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INFLAMMATION AND IMMUNOPHARMACOLOGY
Laboratory of Biopharmaceutics and Pharmaceutical Engineering, University of Franche-Comté, Besançon, France (A.L.); and Laboratory of Pharmaceutical Engineering, Gifu Pharmaceutical University, Gifu, Japan (H.Y., H.T., Y.K.)
Received April 19, 2005; accepted June 15, 2005.
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Abstract |
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), all marketed delivery systems still appear to be insufficiently selective (Lamprecht, 2003). This is due to the fact that the drug release mechanisms are based on physiological parameters that are not related to the inflammation and barely to its location. Drug delivery is usually triggered by the luminal pH in the gastrointestinal tract, the enzymatic activity of colonic bacteria, or the transit time of the drug carrier normally initiating the drug release in the distal ileum. Because the inflamed areas affect only limited sectors of the intestinal tissue and may vary from patient to patient, subsequently, still distinct drug loads are delivered unintentionally to noninflamed regions. This misdirected drug release can be hardly excluded by the state-of-the-art drug delivery systems, thus remaining a general problem responsible for therapy failure and undesired adverse effects.
The mainstays of the drug therapy of IBD are a variety of formulations of 5-aminosalicylic acid, the conventional and newer low bioavailability glucocorticoids, the nitroimidazole antibiotic metronidazole, and certain immunomodulating agents. Increased understanding of the mechanisms of inflammation in IBD has permitted the development of effective designer drugs (Egan and Sandborn, 1998). The proposed use of tumor necrosis factor-
monoclonal antibodies in the treatment of rheumatic arthritis and, more recently, in Crohn's disease showed distinct success of the therapy due to the antibody specificity, which greatly reduces adverse effects. It was found, however, that patients develop antibodies against the treatment with an increased risk of infusion reactions and a reduced duration of response to treatment (Baert et al., 2003). Therefore, low-molecular weight immune-suppressant drugs may still be of interest, however, requiring the design of intelligent drug delivery systems providing a more local treatment. FK506 (tacrolimus), initially developed as an immune-suppressive drug used to inhibit transplantation rejection, was successfully applied in IBD to treat refractory ulcerative colitis where corticoid treatment failed (Hoshino et al., 1995; Aiko et al., 1997; Fellermann et al., 1998; Matsuhashi et al., 2000). These treatments were based on oral FK506 administration with macroscopic carriers (tablets, etc.) or parenteral injections resulting in a therapeutic success after systemic availability. However, these forms of treatment also provoke significant adverse effects (Finn, 1999) known to be a limiting factor of the therapy.
A new therapeutic approach is proposed on the basis of the strong cellular immune response occurring in the inflamed regions, i.e., in general, an increased presence of neutrophils, natural killer cells, mast cells, and regulatory T cells, which play an important role in the pathophysiology of inflammatory bowel disease (Allison et al., 1988; Seldenrijk et al., 1989). Thus, it was hypothesized that particle uptake into those immune-related cells (Tabata et al., 1996) or the disrupted intestinal barrier at ulcerated regions (Stein et al., 1998) could allow the selective accumulation of the particulate carrier system in the desired area.
The increased adhesion of small particulate drug carriers to the inflamed tissue in ulcerative colitis led to a new therapeutic concept allowing a specific drug targeting in this disease (Nakase et al., 2000, 2001; Lamprecht et al., 2001c). This approach is mainly based on two pathophysiological changes in the inflamed tissue, allowing the higher adhesion of the carriers in the inflamed tissue caused by elevated levels of mucus production and an intense particle uptake inside the colitis tissue as a result of an enhanced permeability and the presence of a highly increased number of immune-related cells. This accumulation phenomenon was observed to be particle size dependent with an increasing effect for smaller particle diameters and highest efficiency for nanoparticles (NP) of around 100 nm (Lamprecht et al., 2001a).
FK506-containing polymeric NP were designed and tested for their therapeutic efficiency in two different rat colitis models after oral or rectal administration. To understand the mechanism of action of this new drug delivery strategy more clearly, drug and particle penetration studies allowed the elucidation of the processes on tissue level ex vivo.
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Materials and Methods |
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NP Preparation and Characterization. The preparation of NP was based on an oil/water emulsification-solvent evaporation method. Fix amounts of poly(lactic-coglycolic acid) (210 mg) and FK506 (30 mg) were dissolved in 9 ml of methylene chloride. This solution was poured into 45 ml of 5% (w/w) polyvinyl alcohol, and an oil/water-emulsion was formed by ultrasonification for 3 min. After evaporation of the solvent, NP were recovered by centrifugation at 55,000g for 30 min and washed with distilled water. The washing step was repeated once before NP were resuspended in distilled water and lyophilized overnight.
NP were analyzed for their size distribution and their surface potential using a Photal laser particle analyzer LPA 3100 (Otsuka Electronics, Osaka, Japan) and a Zetasizer II (Malvern Instruments, Worcestershire, UK), respectively. FK506 entrapment within NP as well as the in vitro drug release kinetics was determined by high-performance liquid chromatography as described elsewhere in detail (Akashi et al., 1996; Lamprecht et al., 2001b).
Animal Treatment. All animal experiments were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, Washington, DC). The TNBS and oxazolone rat models were chosen as well recognized experimental models (Morris et al., 1989; Ekström, 1998) that allow induction of colitis at an exact location. This permitted administration of rectal drug/NP in the form of an exclusively local delivery to the inflamed tissue. Moreover, the differences in both models allowed characterizing the efficiency of the NP under varying pathophysiological conditions resembling Crohn's disease and ulcerative colitis, respectively. Male Wistar rats (10 weeks; n = 6/group) were treated by the following procedure to induce the TNBS model colitis. After light narcotizing with ether, the rats were catheterized 8 cm intrarectal, and 500 µl of TNBS (140 mg/kg) in an ethanol/water mixture was applied. Control groups received a rectal installation of an ethanol/saline mixture without TNBS instead. For 48 h, the rats were housed without treatment to maintain the development of a full inflammatory bowel disease model.
The second colitis model was based on the induction with oxazolone (Ekström, 1998). Rats were immunized with 300 µl of a solution containing acetone/ethanol (1:4), and 12 mg oxazolone was applied topically to the skin. The challenge was performed a week later by rectal administration of 10 mg of the haptenating agent, and oxazolone was administered per rectum in a total volume of 450 µlof an ethanol/water mixture. Again, control groups received a rectal administration of ethanol/saline mixture without oxazolone instead. Similar to TNBS, for 48 h, the rats were housed without treatment to maintain the development of a full inflammatory bowel disease model.
During the treatment period, all animals received (orally or rectally) either 0.3 ml of FK506 solution or FK-NP suspension once daily for 10 (TNBS-induced) or 6 (oxazolone-induced) consecutive days at a dose of 1 mg/kg body weight. The oral administration was performed by gavage, whereas rectal administration consisted of an 8-cm intrarectal catheterization providing NP directly to the inflammation site. The control groups received only saline (colitis control), drug-free NP (NP control), or drug-containing particles (healthy control). The animals were sacrificed 24 h after the last drug/particle administration, and colons were resected for the determination of colon/body weight index and myeloperoxidase (MPO) activity.
Clinical Activity Score System, Colon/Body Weight Index, and MPO Activity. Colitis activity was quantified with a clinical score assessing weight loss, stool consistency, and rectal bleeding as previously described elsewhere (Hartmann et al., 2000). Resected distal colon tissue samples were opened longitudinally and rinsed with iced phosphate buffer to remove luminal content and weighed. The colon/body weight index was calculated as a quotient of the colon wet weight compared with the total body weight of each rat. The MPO activity allowed quantifying the severity of the colitis as a reliable index for the infiltration of activated neutrophils into the inflamed tissue. Activities were analyzed according to a standard method (Krawisz et al., 1984).
Creatinine Clearance and Blood Urea Nitrogen Levels. FK506 was administered to a new population of rats daily over 5 weeks by either oral or rectal route (FK506 solution or NP). This treatment period was required to ensure the full manifestation of the nephrotoxic adverse effect. Creatinine clearance and blood urea nitrogen levels were assessed at the end of the treatment. Creatinine in serum or urine and blood urea nitrogen (BUN) levels were determined with the respective kits from Biomérieux (Marcy-l'Etoile, France) according to the supplier's instructions.
Tissue Penetration Experiments. The resected colonic rat tissue samples (healthy or inflamed from TNBS-colitis) were washed with ice-cold phosphate buffer, pH 7.4, and mounted in modified Ussing chambers with an area of 1.13 cm2. The general setup was slightly modified from those described in the literature (Lampen et al., 1996). The tissue samples were preincubated for 30 min before the various drug formulations were added into the apical compartment at final FK506 concentrations of 10–40 µM (see Fig. 5) or 4 to 16 µM (see Fig. 6, A and B); optionally, amiodarone (40 µM) was added as P-glycoprotein (P-gp) inhibitor and/or troleandomycin (10 µM) as cytochrome P450 3A inhibitor and incubated for 1 h. After the incubation was completed, the tissue samples were rinsed carefully and lyophilized. After extraction with acetonitrile from lyophilized tissue, drug content was determined by high-performance liquid chromatography (Akashi et al., 1996).
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Metabolism Experiments. Rat intestine samples were again rinsed with cold phosphate buffer before being cut into small slices of about 200 mg tissue wet weight. Tissue samples were homogenized and 30-min preincubated in gentamicin (10 µg/ml) containing Dulbecco's modified eagle medium. In inhibition experiments, troleandomycin was added prior to the preincubation step. FK506 solutions were added and incubated for 4 h at 37°C. Thereafter, samples were lyophilized, and FK506 was extracted with acetonitrile and quantified by high-performance liquid chromatography (Akashi et al., 1996).
Relative Tissue Penetration Index. A relative penetration index was calculated as the coefficient of drug amounts recovered from intestinal segments of colitis groups versus values from healthy samples at equivalent drug concentrations in the donor compartment. The recovered drug amount was normalized with respect to the tissue sample weight.
Statistical Analysis. The results were expressed as mean values ± S.D. For the pair-wise comparison of healthy and colitis groups, the Mann-Whitney U test was used to investigate differences statistically. The statistical analysis of treatments with more than two groups was performed with Kruskal-Wallis test followed by Dunn's test, except when normality and equal variance were passed, it was followed by the Tukey test. In all cases, P < 0.05 was considered to be significant.
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Results |
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For purposes of testing the therapy concept in a preexisting experimental colitis in rats, FK506 formulations were administered either orally or rectally to determine possible differences occurring by the gastrointestinal passage. In the TNBS model, clinical activity was found to be lower after the administration of FK506-NP compared with the groups receiving FK506 solution or saline (Fig. 1A). Although the observed effect after oral and rectal administration of FK506-NP was similar, a faster response to treatment was found when administered by rectal route (different from untreated control, rectal, day 8; oral, day 10; P < 0.05). MPO activity and the colon/body weight ratio were only slightly reduced when FK506 or FK506-NP were administered by oral route (Fig. 2A). Oppositely, after rectal administration, distinct differences were observed between FK506- and FK506-NP-treated groups (P < 0.05). The rectal administration of FK506-NP reduced both MPO activity and colon/body weight ratio below the 50% level of untreated animals, whereas the mitigating effect was minor for the FK506 solution (Fig. 2B).
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Nephrotoxic effects were assessed by BUN and creatinine clearance. BUN levels were increased after oral administration of FK506 solution, whereas particle formulations reduced BUN values to a level comparable with untreated control (Fig. 3A). Endogenous creatinine clearance was significantly decreased in FK506 solution-treated rats (Fig. 3B), whereas oral and rectal FK506-NP resulted in a creatinine clearance similar to the control group.
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When inhibiting P-gp with amiodarone (FK506 + amiodarone), higher tissue levels were detected in both healthy and inflamed tissue, whereas the increase was more dramatic for the inflamed samples (Fig. 5A). Compared with tissue concentrations after incubation with FK506 + amiodarone, FK506-NP led to increased drug tissue levels in inflamed tissue but lowered the FK506 concentrations in healthy samples.
When colonic tissue samples were homogenized (to exclude influences by P-gp) and incubated together with FK506, higher metabolic drug degradation was observed in the inflamed tissue (Fig. 5B). Addition of CYP3A inhibitor troleandomycin reduced the metabolic activity generally, and in colitis tissue and healthy control, similar metabolism levels were obtained. FK506-NP achieved a higher preservation of the incorporated drug compared with drug solution in colitis group at some drug concentrations, whereas values with NP in healthy tissue were only slightly reduced.
The relative penetration of FK506 solution was higher into healthy compared with inflamed tissue, whereas differences were reduced in the presence of the inhibitors amiodarone and troleandomycin (Fig. 6). FK506-NP were, however, able to completely inverse this penetration phenomenon, leading to about 3-fold higher drug concentrations inside the inflamed tissue than in healthy tissue.
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Discussion |
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; Nakase et al., 2000, 2001; Lamprecht et al., 2001c; Jubeh et al., 2004). The selective adhesion of particulate drug carriers to the inflamed tissue in experimental colitis proved to be a promising therapeutic concept of specific drug targeting in IBD.
Standard drug formulations applied in the clinical studies with FK506 (Fellermann et al., 1998; Matsuhashi et al., 2000) released the drug during the intestinal passage followed by its systemic absorption. After oral administration, the effective dose of FK506 is lowered distinctly due to drug's P-gp-related efflux combined with inactivation from mucosal metabolism by CYP3A, which cause a relatively low bioavailability (Kagayama et al., 1993; Shimomura et al., 2002), subsequently reducing the therapeutic efficiency. Thus, effects achieved in the reference group receiving drug solution orally closely reflect the properties of such a nonspecific formulation.
The rectal administration of FK506 solution, reflecting drug availability of commercialized systems for rectal drug administration or standard colon delivery systems (pH-dependent release, prodrugs, etc.), demonstrated also relative limited efficiency. Rectally administered free FK506 aiming mainly for a local effect undergoes an increased drug efflux due to the higher expression levels of P-gp in the inflamed tissue (Farrell et al., 2000; Farrell and Kelleher, 2003). Moreover, an overall increasing mucosal metabolism in inflamed tissue related to the highly increased levels of lymphocytes and neutrophils, which express CYP3A (Stärkel et al., 1999), further reduces relevant drug concentrations at the site of action.
NP administered by oral route are expected to accumulate in the inflamed tissue to develop a more local effect by their accumulation at the site of action (Lamprecht et al., 2001b,c). Surprisingly, oral NP administration resulted in distinct lack of therapeutic efficiency, which is seemingly based on a combination of several influencing parameters. Although freely available, FK506 resulting from the initial drug release during the passage in the small intestine may be limited due to the slow onset of drug release, other factors such as degradation of the NP building matrix polyester by digestive enzymes may play a role. Besides, a distinct uptake into Peyer's patches and other elements of the gut-associated lymphoid tissue has been reported to lead to a systemic drug availability followed by hepatic metabolism (Florence et al., 1995). All these factors contribute partially to the reduced efficiency of orally administered FK506-NP. However, rectally administered FK506-NP showed a major enhancement of the therapeutic efficiency. Apparently, this is based on the adhesion of NP at the inflammation site. Oppositely to the orally administered NP, those particles were applied directly to the inflammation site, avoiding an early carrier degradation or uptake, and, therefore, did not risk a loss of efficiency.
The study determining the therapeutic efficiency of FK506-NP was performed in the TNBS and oxazolone colitis models in rats. Despite the fact that the relationship of the TNBS model to human disease is imperfect (Neurath et al., 1995; Fiocchi, 1998), it displays several Crohn's disease-like features, especially full-thickness transmural mononuclear inflammation. Oxazolone colitis is a mucosal model of colitis that is an interleukin-4-driven, Th2 inflammation that has histologic features resembling the human disease, ulcerative colitis (Boirivant et al., 1998), being relatively superficial at the microscopic level.
The two colitis models led to generally comparable findings on the therapeutic efficiency. Although the treatment period was set shorter in the oxazolone model due to limited persistence of acute inflammation state, the therapy with FK506 was not less efficient. Because submucosal swelling was reduced in oxazolone compared with TNBS model, less penetration depth was required for the drug or drug carriers to develop a full mitigating effect. Apparently, the lower tissue thickness in the oxazolone model led to general higher therapeutic efficiency of local FK506 treatment. This explains the higher efficiency of rectal FK506 solution and the observation that the rectal administration of FK506-NP approached values of a complete remission. Beside this effect of thickness, the dense infiltration of immune-related cells in mucosal and submucosal tissue may essentially contribute to the metabolism of FK506 being responsible for reduced drug tissue concentrations in TNBS colitis samples.
Rectally administered FK506-NP clearly inhibited the elevation of BUN and prevented the reduction of creatinine clearance compared with solution groups. Moreover, measuring BUN and creatinine clearance revealed that FK506-NP levels did not differ from that of untreated control animals. Although this was also observed for orally given FK506-NP, it should be mentioned that in terms of therapeutic efficiency, the rectal NP group was distinctly superior.
From the therapeutic point of view, in addition to the locally delivered drug concentration, drug penetration into the site of action is a major issue to address. Several factors are known to impede drug penetration into the mucosa besides the general tissue permeability, e.g., P-gp and CYP3A, which are altered in inflamed intestinal tissue. Studies in excised samples of healthy or inflamed tissue elucidated details on the mechanism of action of NP. Although free FK506 exhibited a higher relative tissue concentration in healthy tissue, FK506-NP inversed this phenomenon in favor of increased drug amounts in the inflamed region. It might be concluded that the lower drug penetration into healthy tissue in the case of NP is mainly based on the reduced NP adhesion. When, however, mucus production and NP capture in ulcerations are enhanced, a higher NP accumulation tendency is observed, leading to higher drug concentrations inside inflamed tissues. Moreover, the entrapment of FK506 inside NP obviously prevented exposure of the drug to P-gp efflux and simultaneously to its mucosal metabolism. This protective effect of NP against P-gp and CYP3A in colitis tissue is apparently complementary, which explains the supplementary increase of drug concentration inside the inflamed tissue compared with FK506 when the P-gp is inhibited.
For an increased therapeutic effect, it seems not to be essentially necessary that the majority of NP enter the inflamed tissue to release the entrapped drug. As it became evident by the confocal fluorescence images, most of the NP were located inside the mucus layer and ulcerated regions. The subsequent local drug concentration with NP at this site was much higher compared with concentrations that could be reached with drug solution alone. Subsequently, P-gp and CYP3A capacities could be saturated by a mechanism of high local drug dose that was proposed similarly for nanoparticulate drug delivery across the blood-brain barrier (Pardridge, 2002).
Both factors, the higher selective adhesion to and increased drug penetration in the inflamed region, in combination lead to a distinctly increased therapeutic efficiency of NP. Such a high selectivity is not reached by any other drug delivery approach in colitis.
The described NP demonstrate the possibilities in innovative drug delivery for the treatment of IBD by targeted delivery based on factors directly related to the location and intensity of the inflammation. FK506-NP allow a selective adhesion to the inflamed tissue as some kind of drug reservoir with a reduced drug availability at the healthy surrounding tissue. Additionally, these NP provide a selectively enhanced drug penetration into the inflamed tissue compared with free drug, whereas in healthy tissue, this phenomenon is inversed. The NP deposition and penetration in the inflamed tissue as therapeutic strategy appears to be a promising approach in the design of new carrier systems for the treatment of IBD.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: IBD, inflammatory bowel disease; FK506, tacrolimus; NP, nanoparticle(s); TNBS, 2,4,6-trinitrobenzenesulfonic acid; MPO, myeloperoxidase; BUN, blood urea nitrogen; P-gp, P-glycoprotein.
Address correspondence to: Dr. Alf Lamprecht, EA 3924, Faculty of Medicine and Pharmacy, Place Saint Jacques, 25030 Besançon Cedex, France. E-mail: alf.lamprecht{at}pharma.uhp-nancy.fr
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, colitis control; 
, FK506 solution oral; 
, FK506 NP oral; 
, FK506 solution rectal; 
, FK506 NP rectal; NP controls being similar to the colitis control as well as error bars are not shown for clarity reasons; n = 6). *, P < 0.05, compared with colitis control rats given saline.
