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Lilly Research Laboratories, Indianapolis, Indiana
(Received April 6, 2007; accepted July 19, 2007)
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Abstract |
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; Testa, 2004). In recent years, targeted mechanisms for prodrug delivery and activation have been applied by using specific transport and/or bioactivation processes in vivo (Han and Amidon, 2000; Anand et al., 2002; Sai and Tsuji, 2004; Wu et al., 2006). The human intestinal peptide transporter, hPepT1 (SLC15A1), appears to be an effective target for increasing intestinal absorption of some small molecules. This proton-dependent transporter is responsible for the absorption of several classes of drugs, such as cephalo-sporins, angiotensin-converting enzyme inhibitors, and valacyclovir (the valine-ester prodrug of acyclovir) (Han and Amidon, 2000; Sai, 2005). The primary endogenous function of hPepT1 is to facilitate the absorption of dietary di- and tripeptides. However, it is attractive as a prodrug delivery target because of its high capacity, broad substrate specificity, high level of expression in the intestinal epithelium, and low occurrence of functional polymorphisms (Brandsch et al., 2004; Zhang et al., 2004a,b).
The dietary intestinal transport systems work in concert with numerous hydrolytic enzymes in mammalian enterocytes. The intestinal hydrolases include carboxylesterases, aminopeptidases, carboxypeptidases, and amidases (Satoh and Hosokawa, 1998; Brodin et al., 2002; Imai, 2006; Imai et al., 2006). Many of these enzymes are highly expressed, low affinity enzymes, with affinity for a variety of ester-, thioester-, carbamate-, and amide-containing compounds. Therefore, by using enzymatically hydrolyzable bonds in preparation of PepT1-targeted prodrugs, it is possible to dramatically improve the systemic availability of poorly absorbed drugs, with limited systemic exposure to the intact prodrug. Han and Amidon (2000) described this general strategy as "peptide transport associated prodrug therapy". However, there are still relatively few examples of PepT1-targeted prodrugs in the literature, with valacyclovir being the most widely studied (Balimane et al., 1998).
The glutamate analog (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740; Fig. 1) is a potent and selective agonist of group II/cAMP-coupled metabotropic glutamate (mGlu2/3) receptors, with anxiolytic activity in numerous animal models (Schoepp et al., 1997, 2003). Although LY354740 is highly soluble and not metabolized in any studied species, its bioavailability ranged from only 10% in rats to 45% in dogs (Johnson et al., 2002) and was similarly low in humans (Kellner et al., 2005). The low systemic availability of LY354740 appears to be due to poor intestinal permeability, related to its low molecular weight, zwitterionic character, and polarity. As a result, LY354740 exhibits variable, absorption-limited pharmacokinetic behavior, lacks dose proportionality, and is subject to food effects in some species.
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). An N-linked alanyl prodrug, LY544344 (Fig. 1), emerged from this, based on its active transport and activation profiles. LY544344 has relatively high affinity for hPepT1, is rapidly hydrolyzed in human jejunum and liver homogenates, and displays dramatically improved bioavailability and pharmacologic activity in rodents (Rorick-Kehn et al., 2006). The present experiments were conducted with LY544344 to further elucidate the first-pass activation process and to evaluate dose proportionality in nonclinical species. |
Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Pharmacokinetic Studies. Rat pharmacokinetic dose-response. Male and female Fischer 344 (F344) rats (Harlan, Indianapolis, IN) were administered oral gavage doses of LY544344 (5, 70, 300, and 1000 mg/kg) or LY354740 (1000 mg/kg) in aqueous solution. Rats were anesthetized with isoflurane, and blood samples were collected from the orbital plexus of three rats per sex per time point and placed into EDTA-treated tubes. Samples were collected using a sparse sampling paradigm at time points ranging from 0.25 to 24 h. Only one blood sample was collected from each rat. Plasma was isolated by centrifugation, frozen at -70°C, and subsequently analyzed for LY544344 and LY354740 by LC/MS/MS.
Rat portal vein pharmacokinetics. The systemic and hepatic portal pharmacokinetics of LY544344 and LY354740 were evaluated in male F344 rats (approximately 250 g; n = 3) after a single 10-mg/kg oral gavage dose of LY544344. Rats were obtained from Harlan. Blood samples were collected from three rats per time point and placed into EDTA-treated tubes at 0.12, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h. Animals were anesthetized with isoflurane, and a lateral incision was made to allow access to the aorta and hepatic portal vein. Portal vein and aortic samples were withdrawn simultaneously with individual 1-ml syringes. Plasma was isolated by centrifugation, frozen at -70°C, and analyzed for LY544344 and LY354740 by LC/MS/MS.
Dog pharmacokinetic dose-response. Male and female beagle dogs (Marshall Farms, North Rose, NY) were administered oral doses of either LY544344 (5, 7, 35, 60, 140, and 200 mg/kg) or LY354740 (300 mg/kg) in solution. This dose range was split over two separate studies, but pharmacokinetic parameters are combined for simplicity. Blood samples for pharmacokinetic analysis were collected after four daily doses to allow accommodation to initial emesis. Samples were drawn from three dogs per sex per time point in EDTA-treated tubes at time points ranging from 0.25 to 24 h. Plasma was isolated by centrifugation, frozen at -70°C, and subsequently analyzed for LY544344 and LY354740 by LC/MS/MS.
Dog portal vein and intravenous pharmacokinetics. The pharmacokinetics of LY544344 and LY354740 were evaluated in portal vein-cannulated male beagle dogs (Marshall Farms; n = 3) in a crossover design (three periods). The treatment periods consisted of 1) a single 5-mg/kg i.v. bolus dose of LY354740, 2) a single 7-mg/kg i.v. bolus dose of LY544344, and 3) daily 7-mg/kg oral doses of LY544344 for 4 days. A 1-week washout was allowed between periods. Blood samples were collected in EDTA-treated tubes after each i.v. dose and after the fourth oral dose. Samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, 18, 24, and 36 h. Portal blood samples were collected simultaneously with systemic (femoral) samples in the LY544344 oral dose period. Plasma was isolated by centrifugation, frozen at -70°C, and analyzed for LY544344 and LY354740 by LC/MS/MS.
Pharmacokinetic Analysis. Plasma pharmacokinetic parameters were calculated by noncompartmental analysis, using WinNonlin Professional version 3.1 (Pharsight Corp., Mountain View, CA). Rat pharmacokinetic parameters were determined from the mean concentration value at each time point. Area under the curve (AUC) was calculated using the linear trapezoidal rule, and concentrations below the limit of assay detection were considered to be zero for pharmacokinetic calculations.
Bioanalytical Methods. Plasma concentrations of LY544344 and LY354740 were determined using validated LC/MS/MS methods. The assays were validated over two concentration ranges, with a combined analytical range of 0.5 ng/ml to 100,000 ng/ml. Samples above 100,000 ng/ml were determined by dilution.
In brief, the LY544344 and LY354740 and internal standards ([13C3][15N]-LY544344 and [13C3][15N]-LY354740) were isolated from plasma by formic acid/acetonitrile precipitation. After centrifugation, the sample supernatants were removed and evaporated to dryness. The dry sample residue was dissolved in 1% ammonium hydroxide in methanol/acetonitrile (90:10 v/v). Solid-phase extraction was performed using Isolute SAX cartridges in 96-well format (International Sorbent Technology, Ltd.; Mid Glamorgan, UK). The analytes and internal standards were eluted using 2.5% acetic acid in methanol/water (90:10 v/v), evaporated to dryness, and reconstituted in methanol/water/acetic acid (95:4.5:0.5 v/v).
Liquid chromatographic separation was conducted using a Supelco Discovery Amide RP C16 column (150 mm x 2.1 mm; 5 µm), with a gradient of 0.05% acetic acid in water as mobile phase A and 0.05% acetic acid in methanol/water (95:5 v/v) as mobile phase B. The flow rate was 400 µl/min. Detection was performed using LC/MS/MS with positive ion electrospray ionization (5500 V, source temperature = 500°C) on a PerkinElmerSciex API 3000 mass spectrometer (PerkinElmerSciex Instruments, Waltham, MA). The MS/MS transitions (m/z) monitored were 186.4
151.0 (LY354740), 257.3211.3 (LY544344), 190.5154.1 ([13C3][15N]-LY354740), and 261.1214.4 ([13C3][15N]-LY544344).
In Vivo Excretion and Metabolism. Rat excretion and in vivo metabolism. Male F344 rats (Harlan) were administered a single oral (100 mg/kg) or intravenous (25 mg/kg) dose of [14C]LY544344 (100 µCi/kg) and housed in Nalgene metabolism cages (one rat per cage; N = 3/treatment group) for collection of urine, feces, and cage washings (30 ml of methanol/H2O, 50:50). Urine was collected at 12, 24, 48, 72, and 96 h postdose, whereas feces and cage wash were collected at 24, 48, 72, and 96 h postdose. Animals were euthanized at 96 h postdose for carcass collection.
A second group of nine male F344 rats was given a 100-mg/kg (100 µCi/kg) oral dose of [14C]LY544344 for assessment of plasma metabolite profiles. Whole blood samples were collected from three rats at 0.25, 2, and 12 h postdose into EDTA-treated tubes. Plasma was obtained from blood by centrifugation.
Dog excretion and in vivo metabolism. Female beagle dogs (n = 4/group; Marshall Farms) were given oral or intravenous doses (7 mg/kg, 5 µCi/kg) of [14C]LY544344 and housed in stainless steel metabolism cages (one dog per cage). To reduce the potential for emesis, the orally dosed animals were administered three daily doses of unlabeled LY544344 before the radioactive dose.
Urine, feces, and cage wash (100 ml of methanol/H2O, 50:50) samples were collected at 12, 24, 48, and 72 h postdose. Blood samples were collected at 0.5, 1, 4, and 12 h postdose into EDTA-treated tubes. For the oral dose group only, 0.5-ml aliquots of whole blood were retained in separate containers for radioactivity analysis. Plasma was obtained by centrifugation and stored at -70°C.
Radioactive analysis of plasma, blood, and excreta. The specific activities of dose solutions were confirmed by liquid scintillation counting (LSC) of three 100-µl aliquots of each dose preparation. Triplicate aliquots of each urine and plasma sample (approximately 0.1 g) were transferred into tared scintillation vials and weighed. The weights were recorded, and 12 ml of Beckman Ready Protein+ LSC fluid (Beckman Coulter, Fullerton, CA) was added. The samples were assayed for radioactivity using LSC. Fecal samples were soaked in a 1:1 H2O/methanol solution and then shaken vigorously to produce a slurry. Samples were then frozen at -70°C overnight, thawed, and again shaken for approximately 2 h. Triplicate aliquots of each fecal slurry (approximately 0.5 g) were placed into combustion thimbles and weighed. The samples were allowed to dry overnight and combusted in a TriCarb Oxidizer 307 (Perkin-Elmer Life and Analytical Sciences, Boston, MA). The resulting 14CO2 was trapped and assayed for radioactivity by LSC. Aliquots of each cage wash sample were transferred into scintillation vials and analyzed by LSC. Blood aliquots were weighed (approximately 100 µl) into combustion thimbles. The samples were allowed to dry overnight, and were combusted in a PerkinElmer TriCarb Oxidizer 307. The resulting 14CO2 was trapped and assayed for radioactivity using LSC. Carcasses were gently boiled in a beaker containing ethanol and potassium hydroxide until all tissues were dissolved. Aliquots (approximately 200 µl) of the resulting digests were transferred into scintillation vials, and Ultima Gold scintillation fluid (PerkinElmer) and acetic acid (approximately 200 µl) were added. Samples were assayed for radioactivity by LSC.
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Radiolabeled compounds in plasma and urine were separated by high-performance liquid chromatography, using a Zorbax SB-CN (4.6 mm x 250 mm, 5 µm) column (Rockland Technologies, Newport, DE), connected to a Berthold Technologies (Bad Wildbad, Germany) LB509 radioactive detector (500-µl liquid flow cell, scintillation fluid set to 4 ml/min). Analytes were eluted with water/0.05% TFA, followed by a step gradient to 100% acetonitrile/0.05% TFA at 10 min to remove any additional bound radioactivity.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The absorption and presystemic activation of the prodrug LY544344 in F344 rats was rapid, with peak plasma concentrations of the active drug (LY354740) more than 100-fold higher than those of the prodrug. Concentrations of LY354740 decreased biexponentially, with an apparent terminal half-life of around 3 to 4 h. The half-life of LY544344 could not be accurately determined because of low concentrations and rapid clearance. In rats administered the prodrug, systemic exposure (AUC) to LY354740 increased in a dose-proportional manner up to 1000 mg/kg, whereas peak concentrations (Cmax) values displayed a less than dose-proportional increase with dose. The prodrug-related increase in oral availability of LY354740 was marked, with approximately 17-fold higher dose-normalized AUC in the top prodrug dose group compared with the 1000 mg/kg dose of LY354740.
Portal vein pharmacokinetic studies were conducted to examine the role of prehepatic hydrolysis in the disposition of LY544344. Simultaneous portal and systemic blood samples were drawn from anesthetized F344 rats after a 10-mg/kg oral dose of LY544344 (Fig. 2). As previously noted, oral absorption and activation of the prodrug were rapid, as indicated by the early peak of plasma concentrations (Table 1). The portal vein study revealed that the majority of prodrug conversion occurred before reaching the portal circulation, with portal Cmax and AUC values for LY544344 less than 1% of those for LY354740. Further conversion of LY544344 appeared to occur in the liver or blood, as LY544344 Cmax dropped to approximately 0.2% of the LY354740 value in systemic plasma. The prodrug was rapidly cleared, with an average elimination half-life of 0.4 h in the portal circulation.
Dog Pharmacokinetics. Pharmacokinetic parameters for LY544344 and LY354740 in beagle dogs after oral administration of LY544344 HCl (5, 7, 35, 60, 140, or 200 mg/kg) are shown in Table 2. Emesis was observed after initial dosing, but this effect subsided after several days of daily administration. Therefore, plasma samples for pharmacokinetic analysis were examined after the fourth daily oral dose to reduce emesis-related pharmacokinetic effects. Similar to its behavior in rats, the prodrug was rapidly absorbed and extensively converted to LY354740 after oral dosing, with no observed sex-related differences in pharmacokinetics. Mean LY544344 AUC values were <1% of those for LY354740, and exposure (AUC and Cmax) to LY544344 and LY354740 was increased in proportion to dose.
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To determine the pharmacokinetics and extent of in vivo conversion of LY544344 to LY354740, LY544344 or LY354740 was administered intravenously (Fig. 3; Table 3). In animals given LY544344, prodrug concentrations decreased rapidly, with detectable plasma concentrations for only 4 h postdose. Consequently, the resulting plasma levels of LY354740 peaked at 5 to 15 min, and the pharmacokinetic profile of LY354740 was nearly indistinguishable from that of intravenous LY354740 (Fig. 3). The activation of LY544344 was complete, with 100% relative availability of LY354740 after i.v. administration of the prodrug. Elimination of LY354740 was bicompartmental, with distribution (alpha) half-life values of around 0.75 h. The mean terminal elimination half-life ranged from approximately 10 to 25.
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Radioactive Excretion. After a 100-mg/kg oral dose of [14C]LY544344 to rats, the mean total recovery of radiocarbon was 94% over 96 h, with 73% of the dose recovered in urine, 16% of the dose recovered in feces, 0.4% of the dose recovered in carcass, and 4.5% of the dose recovered in cage wash (Table 4). The mean total recovery of the 25-mg/kg intravenous dose was 90% after 96 h, with 76% of the dose recovered in urine, 5.6% of the dose recovered in feces, 0.7% of the dose recovered in carcass, and 7.9% of the dose recovered in cage wash. The majority of the radiocarbon was excreted within the first 24 h of dosing.
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The mean total recovery (72 h) of radioactivity in female beagle dogs, after an oral dose of [14C]LY544344, was 88% (Table 5). The majority of the oral dose (68%) was recovered in the urine, with 16% of the dose recovered in feces, and 4.2% of the dose recovered in cage wash. The mean total recovery (72 h) of radioactivity after an intravenous dose of [14C]LY544344 was 96%, with 92% of the dose recovered in urine, 0.4% of the dose recovered in feces, and 3.1% of the dose recovered in cage wash. Most of the radioactive dose (approximately 81% p.o. to 91% i.v.) was excreted within the first 24 h after dosing.
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Metabolite Profiling. A single radioactive peak, consistent with the retention time of [14C]LY354740, was detected in rat and dog plasma (Fig. 5) and in rat urine samples (not shown). A second minor peak (<2%), identified as LY544344, was observed in dog urine samples. The calculated chromatographic recovery of radioactivity from the reconstituted plasma and urine samples was 
100%. These data are consistent with the previously described pharmacokinetic studies, indicating that LY544344 is almost completely converted to active LY354740 in vivo with no further metabolism.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Previous reports demonstrated that this combination of active absorption and rapid enzymatic hydrolysis results in a dramatic increase in the oral bioavailability of LY354740 in rats (Rorick-Kehn et al., 2006) and humans (Kellner et al., 2005). However, the doses at which these studies were conducted were relatively low. The current studies examined the absorptive capacity of PepT1-driven absorption and help elucidate the primary site of LY544344 activation in rats and dogs.
PepT1 transporters appear to be highly conserved, in terms of sequence, distribution, and function, between mammalian species. The PepT1 gene sequences for dog (NM_001003036) and rat (NM_057121.1) are 85% and 82% identical to the human gene (NM_005073
[GenBank]
), respectively. Recent data indicate a similar pattern of intestinal distribution of PepT1 between Sprague-Dawley rats and humans. Both species have high expression in the small intestine, with similar levels of transcript detected in duodenum, ileum, and jejunum. Likewise, both rat and human have relatively little PepT1 expressed in the large intestine (Englund et al., 2006; Lu and Klaassen, 2006). Numerous studies in rats have demonstrated functional similarity of in vivo transport of PepT1 substrates between rats and humans (Han et al., 1998; Sinko and Balimane, 1998; Sawada et al., 1999; Thomsen et al., 2004; Tsuda et al., 2006; Radeva et al., 2007). Relatively little work has been published regarding peptide transporter expression and function in dogs. However, it is presumed, based on structural similarity to rodents and humans, that the dog protein shares the same primary function of intestinal absorption of small peptides and related substrates.
LY544344 is rapidly absorbed and extensively converted to LY354740 after oral administration to male F344 rats. Recent studies in a rat intestinal perfusion model have demonstrated the role of pepT1 in the absorption of LY544344 (Eriksson et al., 2006). In both rats and dogs, the hydrolysis of LY544344 occurs primarily within intestinal cells after oral dosing, as evidenced by the relatively small amount of LY544344 detected in the portal circulation. This is consistent with the in vitro results reported by Bueno et al. (2005), which demonstrated almost complete hydrolysis of the prodrug (10 µM) within 1 h in a jejunal homogenate. The small fraction of LY544344 that passes through the enterocytes is further hydrolyzed, as seen by the low concentration and rapid clearance of prodrug in the systemic plasma. The enzymes responsible for LY544344 activation have not been identified. However, we have noted that EDTA will completely inhibit hydrolysis in plasma and tissue homogenates, suggesting the involvement of metallopeptidase enzymes.
The high capacity of PepT1-mediated absorption was demonstrated in the current studies, with dose proportionality of LY354740 AUC up to doses of 1000 mg/kg in rats and 200 mg/kg in dogs. Interestingly, exposure to the prodrug molecule was not strictly dose-proportional. However, this finding is probably due to the low concentrations and limited time course from which its pharmacokinetic parameters were calculated. Saturation of enterocytic hydrolysis during the initial absorptive phase at high doses may also play a role. There was evidence of slight saturation of absorption in rats, as evidenced by the nonlinearity of Cmax at the highest doses. However, this saturation appeared to affect only the extent of maximum transport, with no impact on overall exposure. Due to the low inherent permeability of LY354740, it is possible that movement of LY354740 across the basolateral membrane is the rate-limiting step in the process of its delivery to plasma via the prodrug. Studies to elucidate the mechanisms of LY544344 and LY354740 cellular flux in Caco-2 cells are under way.
After absorption and hydrolysis of the prodrug, the excretion of LY354740 is unchanged compared with direct oral administration of the active drug. The lack of further metabolism and the patterns of excretion are similar to previous data for LY354740 (Johnson et al., 2002). Therefore, due to the nearly complete first-pass hydrolysis of LY544344, the pharmacokinetics of the active LY354740 is the best measure of the prodrug's performance in vivo.
In conclusion, the prodrug LY544344 demonstrates the utility of PepT1-targeted non-ester prodrugs. This compound exhibits near-ideal prodrug properties, with good solubility and chemical stability, extensive and reproducible absorption across species, low concentrations of circulating nontoxic prodrug, and pharmacokinetic linearity across a wide dose range. The initial problems of poor permeability and low bioavailability were overcome, allowing further clinical development and extension of dose-ranging studies in animal models and humans. The parent drug in this case, LY354740, was well suited for this approach to prodrug design, being a constrained amino acid analog. Due to the requirements for transporter recognition and enzymatic conversion, it is likely that applications of PepT1-targeting of prodrugs are relatively limited. Most efforts to use this mechanism have focused on amino acid esters of small polar drug molecules. However, the current data clearly demonstrate the potential of peptide bond-based transporter-associated prodrugs.
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Acknowledgments |
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Footnotes |
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ABBREVIATIONS: hPepT1, human intestinal peptide transporter 1; LY544344, (1S,2S,5R,6S)-2-[(2'S)-(2-amino)propionyl]aminobicyclo[3.1.0.]hexen-2,6-dicarboxylic acid; LY354740, (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid; PepT1, intestinal peptide transporter 1 (SLC15A1); LC/MS/MS, liquid chromatography-tandem mass spectrometry; AUC, area under the curve; LSC, liquid scintillation counting; TFA, trifluoroacetic acid; HPLC, high-performance liquid chromatography.
Address correspondence to: Dr. Everett J. Perkins, Lilly Corporate Center, Indianapolis, IN 46285. E-mail: perkins.e{at}Lilly.com
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