0090-9556/99/2705-0627– 632$02.00/0
`DRUG METABOLISM AND DISPOSITION
`Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
`
`Vol. 27, No. 5
`Printed in U.S.A.
`
`ABSORPTION AND INTESTINAL METABOLISM OF SDZ-RAD AND RAPAMYCIN IN RATS
`
`ANDREW CROWE, ARMIN BRUELISAUER, LOUISE DUERR, PIERRETTE GUNTZ, AND MICHEL LEMAIRE
`
`Compound Formulation and Selection Support/Drug Metabolism and Pharmacokinetics, Novartis Pharma Inc., Basel, Switzerland
`
`(Received August 17, 1998; accepted December 18, 1998)
`
`This paper is available online at http://www.dmd.org
`
`ABSTRACT:
`
`The new immunosuppressive agent, SDZ-RAD, and its analog
`rapamycin were examined for intestinal absorption, metabolism,
`and bioavailability in Wistar rats. Intestinal first-pass metabolism
`studies from rat jejunum showed that at 0.5 mg of SDZ-RAD/kg rat,
`50% of the parent compound was metabolized in the intestinal
`mucosa, and this decreased to around 30% when SDZ-RAD was
`increased to 5.0 mg/kg rat. Results for rapamycin at the low dose
`were similar to those for SDZ-RAD, but at the higher dose only 1 to
`14% of the total rapamycin absorbed was metabolized by the
`intestine. After i.v. administration of 1 mg/kg SDZ-RAD or rapamy-
`cin, the area under the concentration curve (AUC) for rapamycin
`was twice that of SDZ-RAD, resulting in a systemic clearance of 6.2
`ml/min and 3.0 ml/min for SDZ-RAD and rapamycin, respectively.
`However, the AUC for oral absorption was similar for the two
`
`compounds: 140 and 172 ng*h/ml for SDZ-RAD and rapamycin,
`respectively. Because blood clearance was faster for SDZ-RAD
`after i.v. administration, the absolute oral bioavailability for SDZ-
`RAD was 16% compared with 10% for rapamycin. Overall, the data
`suggest that intestinal first pass is a major site of metabolism for
`SDZ-RAD and rapamycin and that intestinal absorption of SDZ-
`RAD was much faster than that of rapamycin. This allowed it to
`counteract the combined actions of faster systemic clearance and
`increased intestinal metabolism, resulting in comparable absolute
`exposure when given orally. Also, the coadministration of cyclo-
`sporin A with SDZ-RAD was shown to dramatically increase blood
`AUCs for SDZ-RAD, probably through saturating intestinal metab-
`olism mechanisms.
`
`Cyclosporin A (CsA)1 is currently the main immunosuppressant
`used in solid organ transplantation. This compound binds to a cyto-
`plasmic cyclophilin, and the resulting complex inhibits calcineurin
`and, hence, interleukin 2, which blocks transcriptional activation of T
`cells (Graham, 1994). This immunosuppressant initially was used in
`the early 1980s. Recent research has been conducted into both im-
`proving the formulation of CsA delivery and developing other com-
`pounds to improve immunosuppression either as replacements for, or
`to work in synergy with, CsA (Kahan et al., 1991; Lake and Canafax,
`1995; Lampen et al., 1995).
`Allograft rejection in organ transplantation is an area that should be
`reduced greatly with the advent of the new, orally active immunosup-
`pressant, SDZ-RAD. This compound is a derivative of rapamycin,
`which inhibits the proliferation of T cells by a different mechanism
`than that of CsA, preventing their entry into the S phase of cell
`division (Goral and Helderman, 1997). However, animal and human
`studies have shown that the absorption and bioavailability of rapamy-
`cin have considerable variability (Granger et al., 1995; Ferron et al.,
`1997).
`One of the significant contributions to intestinal uptake variability
`is active efflux back to the lumen by P-glycoprotein (P-gp) and other
`active efflux proteins present in the apical layer of enterocytes at the
`
`villus tip of intestinal microvilli. P-gp originally was identified as an
`efflux pump responsible for multidrug resistance in tumor cells, but
`since has been found to be expressed in many tissues of normal cells
`including the kidney, blood-brain barrier, and enterocytes of the
`intestine (Cordon-Cardo et al., 1989; Augustijns et al., 1993; Hunter
`et al., 1993). Recent reports suggest that rapamycin is a substrate for
`P-gp, as are many immunosuppressive compounds including CsA and
`tacrolimus (Augustijns et al., 1993; Hoof et al., 1993; Hebert, 1997).
`Current results from our own group suggest that SDZ-RAD also is a
`substrate for P-gp (Crowe and Lemaire, 1998). With the confirmed
`presence of P-gp in the intestine and its links with metabolizing
`enzymes of the CYP3A class (Gan et al., 1996), emphasis is being
`focused on the relevance of intestinal metabolism when determining
`the effect of first-pass metabolism on these compounds and, hence,
`their bioavailability. Other in vitro and in situ studies in our laboratory
`indicated that SDZ-RAD has better intestinal absorption than its
`parent compound, rapamycin (Crowe and Lemaire, 1998). This study
`aims to expand on our early in vitro and in situ observations by
`examining the difference in absorption and disposition between SDZ-
`RAD and rapamycin in a rat model focusing on metabolism at the
`intestine. Potential synergistic action between SDZ-RAD and CsA
`also was explored to determine whether these two P-gp substrates
`could increase each other’s intestinal absorption.
`
`1 Abbreviations used are: CsA, cyclosporin A; P-gp, P-glycoprotein; AUC, area
`under the concentration curve; LC-RID, liquid chromatography-reverse isotope
`dilution; AUMC, area under the mean concentration curve.
`
`Send reprint requests to: Dr. Michel Lemaire, Drug Metabolism and Pharma-
`cokinetics, Novartis Pharma Inc., CH 4002 Basel, Switzerland. E-mail:
`andrew.crowe@pharma.novartis.com
`
`Materials and Methods
`Materials. [3H]SDZ-RAD (44.67 MBq/mmol) and [14C]rapamycin (1.571
`MBq/mmol) were prepared by Novartis’ isotope laboratory and shown to be
`higher than 99% pure by HPLC analysis. Placebo microemulsion (as described
`in Schuler et al., 1997), concentrate for infusion (polyethoxylated castor oil and
`ethanol (65:35, w/w), nonlabeled SDZ-RAD, nonlabeled rapamycin, and CsA
`also were prepared from within Novartis.
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`CROWE ET AL.
`
`Animal Studies. The experiments were performed with male Wistar rats
`weighing approximately 300 g (BRL, Fuellinsdorf, Switzerland). They were
`anesthetized with Metofane (methoxyflurane; Mallinckrodt Veterinary Inc.,
`Mundelein, IL) in a veterinary respirator (model HN64; Holzel). The right
`femoral artery was cannulated with a segment of polyethylene tubing contain-
`ing heparinized saline (100 U/ml) for the collection of blood. Rats that were
`infused i.v. also had the right femoral vein cannulated. The tubes were passed
`s.c. to emerge at the base of the neck. Animals were isolated in metabolic cages
`and allowed to move freely. Full recovery from anesthesia usually occurred
`within 2 h, and the presence of the catheter(s) caused no obvious discomfort to
`the animals. Administration of the drugs was carried out the following day,
`after surgery. Animals had access to food before and after surgery, but this was
`removed 14 h before administration of the compounds. All animals had free
`access to drinking water.
`Dosage and Administration. Intravenous infusion. [3H]SDZ-RAD was
`mixed with nonlabeled SDZ-RAD to obtain the appropriate specific radioac-
`tivity, whereas [14C]rapamycin was used without nonlabeled additions. Both
`compounds were dissolved in microemulsion and diluted in saline (1:2.6, v/v).
`The formulations were administered as a 2-h infusion (0.3 ml/h) in the
`cannulated femoral vein. The dose for both compounds was 1 mg/kg. Blood
`was collected from the cannulated femoral artery 1 h before the end of
`infusion, at the end of infusion (time 0), and at 0.5, 1.0, 2.0, 4.0, 8.0, 24, 32,
`48, 72, and 168 h after drug infusion had ceased.
`Oral administration. [3H]SDZ-RAD and [14C]rapamycin were dissolved in
`microemulsion and diluted to a final volume with saline. The dose ingested
`was 1.5 mg/kg using 5.0 ml/kg of the saline/microemulsion mixture adminis-
`tered by gastric intubation according to the individual body weight on the day
`of application. Blood was collected from the cannulated femoral artery 0.5, 1.0,
`2.0, 4.0, 8.0, 12, 24, 32, and 48 h after gastric intubation of radiolabeled
`compounds. At various time points for both oral and i.v. administered rats, a
`volume of blood equivalent to that taken was replaced through the cannulated
`femoral artery using fresh blood from donor rats.
`CsA, as Neoral containing 100 mg/ml CsA, was administered to rats either
`alone at 2.5 mg/kg by gastric intubation or in combination with 0.6 mg/kg
`[3H]SDZ-RAD for the oral coadministration study. [3H]SDZ-RAD also was
`used alone at 0.6 mg/kg in this study.
`Mesenteric Vein Method. Adult Wistar rats (300 g) were fasted overnight
`before initiating the dosing study. Rats were anaesthetized for the duration of
`the study with i.p. injections of urethane (1.1 g/kg). The middle 10 cm of the
`jejunal segment was ligated in a location that allowed all blood from mesen-
`teric branching along the ligated segment to be collected from one point just
`before entering the superior mesenteric vein. One milliliter of placebo micro-
`emulsion/saline mix (1:13.5), containing either 0.15 mg or 1.5 mg of a
`[3H]SDZ-RAD solution (189 –329 mCi/ml), or a [14C]rapamycin solution
`(7.0 –13.9 mCi/ml), was injected into the segment. The total mesenteric blood
`for the region was collected immediately using a 27-gauge needle and 1-ml
`syringe. Collection continued for as long as possible, usually 3 to 5 min,
`allowing no blood to enter the circulation from the ligated segment. Unlike
`other studies examining mesenteric vein blood that have used lengths of time
`of more than 30 min (Kim et al., 1993), we did not want to perfuse the animals
`with donor blood, limiting our study to the initial absorption phase (approxi-
`mately 5 min). Blood of individual animals was examined for total radioac-
`tivity by liquid scintillation counting and for parent drug by liquid chroma-
`tography-reverse isotope dilution (LC-RID).
`Parent Drug Determinations (LC-RID). Blood aliquots (200 ml) were
`spiked with 200 ml of cold compound (50 mg/ml of either SDZ-RAD or
`rapamycin in acetonitrile). Water (1 ml) and 100 ml of 53 concentrated Merck
`Titrisol, pH 9.0, buffer also were added. Two hundred fifty microliters of
`SDZ-RAD and rapamycin was extracted in diethylether, evaporated, and
`reconstituted in mobile phase consisting of acetonitrile/tertiary butyl methyl-
`ether and 0.1% tetramethylammonium hydrogen sulfate (370:60:500, w/w/w).
`Seventy-five microliters of n-hexane was then added. Samples were vortexed
`vigorously, and, after centrifugation, the hexane layer was removed. SDZ-
`RAD and rapamycin in the remaining phase were separated from their metab-
`olites by HPLC. Chromatography was performed on a HPLC system (Kontron
`Instruments, Zurich, Switzerland). Separation was conducted on a Brownlee
`Spheri-10 RP2 column (4.6 3 220 mm) at 70°C. The mobile phase was as
`described above, and the flow rate was 1.2 ml/min. The effluent was monitored
`
`at 278 nm, and the peak corresponding to either unchanged [3H]SDZ-RAD or
`[14C]rapamycin was collected in a polyethylene vial by fraction collection
`(SuperFrac; Pharmacia LKB, Uppsala, Sweden) and subjected to radioactivity
`determinations. The concentration of parent compound in each sample was
`calculated from the ratio of the amount of radioactivity in the collected fraction
`to the area of the UV absorbance of the nonradiolabeled SDZ-RAD or
`rapamycin as used as internal standards (Everett et al., 1989).
`CsA Determinations (enzyme-linked immunosorbent assay). CsA was
`determined using the whole-blood cyclosporin displacement immunoassay
`procedure. Microtest plates were coated with goat anti-mouse (Fc) in coating
`buffer. Twenty-five microliters of whole blood was mixed with 75 ml of CsA
`displacement/lysis buffer. Biotinylated CsA and a CsA-specific monoclonal
`antibody were then added to the mixed blood, and the resulting samples were
`pipetted into the precoated wells and sealed at 4°C for 150 min. After
`streptavidin-peroxidase and peroxidase substrate additions,
`the microtiter
`plates were examined at 490 nm in a spectrophotometric plate reader.
`Data Analysis. Blood levels of unchanged SDZ-RAD and rapamycin were
`evaluated by nonlinear regression analysis using the noncompartmental model
`of constant infusion for i.v. doses and extravascular input for oral doses using
`the WinNonlin Pro package for Windows NT 4.0 (Scientific Consulting Inc.,
`Cary, NC), using a 166-MHz Pentium computer. AUC and area under the
`mean concentration curve (AUMC) of the blood-drug concentration time
`curves were obtained by the linear trapezoidal rule and extrapolated to infinite
`2 1 C(tm)*tm/l
`
`time by the additions of C(tm)/lz (AUC) and C(tm)/lz
`
`z
`(AUMC), where C(tm) is the last concentration above the limit of quantifica-
`
`tion at time tm and lz is the slope of the terminal elimination phase. Total
`clearance (CL) was calculated as dose/AUCi.v. The volume of distribution at
`5 CL*AUMC/AUC. Results expressed in
`steady state was calculated as Vss
`this study are presented as the mean 6 S.E.M. Significant differences between
`values were examined using Student’s two-tailed unpaired or paired t test as
`appropriate. Results were considered significant if P , .05.
`
`Results
`The mean blood concentrations of both SDZ-RAD and rapamycin
`after i.v. and oral administration can be seen in Fig. 1. Blood con-
`centrations of intact SDZ-RAD from a 2-h infusion of 1 mg/kg
`SDZ-RAD were shown to be cleared more rapidly than that of an
`equivalent dose of rapamycin (Fig. 1A). When the AUCs were cal-
`culated to infinity (Table 1), it was established that rapamycin had an
`AUC which was double that of SDZ-RAD (1140 compared with 573
`ng*h/ml). However, when applied via the oral route, blood concen-
`trations of intact SDZ-RAD and rapamycin were very similar. A
`comparison of blood levels obtained after i.v. and oral administration
`indicated a low absorption of both compounds; however, the absorp-
`tion that did occur proceeded very rapidly—the highest blood con-
`centrations were observed after only 30 min (Fig. 1B). A clear
`biphasic response in elimination of orally presented rapamycin was
`observed in this study, which was much more pronounced than that of
`SDZ-RAD (Fig. 1B). Up to 12 h of elimination of rapamycin was
`quite rapid whereas elimination after 12 h was significantly lower.
`The first phase would correspond to rapid tissue distribution whereas
`the second was most likely limited by systemic metabolism (Fig. 1A).
`Blood samples from the oral dose rapamycin experiment was col-
`lected only for 48 h; therefore, for consistency, all results in Table 1
`were calculated from the extrapolation of 0- to 48-h data. It can be
`seen that very little difference existed in the primary half-life of
`rapamycin and SDZ-RAD regardless of the route of administration.
`However, the terminal half-life of rapamycin was 25 h using the 0- to
`48-h data compared with only 15 h for SDZ-RAD (Table 1). Again,
`no difference in half-life was apparent between oral and i.v. doses.
`The systemic bioavailability of SDZ-RAD, estimated by the ratio of
`dose-normalized blood AUCs, amounted to more than 16% whereas
`the bioavailability of rapamycin was only 10% in comparison.
`The comparison between total radioactivity and parent compound
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`SDZ-RAD AND RAPAMYCIN INTESTINAL ABSORPTION
`
`629
`
`FIG. 1.Mean blood concentration in adult male rats of rapamycin and SDZ-RAD after a 2-h infusion of either 1 mg/kg rapamycin or SDZ-RAD (A; inset shows a
`magnification of blood concentrations over the first 8 h) and an oral gavage injection of 1.5 mg/kg rapamycin or SDZ-RAD (B).
`Results are the mean 6 S.E.M. of four rats.
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`TABLE 1
`
`Pharmacokinetic parameters for SDZ-RAD and rapamycin in adult rats after single oral administrations (1.5 mg/kg) and 2-h i.v. infusions (1.0 mg/kg)
`
`Compound
`
`Route
`
`Rapamycin
`SDZ-RAD
`Rapamycin
`SDZ-RAD
`
`Oral
`Oral
`i.v.
`i.v.
`
`AUC02‘
`
`a
`
`ng2eq*h/mlb
`428 6 12
`606 6 36
`1760 6 30
`1350 6 90
`
`AUC02‘
`
`a
`
`ng*h/mlc
`172 6 10
`141 6 43
`1140 6 90
`573 6 32
`
`CL
`
`ml/min
`
`VSS
`
`l/kg
`
`3.0 6 0.2
`6.2 6 0.3
`
`27 6 3
`52 6 4
`
`T1/2l1
`h
`2.4 6 0.5
`2.7 6 0.5
`1.9 6 0.1
`3.4 6 1.1
`
`T1/2term
`h
`24.3 6 4.6
`15.5 6 1.5
`25.2 6 2.6
`14.2 6 0.4
`
`MRT
`
`h
`23.8 6 5.3
`16.4 6 1.3
`34.5 6 4.2
`15.9 6 0.5
`
`F
`
`%
`10.0
`16.4
`
`T1/2l1 is half-life of main elimination phase, and T1/2term is half-life of the terminal phase based on the 0- to 48-h data only. Results are expressed as means 6 S.E.M. of four to five rats.
`a AUC02‘ is extrapolated to infinity from 0- to 48-h data.
`b ng2eq*h/ml refers to the total radioactivity measured in blood.
`c ng*h/ml refers to the parent compound.
`
`retained in the AUCs for blood concentration indicated that when the
`compounds were given as a 2-h i.v. infusion almost 65% and 42.5%
`of the AUCs for rapamycin and SDZ-RAD, respectively, were re-
`tained as parent compound, yet when the compounds were given
`orally, the relative amount of parent compound decreased such that
`only 40% of rapamycin and 23% of SDZ-RAD were present as parent
`compound. These results suggested that intestinal metabolism could
`be important in the first-pass effect for these compounds. Therefore,
`we examined the metabolism of SDZ-RAD and rapamycin immedi-
`ately after passage through the mesenteric vein.
`The average volume of mesenteric vein blood collected was around
`0.8 to 1.2 ml per rat. Collection of total blood from the mesenteric
`vein immediately after administration into a ligated intestinal segment
`ensured that none of the compound could enter the main circulation,
`thereby removing the possibility of metabolized compound recircu-
`lating through the mesenteric arteries. Hence, any metabolism ob-
`served in the samples had come from the intestine only. In the animals
`given 0.5 mg/kg of either [3H]SDZ-RAD or [14C]rapamycin, 0.05 6
`0.01% of the total amount of radioactivity available for absorption
`(drug and metabolites) was collected. In comparison, the proportion
`collected from the animals given 5 mg/kg of either compound was
`0.06 6 0.01%. Only a qualitative assessment regarding the initial
`jejunal absorption phase could be made from the mesenteric vein
`study, but the similar percentages of total collected material did
`suggest that there was no difference in very early absorption rates
`between SDZ-RAD and rapamycin and that effectively the same
`proportion of compound was being absorbed across the jejunal mem-
`brane into the mesenteric system regardless of the concentration
`applied.
`The results of parent drug uptake for animals given 0.5 mg/kg
`[3H]SDZ-RAD (Table 2) showed that in the initial uptake phase, 60%
`of the transcytosed compound was metabolized; this dropped to 47%
`as absorption continued during the first 5 min. In contrast, the me-
`tabolism of SDZ-RAD by the gastrointestinal tract in animals given
`10 times this amount (5 mg/kg) was lower; only 43% of initially
`absorbed SDZ-RAD was metabolized and this dropped to 29% as
`absorption continued. Hence, a greater percentage of parent com-
`pound was able to cross the intestinal barrier into the mesenteric blood
`with a combination of increasing concentration and increasing time in
`the luminal environment. Upward of 900 ng of unchanged SDZ-
`RAD/ml blood was being absorbed during the first 5 min of ligated
`jejunal administration (Table 2), when rats were given the higher
`5-mg/kg dose. Qualitatively, this confers a reasonably good absorp-
`tion profile.
`The results for parent drug uptake of rapamycin were very similar
`to results for that of SDZ-RAD, in that the amount retained as parent
`compound at the low dose was significantly lower than that at the
`higher dose (Table 2). Also, when directly comparing the amount of
`intact rapamycin and SDZ-RAD together at the low 0.5-mg/kg dose,
`there was no significant difference between the two compounds.
`
`However, at the higher dose, rapamycin metabolism through the
`intestine was reduced greatly. During the initial 2.5 min, more than
`25% of the rapamycin transported across the intestine was metabo-
`lized by the intestine, but during the next few minutes, 99% of the
`absorbed compound was in its original form.
`It has been shown recently that the extent of intestinal metabolism
`by the CYP3A subtype could be related to the affinity of P-gp toward
`a particular compound (Gan et al., 1996; Schuetz et al., 1996).
`Previous results from our laboratory have shown that in the in vitro
`intestinal Caco-2 cell model, SDZ-RAD and rapamycin were shown
`to be acted upon by active efflux pumps that were most likely related
`to P-gp (A.C. and M.L., unpublished observations). We continued this
`study by examining the blood levels of SDZ-RAD and CsA when both
`compounds were given simultaneously as an oral gavage to rats (Fig.
`2 and Table 3). The AUC for blood concentrations of CsA was
`increased by 22% when orally coadministered with SDZ-RAD. SDZ-
`RAD contributed only 20% of the total drug dose in this study, which
`appeared to suggest a direct concentration-based response.
`SDZ-RAD blood-concentration AUC was increased 5-fold when
`coadministered with CsA (Fig. 2; Table 3), which also was related
`directly to the increase in the amount of CsA over SDZ-RAD added
`for oral absorption. When compared, as a percentage, with the total
`radioactivity that was found in the blood, it was also noticed that
`unchanged SDZ-RAD increased from 30 to 40% of the total pool of
`drug and metabolites found in the blood after coadministration with
`CsA.
`
`Discussion
`SDZ-RAD is a structural analog of rapamycin, sharing the same
`mechanisms of action (Schuler et al., 1997). It was of interest to
`determine the oral absorption profile of the new immunosuppressant
`SDZ-RAD and compare it with rapamycin because a previous report
`by Schuler et al. (1997) reported that even though the in vitro activity
`of SDZ-RAD was less than half that of rapamycin, in their rat model,
`oral activity was equivalent for the two compounds.
`The results of this study showed that when both compounds were
`given i.v., rapamycin had a much longer residence time in the blood
`compared with SDZ-RAD. However, it was shown clearly that when
`both compounds were administered orally, the blood AUCs for SDZ-
`RAD and rapamycin were similar, consistent with the oral equipo-
`tency of these two immunosuppressants described recently (Schuler et
`al., 1997). Hence, the bioavailability of SDZ-RAD was 60% higher
`than that of rapamycin, most likely because of more rapid absorption
`of SDZ-RAD across the intestinal wall. This study used normal,
`healthy rats, but a recent study has examined the pharmacokinetics of
`rapamycin in human kidney transplant patients and concluded that the
`oral absorption was 20% (Ferron et al., 1997), about double our
`estimate in rats from this study. However, the variability seen in their
`study suggests that interpatient differences in rapamycin bioavailabil-
`ity could range from 3 to 35%, just as CsA is reported to have a
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`SDZ-RAD AND RAPAMYCIN INTESTINAL ABSORPTION
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`TABLE 2
`[3H]SDZ-RAD and [14C]rapamycin recovery from adult rat mesenteric vein after in situ jejunal administration at either 0.5 or 5.0 mg/kg
`
`Compound
`
`SDZ-RAD
`
`Rapamycin
`
`Dose
`
`mg/kg
`0.5
`
`5
`
`0.5
`
`5.0
`
`Collection
`
`min
`0–2.5
`2.5–5
`0–2.5
`2.5–5
`0–2.5
`2.5–5
`0–2.5
`2.5–5
`
`Total Activity
`
`ng2eq/ml
`30 6 9
`95 6 22
`520 6 129
`1300 6 70
`48 6 16
`135 6 43
`402 6 123
`1420 6 450
`
`Results are the mean 6 S.E.M. of four to six samples.
`
`Parent Drug
`
`ng/ml
`15 6 7
`47 6 9
`313 6 88
`913 6 71
`22 6 8
`88 6 28
`319 6 102
`1450 6 520
`
`Fraction of
`Parent Drug
`
`%
`40 6 6
`53 6 9
`57 6 4
`71 6 8
`43 6 3
`64 6 2
`76 6 3
`99 6 7
`
`The first 12 h of oral absorption showed rapamycin to have a
`slightly faster rate of blood drug level decrease than SDZ-RAD. The
`opposite effect was observed after i.v. administration, which could
`indicate that gastrointestinal first-pass metabolism was the initial
`limiting factor in circulating blood levels of rapamycin, but that once
`most of the compound had been absorbed, then systemic clearance
`became the limiting factor.
`Of the current immunosuppressants, P-gp’s role in CsA activity and
`absorption has been examined by many studies (Zacher et al., 1994;
`Fricker et al., 1996; Tanaka et al., 1996; Terao et al., 1996). It was
`found that P-gp efflux at the intestinal wall accounts for almost half of
`the variability in bioavailability of CsA (Lown et al., 1997). After oral
`application in this study, the percentage of parent compound remain-
`ing in the blood was lower for both SDZ-RAD and rapamycin in
`comparison to i.v. dosing, suggesting that intestinal metabolism was
`occurring, and this was confirmed from analysis of mesenteric vein
`blood immediately after absorption. Therefore, we also would con-
`clude from our animal studies that significant proportions of both
`rapamycin and SDZ-RAD, especially at low oral doses, are affected
`by intestinal first-pass metabolism and that P-gp efflux may be a
`factor in this effect along with CYP3A4 and other cytochrome P-450s.
`The mesenteric vein study showed that SDZ-RAD was metabolized
`by the intestine to a greater extent than rapamycin. SDZ-RAD also
`was eliminated from the blood faster than rapamycin after i.v. admin-
`istration because of the combined effect of increased clearance and a
`much higher volume of distribution. However, the AUCs after oral
`application were similar for both compounds, which suggests that
`SDZ-RAD has a very high rate of absorption to counteract its metab-
`olism and elimination pathways. This conclusion is supported by other
`recent results in our laboratory that showed SDZ-RAD to have almost
`double the intrinsic permeability of rapamycin through our in vitro
`Caco-2 intestinal transport system (Crowe and Lemaire, 1998). It is
`interesting that SDZ-RAD differs from rapamycin solely by the ad-
`dition of a 40-O-(2-hydroxymethyl) group. This alteration appears to
`be enough to completely alter membrane absorption, metabolic en-
`zyme affinity, and pharmacokinetic profiles, as suggested here and
`elsewhere (Schuler et al. 1997; Crowe and Lemaire, 1998), yet with
`these differences, systemic exposure after oral dosing is similar for
`rapamycin and SDZ-RAD. Because absorption is rapid for SDZ-
`RAD, it may be possible to inhibit intestinal metabolism by allowing
`greater amounts of the compound to enter the circulation intact.
`It has been shown that the addition of SDZ-RAD in conjunction
`with the P-gp and CYP3A inhibitor, CsA, can result in an increase in
`blood levels of CsA, just as CsA can equally increase blood levels of
`SDZ-RAD. A recent publication examined the blood concentrations
`of CsA and rapamycin after i.v. coadministration in rats, and rapa-
`mycin was able to increase CsA blood concentrations but CsA was not
`
`FIG. 2.Blood concentrations of CsA (A) and SDZ-RAD (B) after administration
`alone at 2.5 mg/kg (CsA), 0.6 mg/kg (SDZ-RAD), and coadministration at the
`same doses.
`Results are the mean 6 S.E.M. of four rats.
`
`bioavailability of between 2 and 89% (Ptachinski et al., 1985), which
`is likely a result of different expression levels of MDR1, the intestinal
`P-gp efflux pump, in the villus tip of human enterocytes (Kolars et al.,
`1991; Hebert, 1997; Lown et al., 1997).
`
`Roxane Labs., Inc.
`Exhibit 1032
`Page 005
`
`

`
`632
`
`CROWE ET AL.
`
`TABLE 3
`
`Comparison of absorption parameters of CsA and SDZ-RAD after oral administration of either 2.5 mg/kg CsA or 0.6 mg/kg SDZ-RAD alone and
`as a coadministration
`
`Compounds
`
`CsA
`
`CsA
`
`RAD
`RAD
`
`Results are the mean 6 S.E.M. of four samples.
`
`Cmax
`
`ng/ml
`
`CsA
`
`800 6 146
`
`743 6 88
`
`RAD
`
`7 6 2
`34 6 4
`
`AUC
`
`CsA
`
`RAD
`
`ng z ml21 z h
`
`410 6 530
`
`5020 6 420
`
`43 6 12
`217 6 30
`
`able to increase rapamycin levels, even though it was present at 50
`times the concentration of rapamycin (Stepkowski et al., 1997). How-
`ever, a recent peroral study in rats examining rapamycin tissue and
`blood levels after a 14-day coadministration with CsA did show
`increased blood rapamycin levels after 14 days when CsA was coad-
`ministered at 6.5 times the concentration of rapamycin (Napoli et al.,
`1998). One of their dosages (2.5:0.4 mg/kg, CsA/rapamycin) was very
`similar to ours (2.5:0.6 mg/kg, CsA/SDZ-RAD), so it was interesting
`to see that this dosage of CsA could double mean rapamycin blood
`levels after 14 days, whereas our results show this concentration of
`CsA increased the SDZ-RAD AUC by 5-fold and the maximum blood
`concentration by 4.5-fold. Even though their study was a sustained-
`exposure protocol for 2 weeks and ours was a short-term, 24-h
`pharmacokinetic study, it clearly can be seen that coadministration of
`these immunosuppressants does lead to higher concentrations in the
`blood after oral application. Ferron and coworkers (1997) also exam-
`ined whether CsA blood concentrations were changed after giving
`steady-state doses to patients followed by a single dose of rapamycin,
`but no alterations in CsA blood levels were noted. Other groups have
`examined the synergistic activity of rapamycin with either tacrolimus
`(Arceci et al., 1992) or CsA (Kahan et al., 1991) together, showing
`that combinations of immunosuppressants have an additive effect on
`intracellular accumulation of target drugs and increase survival of
`allograft transplanted animals at lower levels than could be effective
`singularly. Apart from showing that CsA can increase blood concen-
`trations of SDZ-RAD, our combined results with SDZ-RAD and CsA
`add weight to these previous reports and suggest encouragement for
`synergistic action in immunosuppression therapy (Schuurman et al.,
`1997).
`In conclusion, it has been shown that SDZ-RAD has a better oral
`absorption profile than rapamycin in this study, which counteracts its
`high rate of intestinal metabolism and systemic elimination so that
`both rapamycin and SDZ-RAD have similar blood levels after oral
`application. Also, that CsA was able to increase blood levels of
`SDZ-RAD shows that the additive effect of these two compounds can
`benefit the pharmacokinetics of both compounds.
`
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