder A, Burger A, Guven N, Loffler H, Bamberg M. The underlying cellular mechanism of fibrosis. Kidney Int Suppl. 1996;54:S32-S36. 8. Friedman SL. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N Engl J Med. 1993;328:18281835. 9. Haber PS, Keogh GW, Apte MV, et al. Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis. Am J Pathol. 1999;155:1087-1095. 10. Kissin E, Korn JH. Apoptosis and myofibroblasts in the pathogenesis of systemic sclerosis. Curr Rheumatol Rep. 2002;4:129135. 11. Safadi R, Friedman SL. Hepatic fibrosis: role of hepatic stellate cell activation. Med Gen Med. 2002;4:27. 12. Schmitz N, Beksac M, Hasenclever D, et al. Transplantation of mobilized peripheral blood cells to HLA-identical siblings with standard-risk leukemia. Blood. 2002;100:761-767. 13. Beatty PG, Hansen JA, Longton GM, et al. Marrow transplantation from HLA-matched unrelated donors for treatment of hematologic malignancies. Transplantation. 1991;2:443-447. 14. Storek J, Gooley T, Siadak M, et al. Allogeneic peripheral blood stem cell transplantation may be associated with a high risk of chronic graft-versus-host-disease. Blood. 1997;12:47054709. 15. Rocha V, Wagner JE Jr, Sobocinski KA, et al. Graft-versushost disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling: Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources. N Engl J Med. 2000;342:1846-1854. 16. Aversa F, Tabilio A, Velardi A, et al. Treatment of high-risk acute leukemia with T cell depleted stem cells from related donors with one fully mismatched HLA haplotype. N Engl J Med. 1998;339:1186-1193. 17. Sullivan KM, Shulman HM, Storb R. Chronic graft versus host disease in 52 patients: adverse natural course and successful treatment with combination immunosuppression. Blood. 1981; 57:267-276. 18. Shimoni A, Nagler A. Non-myeloablative stem cell transplantation (NST): chimerism testing as guidance for immune-therapeutic manipulation. Leukemia. 2001;15:1967-1975. 19. Shulman HM, Sale GE, Lerner KG, et al. Chronic cutaneous graft-versus-host disease in man. Am J Pathol. 1978;91:545-570. 20. Janin-Mercier A, Devergie A, Van Cauwenberge D, et al. Immunohistologic and ultrastructural study of the sclerotic skin in chronic graft versus host disease in man. Am J Pathol. 1984; 115:296-306. 21. Nagler A, Pines M, Abadi U, et al. Oral tolerization ameliorated liver disorders associated with chronic graft versus host disease in mice. Hepatology. 2000;13:641-648. 22. Ilan Y, Gotsman I, Pines M, et al. Induction of oral tolerance in 423


M. Pines et al.


24. 25. 26. 27. 28.


30. 31.












splenocytes recipients towards pre-transplant splenocyte antigens ameliorates chronic graft versus host disease in murine model. Blood. 2000;95:3613-3619. Gaziev D, Galimberti M, Lucarelli G, Polchi P. Chronic graft versus host disease: is there an alternative to the conventional treatment? Bone Marrow Transplant. 2000;25:689-696. Sapadin AN, Fleischmajer R. Treatment of scleroderma. Arch Dermatol. 2002;138:99-105. Haustein UF. Systemic sclerosis-scleroderma. Dermatol Online J. 2002;8:3-73. Trojanowska M. Molecular aspects of scleroderma. Front Biosci. 2002;7:d608-d618. Kahari VM. Activation of dermal connective tissue in scleroderma. Ann Intern Med. 1994;25:511-518. Tyndall A. Haematopoietic stem cell transplantation (HSCT) for severe autoimmune disease: an update of results and planned trials. Arthritis Rheum. 2000;43:1999. Clements PJ, Furst DE. Choosing appropriate patients with systemic sclerosis for treatment by autologous stem cell transplantation. J Rheumatol. 1997;48:85-88. Denton CP, Black CM. Novel therapeutic strategies in scleroderma. Cur Rheumatol Rep. 1999;1:22-27. Kikuchi H, Tasaka H, Hirai S, et al. Potent antimalarial febrifugine analogues against the plasmodium malaria parasite. J Med Chem. 2002;45:2563-2570. McDougal LR. Control of coccidiosis chemotherapy, in Long PL, (ed): Coccidiosis of man and domestic animals. Boca Raton, FL, CRC Press, 1990, 307-320 Granot I, Hurwitz S, Halevy O, Pines M. Halofuginone: an inhibitor of collagen type I synthesis. Biochim Biophys Acta. 1993;1156:107-112. Choi ET, Callow AD, Sehgal NL, Brown DM, Ryan US. Halofuginone, a specific collagen type I inhibitor, reduces anastomotic intima hyperplasia. Arch Surg. 1995;130:257-261. Nagler A, Miao HQ, Eingorn H, et al. Inhibition of collagen synthesis, smooth muscle cell proliferation and injury-induced intimal hyperplasia by halofuginone. Arterio Thromb Vascular Biol. 1997;17:194-202. Bruck R, Genina O, Aeed H, Alexiev R, Nagler A, Pines M. Halofuginone to prevent and treat thioacetamide-induced liver fibrosis in rats. Hepatology. 2001;33:379-386. Nagler A, Rivkind AI, Raphael J, et al. Halofuginone: an inhibitor of collagen type I synthesis: prevents post-operation abdominal adhesions formation. Ann Surg. 1998;227:575-582. Nagler A, Genina O, Lavelin I, Ohana M, Pines M. Halofuginone: an inhibitor of collagen type I synthesis: prevents formation of post-operative adhesions formation in the rat uterine horn model. Am J Obstet Gyn. 1999;180:558-563. Nagler A, Gofrit O, Ohana M, Pode D, Genina O, Pines M. The effect of halofuginone, an inhibitor of collagen type I synthesis, on urethral stricture formation in vivo and in vitro study in a rat model. J Urol. 2000;164:1776-1780. Pines M, Knopov V, Genina O, Lavelin I, Nagler A. Halofuginone, a specific inhibitor of collagen type I synthesis, prevents dimethylnitrosamine-induced liver cirrhosis. J Hepatol. 1997; 27:391-398. Nagler A, Firman N, Feferman R, Cotev S, Pines M, Shoshan S. Reduction in pulmonary fibrosis in vivo by halofuginone. Am J Resp Crit Care Med. 1996;154:1082-1086. Levi-Schaffer F, Nagler A, Slavin S, Knopov V, Pines M. Inhibition of collagen synthesis and changes in skin morphol-




46. 47. 48.













ogy in murine graft-versus-host disease and tight skin mice: effect of halofuginone. J Invest Dermatol. 1996;106:84-88. Pines M, Domb A, Ohana M, et al. Reduction in dermal fibrosis in the tight-skin (Tsk) mouse after local application of halofuginone. Biochem Pharmacol. 2001;62:1221-1227. McGaha TL, Phelps RG, Spiera H, Bona C. Halofuginone, an inhibitor of type-I collagen synthesis and skin sclerosis, blocks transforming-growth-factor-beta-mediated Smad3 activation in fibroblasts. J Invest Dermatol. 2002;118:461-470. Halevy O, Nagler A, Levi-Schaffer F, Genina O, Pines M. Inhibition of collagen type I synthesis by skin fibroblasts of graft versus host disease and scleroderma patients: effect of halofuginone. Biochem Pharmacol. 1996;52:1057-1063. Zhang Y, Gilliam AC. Animal models for scleroderma: an update. Cur Rheumatol Rep. 2002;4:150-162. Bocchieri MH, Jimenez SA. Animal models of fibrosis. Rheum Dis Clin North Am. 1990;16:153-167. Jaffee BD, Claman HN. Chronic graft-versus-host disease (GVHD) as a model for scleroderma: I. Description of model systems. Cell Immunol. 1983;77:1-12. Siracusa LD, McGarth R, Ma Q, et al. A tandem duplication within the fibrillin 1 gene is associated with the mouse tight skin mutation. Genome Res. 1996;6:300-313. Green MC, Sweet HO, Bunker LE. Tight skin, a new mutation of the mouse causing excessive growth of the connective tissue and skeleton. Am J Pathol. 1975;82:493-512. Osborn TG, Bashey RI, Moore T, Fischer VW. Collagenous abnormalities in the heart of tight-skin mouse. J Mol Cell Cardiol. 1987;19:581-587. Jimenez SA, Williams CJ, Myers JC, Bashey RI. Increased collagen biosynthesis and increased expression of type I and type III procollagen genes in tight skin (Tsk) mouse fibroblasts. J Biol Chem. 1986;261:657-662. Pablos JL, Everett ET, Harley R, LeRoy EC, Norris JS. Transforming growth factor- and collagen gene expression during postnatal skin development and fibrosis in tight-skin mouse. Lab Invest. 1995;72:670-678. Siracusa LD, Christner P, McGrath R, Mowers SD, Nelson KK, Jimenez SA. The tight skin (Tsk) mutation in the mouse, a model for human fibrotic diseases, is tightly linked to the beta 2-microglobulin (B2m) gene on chromosome 2. Genomics. 1993;17:748-751. McGaha T, Kodera T, Phelps R, Spiera H, Pines M, Bona C. Effect of halofuginone on the development of tight skin (TSK) syndrome. Autoimmunity. 2002;35:277-282. Chen W, Wahl SM. Manipulation of TGF-beta to control autoimmune and chronic inflammatory diseases. Microbes Infect. 1999;1:1367-1380. Scharffetter K, Lankat-Buttgereit B, Krieg T. Localization of collagen mRNA in normal and scleroderma skin by in situ hybridization. Eur J Clin Invest. 1988;19:9-17. Jelaska A, Arakawa M, Broketa G, Korn JH. Heterogeneity of collagen synthesis in normal and systemic sclerosis skin fibroblasts: Increase proportion of high collagen-producing cells in systemic sclerosis fibroblasts. Arthritis Rheum. 1996;39:13381346. Needelman BW, Ordonez JV, Taramelli D, Alms W, Gayer K, Choi J. In vitro identification of a subpopulation of fibroblasts that produces high levels of collagen in scleroderma patients. Arthritis Rheum. 1990;33:842-852. Verrecchia F, Mauviel A. Transforming growth factor-beta signaling through the Smad pathway: role in extracellular ma-


Halofuginone, cGvHD, and SSc










trix gene expression and regulation. J Invest Dermatol. 2002; 118:211-215. Ghosh AK. Factors involved in the regulation of type I collagen gene expression: implication in fibrosis. Exp Biol Med (Maywood). 2002;227:301-314. Kulozik M, Hogg A, Lankat-Buttgereit B, et al. Co-localization of transforming growth factor beta 2 with alpha 1(I) procollagen mRNA in tissue sections of patients with systemic sclerosis. J Clin Invest. 1990;86:917-922. Sfikakis PP, McCune BK, Tsokos M, et al. Immunohistological demonstration of transforming growth factor-beta isoforms in the skin of patients with systemic sclerosis. Clin Immunol Immunopathol. 1993;69:199-204. Zhang Y, McCormick LL, Desai SR, et al. Murine sclerodermatous graft-versus-host disease, a model for human scleroderma: cutaneous cytokines, chemokines, and immune cell activation. J Immunol. 2002;168:3088-3098. Holweg CT, Baan CC, Balk AH, et al. The transforming growth factor-beta1 codon 10 gene polymorphism and accelerated graft vascular disease after clinical heart transplantation. Transplantation. 2001;71:1463-1467. Leffell MS, Vogelsang GB, Lucas DP, et al. Association between TGF-beta expression and severe GvHD in allogeneic bone marrow transplantation. Transplant Proc. 2001;33:485-486. Verrecchia F, Chu ML, Mauviel A. Identification of novel TGF-beta/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach. J Biol Chem. 2001;276:17058-17062. Chen K, Wei Y, Sharp GC, Braley-Mullen H. Inhibition of TGF beta 1 by anti-TGF beta 1 antibody or lisinopril reduces thyroid fibrosis in granulomatous experimental autoimmune thyroiditis. J Immunol. 2002;169:6530-6538. Zhang W, Ou J, Inagaki Y, Greenwel P, Ramirez F. Synergistic









cooperation between Sp1 and Smad3/Smad4 mediates transforming growth factor beta1 stimulation of alpha 2(I)-collagen (COL1A2) transcription. J Biol Chem. 2000;275:39237-39245. Varga J. Scleroderma and Smads: dysfunctional Smad family dynamics culminating in fibrosis. Arthritis Rheum. 2002;46: 1703-1713. Prud’homme GJ, Piccirillo CA. The inhibitory effects of transforming growth factor-beta-1 (TGF-beta1) in autoimmune diseases. J Autoimmun. 2000;14:23-42. Mallat Z, Gojova A, Marchiol-Fournigault C, et al. Inhibition of transforming growth factor-beta signaling accelerates atherosclerosis and induces an unstable plaque phenotype in mice. Circ Res. 2001;89:930-934. Chen W, Wahl SM. Manipulation of TGF-beta to control autoimmune and chronic inflammatory diseases. Microbes Infect. 1999;1:1367-1380. Kawakami T, Ihn H, Xu W, Smith E, LeRoy C, Trojanowska M. Increased expression of TGF-beta receptors by scleroderma fibroblasts: evidence for contribution of autocrine TGF-beta signaling to scleroderma phenotype. J Invest Dermatol. 1998; 110:47-51. Spira G, Mawasi N, Paizi M, et al. Halofuginone, a collagen type I inhibitor improves liver regeneration in cirrhotic rats. J Hepatol. 2002;37:331-339. Nagler A, Pines M. Topical treatment of cutaneous chronic graft versus host disease (cGvHD) with halofuginone: a novel inhibitor of collagen type I synthesis. Transplantation. 1999;68: 1806-1809. Silman A, Harrison M, Brennan P. Is it possible to reduce observer variability in skin score assessment of scleroderma? The ad hoc International Group on the Assessment of Disease Outcome in Scleroderma. J Rheumatol. 1995;22:12771280.