Keratinocyte Growth Factor

Keratinocyte Growth Factor Catherine L. Farrell*, Sheila Scully and Dimitry M. Danilenko Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA * corresponding author tel: (805) 447-3818, fax: (805) 498-1425, e-mail: [email protected] DOI: 10.1006/rwcy.2002.0811.

SUMMARY Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family whose specificity for epithelial tissues is a consequence of the restricted expression of its receptor, KGF receptor (also known as FGFR2IIIb), on epithelial cells. KGF is typically expressed in stromal or mesenchymal cells adjacent to epithelia and is thought to be an endogenous paracrine regulator of epithelial cell growth, differentiation, and repair. Administration of recombinant human KGF (rHuKGF) has growth, differentiating, and cytoprotective effects on epithelium in animals and humans, and may be a potential therapeutic agent for a variety of conditions involving epithelial injury and/or inflammation. Clinical studies in cancer patients provide evidence that rHuKGF is therapeutically effective in ameliorating chemoradiotherapyinduced oral epithelial injury.

acid sequence was 30±45% related to the six already known members of the fibroblast growth factor (FGF) family (Finch et al., 1989). It has since been assigned the alternate designation of FGF-7. A more highly related family member was subsequently cloned using homology-based PCR (Yamasaki et al., 1996). This factor was designated FGF-10, but is also known as KGF-2 because its structure and specificity of mitogenic activity appear similar to that of KGF (Emoto et al., 1997; Igarashi et al., 1998). More recently, another FGF family member, designated FGF-22, was cloned and found to be most similar to FGF-10 and FGF-7 (46% and 40% amino acid identities, respectively) (Nakatake et al., 2001).

Alternative names KGF, FGF-7

BACKGROUND

Discovery KGF was first described in 1989 as a human growth factor that stimulated epithelial cell proliferation. It was isolated from M426 human embryonic lung fibroblast-conditioned medium, purified by a combination of techniques including heparin-sepharose affinity, and identified as a heparin-binding factor with unusual target specificity for epithelial cells (Rubin et al., 1989). Subsequently, cloning from the M426 cDNA library showed that the primary amino

Cytokine Reference

Structure Expression studies where rHuKGF was produced from mammalian cells indicate that the mature native KGF molecule is a 163 amino acid monomeric polypeptide of approximately 25±29 kDa (Hsu et al., 1998). Deletion and mutation experiments showed that the first 23 amino acids played limited structural and functional roles, that the receptor-binding specificity was conferred by residues 125±127, and that residues 152, 138, and 42 were implicated in heparin binding (Osslund et al., 1998).

Copyright # 2002 Published by Elsevier Science Ltd

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Catherine L. Farrell, Sheila Scully and Dimitry M. Danilenko

Main activities and pathophysiological roles KGF is an epithelial-specific growth factor because it binds exclusively to a receptor whose expression is restricted to epithelial cells. Since KGF is expressed solely in stromal/mesenchymal cells that are often localized adjacent to epithelial cells where the receptor is expressed, it is believed that KGF is a paracrine regulator of mesenchymal±epithelial interactions during development and in adult life (Finch et al., 1989, 1995; Rubin et al., 1989, 1995). This may include roles in growth, development and patterning, and tissue homeostasis. KGF is also markedly upregulated in epidermal wounds (Werner et al., 1992) and in inflammatory bowel disease (Brauchle et al., 1996; Finch et al., 1996), suggesting that it may also have a reparative role in epithelial injury. Administration of rHuKGF has been shown to be therapeutic in numerous models of epithelial injury, indicating that KGF is cytoprotective, and that it may be an important mediator of epithelial repair (Werner, 1998; Danilenko, 1999).

GENE AND GENE REGULATION

Accession numbers Human: M60828, M25295, S81661 Mouse: Z22703, U58503 (genomic) Rat: X56551, AF295300 (partial)

Chromosome location 15

Cells and tissues that express the gene RNase protection assays demonstrate that the mRNA transcript of KGF can be identified in extracts from almost all organs in the body (Figure 1). In situ hybridization analysis localizes expression of KGF mRNA predominantly in stromal mesenchymal cells (Figures 2 and 3), often directly adjacent to epithelial cells that express KGF receptor mRNA (Figure 3). KGF expression has never been reported in epithelial cells.

PROTEIN

Accession numbers Human: P21781 Mouse: P36363 Rat: Q02195

Discussion of crystal structure Structural analysis of recombinant KGF shows that KGF folds into a beta-trefoil motif (Figure 4) similar to other members of the FGF family (Osslund et al., 1998). The N-terminus `tail' appears to differentiate KGF from other FGF members (Ron et al., 1993).

Posttranslational modifications There are several posttranslational modifications of the KGF protein, all of which occur in the Nterminal region. These include partial oxidation at Met28, partial sulfation at Tyr27, and glycosylation at Asn14 (N-linked heterogeneous structures) and Thr22 (O-linked mucin-type structures) (Hsu et al., 1998).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce KGF is produced in a variety of stromal/mesenchymal cells in vitro including fibroblasts from a variety of tissue sources (Rubin et al., 1995). There is also a report of KGF expression by the dendritic epidermal T cell line `7-17', by activated isolates of dendritic epidermal T cells (Boismenu and Havran, 1994), by microvascular endothelial cells (Smola et al., 1993), and by smooth muscle cells (Winkles et al., 1997).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators The induction of KGF has been studied in murine and human fibroblasts. Serum and purified serum

Keratinocyte Growth Factor 3 Figure 1 RNase protection assay in normal adult tissues: Expression of mRNA for both KGF and its receptor are detectable in most tissues. (A) Phosphor imager scan of the gels; (B) and (C) quantified levels of KGF and KGF receptor mRNA normalized to levels of cyclophilin mRNA.

growth factors increased levels of KGF transcript, followed by accumulation of protein in the medium (Brauchle et al., 1994). Specific growth factors that have been found to upregulate KGF include PDGF and EGF (Brauchle et al., 1994). The proinflammatory cytokines IL-1 and IL-6 also upregulate KGF expression in these systems, whereas TNF was observed to upregulate KGF in mouse, but not human, fibroblasts (Werner, 1998).

RECEPTOR UTILIZATION The KGF receptor is FGFR2IIIb ± a splice variant of the FGFR2 gene that is a member of a family of four FGF receptors (Miki et al., 1991, 1992). This receptor has only been found on epithelial cells, and is the only known high-affinity receptor for KGF. FGF receptors have two or three immunoglobulin (Ig) loops in

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Catherine L. Farrell, Sheila Scully and Dimitry M. Danilenko Figure 2 In situ hybridization for KGF and KGF receptor in an E17.5 day rat embryo: Note strong expression of the receptor in lung, intestine, and epidermis.

the extracellular domain, a transmembrane segment, and a two-part cytoplasmic tyrosine kinase domain separated by a small insert. The KGF receptor has a unique 50 amino acid sequence extending from the Cterminal half of the Ig loop adjacent to the cell membrane that confers specificity of binding to KGF (Miki et al., 1992; Rubin et al., 1995). KGF appears to exhibit absolute specificity for this splice variant of the FGF receptor family, whereas FGF-10/KGF-2 exhibits similar ability to bind to KGF receptor but also binds the FGFR1IIIb isoform (Luo et al., 1998).

IN VITRO ACTIVITIES

In vitro findings In contrast to most other members of the FGF family, KGF exhibits relatively restricted specificity of action for epithelial cells, and does not appear to have direct KGF receptor-mediated effects on other types of cells. Most types of normal epithelial cells (i.e. nontumor) that express KGFR have been shown to respond to KGF in vitro in a variety of ways. KGF stimulates the in vitro proliferation of epithelial cells from a variety of sources, including Balb/MK mouse epidermal embryonic keratinocytes, CCL 208 rhesus monkey bronchial epithelial cells, B5/589 human mammary epithelial cells, rat hepatocytes (Rubin et al., 1995), and type II alveolar

epithelial cells (Panos et al., 1993). In the same studies by Rubin et al. (1995), KGF had no effect on human or mouse fibroblasts, nor on human large-vessel endothelial cells, melanocytes, and hematopoietic colony-forming cells. In contrast, a recent study reported that KGF was chemotactic, mitogenic, activated mitogen-activated protein (MAP) kinase, and maintained monolayer barrier function of microvascular endothelial cells (Gillis et al., 1999). However, the authors were unable to demonstrate crosslinking of KGF to its receptor on these cells, nor could they detect the transcript for the KGF receptor by northern blot analysis or RT-PCR. Therefore, the effect of KGF on microvascular endothelial cells as reported in this study does not appear to be KGF receptor-mediated, and the mechanism responsible for this KGF effect has yet to be determined. In human keratinocytes in vitro, KGF acts as a potent differentiation agent (Marchese et al., 1990) that also stimulates migration (Tsuboi et al., 1993). KGF upregulates proliferation-related genes, such as those of enzymes involved in nucleotide biosynthesis (Gassmann et al., 1999) and others related to the cell cycle (Frank and Werner, 1996). In the same model, similar experiments showed that KGF upregulated gene expression of a protein with putative antioxidant function (Frank et al., 1997), as well as Neudifferentiation factor (Castagnino et al., 2000). In postconfluent cultures of keratinocytes, KGF caused increases in cell numbers relative to controls, apparently by inhibiting terminal differentiation

Keratinocyte Growth Factor 5 Figure 3 In situ hybridization for KGF and KGF receptor in adult rat hair follicles: (A) Hematoxylin and eosin-stained photomicrograph of the in situ images in B and C. Strong expression of KGF within the follicular dermal papilla (B) and strong expression of KGF receptor in the adjacent follicular epithelium (C) illustrates the general principle that KGF is a paracrine tissue growth factor.

Figure 4 The secondary structure of KGF: The backbone of the structure has been traced with a ribbon diagram. The secondary structure of KGF is primarily made of Beta sheets which are illustrated as flat yellow ribbons. Disordered loops are colored white and other structural elements such as a beta bulge or a four turn are colored green and orange respectively.

Many epithelial cell lines of neoplastic origin also express KGFR, but the extent of expression is variable and does not necessarily predict an in vitro proliferative response (Ning et al., 1996; Drugan et al., 1997).

Bioassays used The Balb/MK keratinocyte mitogenesis bioassay was used in the initial purification and characterization of KGF (Finch et al., 1989; Rubin et al., 1989). However, cell lines originating from numerous epithelial sources have been used in published studies. In order to use any cell line to assay effects of growth factors, it is necessary to characterize the conditions under which an appropriate growth curve can be obtained, as these will vary with source, passage number and culture conditions.

processes and by increasing cell survival (Hines and Allen-Hoffman, 1996). All of these data suggest that KGF is not only a factor that induces proliferation, but that it also affects functions such as gene regulation and cell survival, presumably on postmitotic cells.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles The expression pattern and the relatively high level of expression of KGF and its receptor during

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Catherine L. Farrell, Sheila Scully and Dimitry M. Danilenko

development (Figure 2) support a role for KGF as a paracrine mediator of epithelial activity during pattern formation, organogenesis, and growth. The widespread presence of KGF within normal tissues of adult animals suggests a role in tissue homeostasis. For example, the squamous epithelium of the oral mucosa is a self-renewing epithelium. The dead cells that are shed at the surface are continuously replaced by offspring of proliferating stem cells in the basal layers. In oral mucosa, KGF is expressed in the cells of the submucosa, and the KGF receptor is expressed in keratinocytes of several layers of epithelium including the basal proliferating and suprabasal postmitotic layers (Figure 5). KGF may have multiple local roles in this system. It may provide a proliferation stimulus to basal layer stem cells, a

differentiating stimulus to postmitotic cells in the suprabasal layers, and it may possibly also function as a survival factor for terminally differentiated cells.

Species differences No species differences characterized.

have

been

thus

far

Knockout mouse phenotypes Table 1 summarizes the published data on mouse KGF and targeted KGF receptor knockouts. These studies show that while some abnormalities in

Figure 5 Localization of mRNA for KGF and KGF receptor in mouse tongue epithelium. This panel of photomicrographs shows the in situ distribution of the mRNA transcript for KGF (B) in scattered cells of the subepithelial mucosa. The cells can be seen more clearly in the adjacent hematoxylin and eosin-stained image of the same section (A) where arrows point to cells expressing the signal. The mRNA transcript for the KGF receptor (D) is localized to cells in the basal and suprabasal layers of the epithelium. These epithelial layers can be seen more clearly in the corresponding hematoxylin and eosin-stained image of the same section (C).

Keratinocyte Growth Factor 7 Table 1 KGF knockout constructs Knockout construct

Citation

Major findings

KGF receptor dominant-negative `knockout' of KGF on basal keratinocytes

Werner et al. (1994)

Epidermal and hair follicle atrophy/defects Delayed wound re-epithelialization Dermal fibrosis Loss of adipose tissue

KGF knockout (kgf/kgf

/

)

Guo et al. (1996)

Rough hair coat/hair follicle defects Small kidneys

development and wound repair have been observed, these mice do develop to adulthood. For the KGF knockout mice in particular, these results suggest that the lack of the KGF gene is compensated for by other KGF receptor ligands (Werner, 1998).

Transgenic overexpression KGF overexpression has been induced in transgenic mice using a variety of promoters, and the results of these published reports are summarized in Table 2. These studies demonstrate local and systemic effects of KGF in a variety of tissue systems during development.

Pharmacological effects Most reports on the pharmacology of KGF published to date are of studies that have been performed in rodents using rHuKGF. The data from these studies shows that pharmacologic doses of rHuKGF have multiple tissue and functional effects in most major organ systems that are consistent with the pattern of expression of the receptor. Administration of a pharmacologic dose of rHuKGF to rats has profound effects on epithelia within the gastrointestinal tract and liver. These effects include proliferation of epithelial cells in the stomach, small intestines, colon (Figure 6), and liver that was observable after a single dose. Multiple doses of rHuKGF induced increased gastrointestinal and liver wet weights, and produced measurable increases in the depth of gastric glands and duodenal and colonic crypts. Recombinant HuKGF also induced a

dose-dependent increase in the number of intestinal mucin-producing goblet cells and in the amount of mucin produced (Housley et al., 1994), as well as an upregulation of intestinal trefoil protein mRNA (Johnson et al., 2000). Both of these goblet cell secretory products (mucin and intestinal trefoil protein) are thought to contribute to intestinal barrier and defense functions. In the initial rat studies, the squamous foregut of the stomach (but not the esophagus or oral cavity) showed thickening and hyperkeratinization (Housley et al., 1994). In later studies in mice, pharmacologic doses of rHuKGF had a trophic effect on the squamous epithelium of the upper aerodigestive tract, including tongue and esophagus, as measured by increased thickness (Farrell et al., 1999) (Figure 7). There was evidence of enhanced proliferation, but there was also an increase in the size and number of keratohyalin granules and in the number of intraepithelial desmosomal attachments (Farrell et al., 1999). Keratohyalin granules and desmosomes are thought to contribute to the barrier function of the mucosa in the oral cavity and esophagus, but could also contribute to the thickness of the epithelium independent of changes in cell number. The lower gastrointestinal tract of mice responded similarly to that of the rat, with increased intestinal crypt cell proliferation (Farrell et al., 1999; Potten et al., 2001), increased crypt depth and increased villus height (Farrell et al., 1998). These data suggest that the trophic effect of rHuKGF on the epithelium of the upper and lower digestive tract is due in part to proliferation, but that other mechanisms may also play a role. The effect of rHuKGF on the rate of cell loss in these continuously renewing epithelia has not been examined experimentally. The pharmacologic effect of rHuKGF on the liver was also measurable systemically. Housley et al.

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Table 2 KGF overexpression transgenics Promotor ± tissue target

Citation

Major findings

Keratin 14 ± Epidermal and hair follicle

Guo et al. (1993)

Epidermal hyperplasia Hair follicle defects Tongue epithelial hyperplasia Salivary gland defects

Surfactant protein C ± Lung

Simonet et al. (1995)

Embryonic lethal Pulmonary cystadenomas

Apolipoprotein E ± Liver, also secreted systemically

Nguyen et al. (1996)

Embryonic lethal Multisystemic epithelial hyperplasia Polycystic kidneys Hair follicle defects Pancreatic ductal nesidioblastosis

Mouse mammary tumor virus ± Mammary and prostatic expression

Kitsberg and Leder (1996)

Alpha crystallin ± Eye lens expression

Lovicu et al. (1999)

Insulin ± Pancreatic islet expression

Krakowski et al. (1999a,b)

Mammary and prostatic hyperplasia Mammary adenocarcinoma Cataracts Ectopic corneal lacrimal gland formation Intra-islet hepatocyte formation Pancreatic ductal proliferation

(1994) reported that molecules that are synthesized or metabolized by the liver, such as albumin, cholesterol, and triglycerides, were significantly elevated in the sera of rats treated with rHuKGF. Nonogaki et al. (1995) showed that this hypertriglyceridemia was a result of increased hepatic triglyceride secretion, with the fatty acids provided by lipolysis making a major contribution to the elevation. Pancreatic ductal proliferation occurred in rats in response to administration of rHuKGF for one to two weeks, and was most prominent in the interlobular ducts adjacent to and within the islets of Langerhans (Yi et al., 1994a). Pharmacologic effects of rHuKGF reported for lung include induction of proliferation of type II pneumocytes (Ulich et al., 1994a), increased lung surfactant recoverable from lung lavage (Yano et al., 2000), and upregulation of fluid transport (Wang et al., 1999). Proliferation of the transitional urothelium of the urinary bladder and renal pelvis have also been documented (Yi et al., 1995). In

neonatal female mice, rHuKGF stimulated hyperplasia and hypertrophy of the uterine surface epithelium and uterine glands as well as the vaginal epithelium (Hom et al., 1998). KGF has also been described as both a potential andromedin in the normal development of the male accessory reproductive tract (prostate, accessory glands) (Alarid et al., 1994; Fasciana et al., 1996), and as a potential progestomedin in the primate endometrium (Koji et al., 1994). In the mammary gland, the reported effects of rHuKGF varied by species and lactational status. In the rat, rHuKGF stimulated hyperplasia of both the mammary acini and ducts, except in lactating rats, whose ducts appeared resistant to the proliferative effects (Ulich et al., 1994b). In mice, rHuKGF induced marked ductal cystic dilation secondary to proliferation of the lining ductal epithelium (Yi et al., 1994b). Thus far, the pharmacologic effects of rHuKGF appear to be transient. For example, in a rat model of

Keratinocyte Growth Factor 9 Figure 6 Hematoxylin and eosin-stained sections of mouse colon demonstrating the effects of rHuKGF on colon morphology. Mice were treated with 5 mg/kg rHuKGF daily for 6 days. Note that rHuKGF induces a marked increase in mucosal thickness (B) relative to normal, untreated controls (A) with an accompanying marked increase in the number of goblet cells (D) compared to normal (C). A and B, C and D are the same magnification.

rHuKGF-induced pulmonary hyperplasia, intratracheal administration of rHuKGF caused a reversible increase in proliferation of type II pneumocytes within 24±48 hours. The resolution of this hyperplasia appeared to be mediated in part by apoptosis, and in part by differentiation of the type II cells into type I cells as lung architecture resumed its normal appearance (Fehrenbach et al., 2000). Similarly, pancreatic ductal proliferation was no longer evident 7±10 days after cessation of rHuKGF dosing (Yi et al., 1995) and in both mice and rats, these mammary gland changes were reversible within 5 days following cessation of rHuKGF treatment.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects The pathophysiological role of KGF in normal humans or in disease states has not been established. However, KGF can be detected systemically in tissues in normal and disease states and injury models,

10 Catherine L. Farrell, Sheila Scully and Dimitry M. Danilenko Figure 7 Hematoxylin and eosin-stained sections of mouse oral mucosa demonstrating the effect of rHuKGF and chemoradiotherapy on mucosal epithelial thickness. Panel B illustrates that rHuKGF induces a marked thickening of the mucosal epithelium in control mice compared to mice that did not receive rHuKGF (A), while panel D illustrates that pretreatment of the mice with 5 mg/kg rHuKGF for three consecutive days protects against the epithelial attenuation induced by chemotherapy plus irradiation (C).

lending indirect support to the idea that it is a mediator of growth, homeostasis, and epithelial injury responses. Table 3 summarizes the results of studies where levels of KGF were measured in tissues or sera. Although some studies suggest that KGF levels appear to be elevated in certain disease conditions, the data are highly variable and the diagnostic utility of the measurement of KGF levels remains to be determined.

IN THERAPY

Preclinical ± How does it affect disease models in animals? Preclinical studies in a wide variety of injury and disease models suggest that KGF is a broad epithelial protectant and reparative agent. This effect does not appear to be injury-mechanism specific, with multifactoral mechanisms involving trophic, differentiating, and cytoprotective activities likely. Model systems studied include gastrointestinal, pulmonary, epidermal, ocular, reproductive, and genitourinary. Since clinical data from studies in which the

Keratinocyte Growth Factor

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Table 3 KGF in tissues and body fluids Tissue or organ

Citations

Major findings

Rhesus endometrium

Koji et al. (1994)

KGF tissue levels were related to progesterone levels: 0.26 ng/g follicular phase alone, 1.1 ng/g luteal phase 0.12 ng/mg tissue protein during follicular phase with estradiol treatment 2.1±4.0 ng/g treated with estradiol and progesterone implants

Human prostate

Giri and Ittmann (2000)

KGF tissue levels were elevated in subjects with benign prostatic hyperplasia (BPH): 20±180 pg/g tissue protein in BPH tissue Three-fold lower in normal prostate

Human prostate

Ropiquet et al. (1999)

KGF tissue elevations were related to proliferation marker status: 28 ng/g wet tissue in normal peripheral zone 50 ng/g in normal transition zone 91 ng/g in hyperplastic tissue No correlation with PSA content Good correlation with Ki67+ nuclei

Serum in subjects with prostate cancer and BPH

Mehta et al. (2000)

KGF serum levels were not correlated with tumor grade: Serum levels were higher in BPH patients than in cancer patients (mean of 1242 versus 828 pg/mL). Strong correlation between PSA and KGF levels in low-grade tumor patients.

Human pulmonary edema fluid

Verghese et al. (1998)

Similar levels of KGF in fluid from acute lung injury patients and patients with hydrostatic pulmonary edema: 0.3±2.1 ng/mL in acute lung injury versus 0.0±2.6 ng/mL in hydrostatic edema

Human preserved amnionic membrane

Koizumi et al. (2000)

KGF levels were higher in amnionic membranes with amnionic epithelium than without amnionic epithelium

Rat BAL following injury with bleomycin

Adamson and Bakowska (1999)

KGF level peaked at 160 pg/mL 7±14 days post injury then dropped sharply Control rats had 50 pg/mL KGF in BAL

Rat urine following i.v. KGF

Ulich et al. (1997)

KGF not detectable in normal rat urine KGF measurable in urine with peak of 10 ng/mL 8 hours after single i.v. injection of 5 mg/kg

Porcine full-thickness wound fluid

Slama et al. (1994)

KGF levels in wound fluid up to 800 pg/mL on day 6 post-wounding Undetectable levels by day 10

Mouse mammary gland

Pedchenko and Imagawa (2000)

Estrogen increased KGF levels in the mammary glands of both peripuberal and mature mice Control: 18±24 pg/gland; Estrogen-treated: 30±35 pg/gland

12 Catherine L. Farrell, Sheila Scully and Dimitry M. Danilenko therapeutic activity of rHuKGF on oral mucosal injury are available, this section will focus on preclinical models of gastrointestinal injury. For reviews of effects of KGF in other preclinical model systems see Werner (1998) and Danilenko (1999). The therapeutic activity of rHuKGF has been extensively studied in rodent models of gastrointestinal injury. In models where mice were treated with various types of systemic chemotherapy, rHuKGF used as a pretreatment significantly improved survival by 55% or greater relative to control injured mice. In addition, the transient weight loss commonly associated with chemotherapy was ameliorated. Similar results were seen when a combination of chemotherapy and radiotherapy was used (Farrell et al., 1998). These systemic benefits appeared to be due in part to the trophic effects of rHuKGF on the intestinal epithelium, possibly via a direct effect on crypt stem cells (Khan et al., 1997; Farrell et al., 1998; Potten et al., 2001). Other studies have shown that rHuKGF given before, during, and/or after radiation therapy enhances the radioresistance of the mucosal surfaces of the upper aerodigestive tract. In mice subjected to total body irradiation, rHuKGF reversed epithelial atrophy of the oral and esophageal mucosa (Farrell et al., 2000) (Figure 7). Similarly, in a mouse model of oral mucositis in which fractionated radiation therapy was administered for 5 consecutive days, treatment with rHuKGF greatly increased the dose of subsequent test radiation required to cause tongue ulceration (DoÈerr et al., 2001). In experiments where cytoablative therapy was used to condition mice to receive allogeneic bone marrow transplants, rHuKGF treatment decreased the damage caused by the graftversus-host reaction to the gastrointestinal mucosa as well as to other host epithelial tissues, including liver, skin, lung, and thymus. Mortality was also reduced in the rHuKGF-treated mice, importantly without affecting T cell responses to host antigens (i.e. the potential graft-versus-leukemia effect) (PanoskaltsisMortari et al., 1998, 2000a,b; Ziegler et al., 2001). In a rat model of intestinal stress induced by malnutrition, rHuKGF administration prevented the malnutrition-induced changes in glutathione levels measured in the intestinal mucosa. Glutathione is an important endogenous antioxidant that detoxifies cellular free radicals, toxins, and carcinogens, and may also be involved in cell growth (Jonas et al., 1999). One of the primary pharmacological effects of rHuKGF in many of these models appears to be enhancement of the structural integrity of the upper and lower gastrointestinal tract epithelium. This enhancement of integrity may lead to the

maintenance of gut barrier and absorptive functions, and improved glandular function in settings of acute gastrointestinal injury or dysfunction. Recombinant HuKGF also appears to be cytoprotective and reparative through a variety of mechanisms including enhancement of antioxidant capacity (Jonas et al., 1999) and enhancement of the injury healing response (Danilenko, 1999). These potent proliferative, differentiative, and cytoprotective effects on gastrointestinal tissues suggest that KGF may be especially useful in reducing or preventing oral and intestinal mucositis in clinical settings where chemotherapy and radiation therapy are indicated for the primary treatment of malignancies.

Pharmacokinetics Pharmacokinetics of rHuKGF was studied in healthy volunteers after intravenous administration of doses between 0.2 and 20 mg/kg/day. The rHuKGF serum concentration profiles were observed to be triphasic, characterized by an initial rapid distribution, redistribution 1±6 hours post dose, and log-linear elimination with a half-life of approximately 4 hours. Pharmacokinetic parameters appeared dose-linear over the range studied and accumulation was observed with the multiple-dose dosing regimen (Serdar et al., 1997).

Toxicity There are no preclinical toxicology studies of rHuKGF published to date. The safety of rHuKGF has been assessed in clinical studies in healthy volunteers and cancer patients, and the emerging profile suggests that the recombinant protein is well tolerated. Reported adverse events include mild-tomoderate skin and oral events, and asymptomatic elevations in serum amylase and lipase (Serdar et al., 1997; Durrant et al., 1999; Meropol et al., 2000; Spielberger et al., 2001; Clarke et al., 2001; Brizel et al., 2001). These events may be systemic evidence of the pharmacological effects of rHuKGF on different organ systems where the receptor is expressed, e.g. epidermis, oral mucosa, salivary glands, pancreas, and stomach.

Clinical results Clinical studies have been performed in normal healthy volunteers and in cancer patients in three

Keratinocyte Growth Factor different oncology settings. The normal volunteer study was a phase 1 study designed to examine the safety, tolerability, pharmacokinetics (see above), and biologic activity of rHuKGF in a dose escalation of a multiple dosing regimen. In a dose range of 0.2±20 mg/ kg/day, rHuKGF appeared safe and well tolerated, and induced biologic activity as evidenced by measurable increases in markers of oral epithelial proliferation (Serdar et al., 1997). Mucositis is a side-effect of cancer chemotherapy and radiotherapy. These treatments are aimed at killing rapidly proliferating tumor cells, but they also damage proliferating cells of the oral cavity and intestinal epithelium. This damage causes inflammation, atrophy, and mucosal ulceration (Sonis, 1998). Mucositis occurs in many cancer settings and is regimen driven. For example, mucositis incidence can be high in settings where high-dose cytoablative therapy, localized radiation therapy, and multicycle chemotherapy with mucotoxic agents are the cancer therapeutic modalities. Accordingly, rHuKGF studies have been performed in the settings of hematologic malignancies where high-dose therapy is often used (Durrant et al., 1999; Spielberger et al., 2001), in advanced head and neck cancer where localized radiation combined with chemotherapy is the standard of care (Brizel et al., 2001), and in advanced colorectal cancer (Meropol et al., 2000; Clarke et al., 2001) in patients receiving multicycle chemotherapy using 5-fluorouracil. In a phase 2 study in patients with advanced colorectal cancer, treatment with rHuKGF prior to the initiation of chemotherapy significantly reduced the incidence and duration of ulcerative oral mucositis (Clarke et al., 2001) compared to placebo. In a phase 2 study in patients with hematologic malignancies whose cancer therapy was a combination of chemotherapy and total body radiotherapy, administration of rHuKGF before and after cancer therapy significantly reduced the duration of severe oral mucositis relative to placebo. In this rHuKGF treatment group, there was less mouth and throat soreness reported by the patients themselves, and improved functionality as assessed by improvement in the ability to swallow, eat, and drink. Narcotic analgesia was reduced as well (Spielberger et al., 2001). These phase 2 data suggest that rHuKGF may be a useful therapeutic for this painful and debilitating condition. Phase 3 studies have been initiated in patients with hematologic malignancies.

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ACKNOWLEDGEMENTS The authors are grateful to Tim Osslund for Figure 4, and Pamela Holland for her assistance in preparing the manuscript.

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