Pharmacokinetics, Biodistribution, Stability and Toxicity of a Cell

Bioconjugate Chem. 2007, 18, 1325−1331

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Pharmacokinetics, Biodistribution, Stability and Toxicity of a Cell-Penetrating Peptide-Morpholino Oligomer Conjugate Adams Amantana,* Hong M. Moulton, Melissa L. Cate, Muralimohan T. Reddy, Tom Whitehead, Jed N. Hassinger, Derek S. Youngblood, and Patrick L. Iversen AVI BioPharma, Inc., Corvallis, Oregon. Received February 22, 2007; Revised Manuscript Received April 27, 2007

Objective: Conjugation of arginine-rich cell-penetrating peptide (CPP) to phosphorodiamidate morpholino oligomers (PMO) has been shown to enhance cytosolic and nuclear delivery of PMO. However, the in ViVo disposition of CPP-PMO is largely unknown. In this study, we investigated the pharmacokinetics, tissue distribution, stability, and safety profile of an anti-c-myc PMO conjugated to the CPP, (RXR)4 (X ) 6-aminohexanoic acid) in rats. Methods: The PMO and CPP-PMO were administrated intravenously into rats. The concentrations of the PMO and the CPP-PMO in plasma and tissues were monitored by HPLC. The stability of the CPP portion of the CPP-PMO conjugate in rat plasma and tissue lysates was determined by mass spectrometry. The safety profile of the CPP-PMO was assessed by body weight changes, serum chemistry, and animal behavior. Results: CPP conjugation improved the kinetic behavior of PMO with a 2-fold increase in the estimated elimination half-life, a 4-fold increase in volume of distribution, and increased area under the plasma concentration vs time curve. Consistent with the improved pharmacokinetic profile, conjugation to CPP increased the uptake of PMO in all tissues except brain, varied between organ type with greater uptake enhancement occurring in liver, spleen, and lungs. The CPP-PMO conjugate had greater tissue retention than the corresponding PMO. Mass spectrometry data indicated no observable degradation of the PMO portion, while there was identifiable degradation of the CPP portion. Time-dependent CPP degradation was observed in plasma and tissue lysates, with the degradation in plasma being more rapid. The pattern of degraded products differed between the plasma and lysates. Safety evaluation data showed that the CPP-PMO was well-tolerated at the dose of 15 mg/kg with no apparent signs of toxicity. In contrast, at the dose of 150 mg/kg, adverse events such as lethargy, weight loss, and elevated BUN (p < 0.01) and serum creatinine (p < 0.001) levels were recorded. Supplementation with free L-arginine ad libitum showed improved clearance of serum creatinine (p < 0.05) and BUN (p < 0.01) at the toxicological dose, suggesting that the CPP caused toxicity in kidney. Conclusion: This study demonstrates that conjugation of CPP to PMO enhances the PMO pharmacokinetic profile, tissue uptake, and subsequent retention. Therefore, when dosed at e15 mg/kg, CPP is a promising transporter for enhancing PMO delivery in therapeutic settings.

INTRODUCTION Phosphorodiamidate morpholino oligomers (PMO1) are uncharged steric-blocking antisense compounds that can interfere with protein translation, pre-mRNA splicing, and RNA synthesis (1-5). The structure of PMO differs from DNA, with two major structural changes in the phosphorodiester internucleoside linkages and the five-membered ring of the deoxyribose sugar of DNA, as shown in Figure 1. Several animal models have demonstrated the antisense specificity and efficacy of PMOs against their targets (6-15). Like many macromolecules, the cellular uptake of PMOs is limited. Recently, several studies have reported success using arginine-rich cell-penetrating peptides (CPPs) to improve cytosolic and nuclear delivery of PMO in cell culture and in ViVo. For instance, conjugation of CPPs to PMOs enhanced the ability of PMOs to inhibit replication of various viruses in cell culture (3, 16-22) and in ViVo (19, 23), as well as to alter pre-mRNA splicing (24-27). The internalization of CPP-PMO conjugates may be through an * Corresponding author. Adams Amantana, Ph.D. Mailing Address: AVI BioPharma, Inc., 4575 SW Research Way, Corvallis, OR 97333. Phone (541) 753-3635; Fax (541) 754-3545. E-mail: ([email protected]). 1 Abbreviations: CPP, cell-penetrating peptide; DMD, Duchene muscular dystrophy; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; m/z, mass-to-charge ratio; PBS, phosphate buffered saline; PMO: phosphorodiamidate morpholino oligomer; PNA, peptide nucleic acid.

Figure 1. Structures of PMO and CPP-PMO.

endocytosis mechanism involving binding of cell surface proteoglycans through electrostatic interactions between cationic charges of the CPP and anionic charges of the proteoglycans (28). These successes point to the potential of CPPs for the intracellular delivery of therapeutic PMO; however, there are no reports on the in ViVo disposition of any CPP-PMO. Information regarding pharmacokinetics (PK), tissue distribution, dosing, and the safety profile of a CPP-PMO would be useful in designing animal experiments for various disease models.

10.1021/bc070060v CCC: $37.00 © 2007 American Chemical Society Published on Web 06/21/2007

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Therefore, the objective of this study was to evaluate the in ViVo disposition of a PMO conjugated to a CPP having the (RXR)4XB sequence (X ) 6-aminohexanoic acid, B ) β-alanine, R ) L-arginine). We chose this CPP for our study on the basis of the following. First, this CPP demonstrated greater serum stability compared to CPPs such as Tat and an oligoarginine (29). Second, PMO conjugated to this CPP had greater antisense activity than PMO conjugated to Tat and the oligoarginine CPP (28). Last, this CPP has been shown to successfully deliver PMO and caused specific antisense activities in several virus models (3, 17, 18, 23) and in pre-mRNA splicing models (24, 25, 28). Here, we report the PK, tissue distribution, biological stability, and safety profile of the (RXR)4XB-PMO conjugate (CPP-PMO) following intravenous injection in rats.

Amantana et al.

the animals were harvested to assess the tissue distributions of the PMO and CPP-PMO. Pharmacokinetic Evaluation. PK was characterized by compartmental analysis from the blood samples collected at different time points following the first administration. Plasma concentration versus time profiles were fitted into a twocompartmental open model with bolus intravenous input using the PK Solutions Software (Summit Research Services, Montrose, CO). The fitted PK parameters include the apparent elimination half-life (T1/2β), plasma clearance (CL), volume of distribution (Vd), and the area under the concentration versus time curve (AUC). The AUC was estimated using the log-linear trapezoidal method. The plasma data were fitted to eq 1

C(t) ) A e-Rt + B e-βt

EXPERIMENTAL PROCEDURES Synthesis of PMO, CPP, and CPP-PMO Conjugate. The PMO, CPP, and CPP-PMO conjugates were synthesized by AVI BioPharma (Corvallis, OR). The PMO with sequence 5′ACGTTGAIIIGCATCGTCGC-3′ (I ) inosine) was synthesized and purified to >90% by the previously described method (1, 30). The peptide was synthesized and purified to >95% purity using the standard FMOC chemistry and HPLC method. The CPP-PMO conjugate was synthesized and purified according to a method described previously (28). The purity of the final conjugate was >90%. PMO Quantitation. The concentration of PMO in tissue homogenates and plasma from the treated animals was evaluated by HPLC as described previously (8). An extra trypsin-digestion step was performed for the samples harvested from CPP-PMO treated rats. The trypsin-digestion step was used for removal of the CPP portion of the conjugate (31) and left the PMO portion for quantification. Briefly, a 100 µL aliquot of the plasma or tissue homogenate was mixed with 100 µL of trypsin solution (1.0 mg/mL in 1× PBS; Sigma Aldrich, St Louis, MO). The mixture was then incubated in a water bath at 37 °C overnight to facilitate the removal of the peptide from the CPPPMO conjugate. Animals and Husbandry. Experimentally naı¨ve catheterized male Sprague-Dawley rats (Zivic Laboratories, Zelienople, PA) were used for this study. At the time of the experiment, the rats were 9-11 weeks old and weighed 200-250 g. On arrival, the animals were individually housed in stainless steel cages during the acclimatization and treatment periods in the laboratory animal resource center at Oregon State University (OSU), Corvallis, OR. The animals were exposed to a 12 h light/dark cycle in a temperature- and humidity-controlled environment and allowed access to commercially available rodent diet and city-supplied tap water. Temperature was maintained at 1826 °C. The light/dark cycle was allowed to be interrupted for study-related activities. The animal studies were used in accordance with the guidelines established in the Guide for the Care and the Use of Laboratory Animals of National Research Council and was monitored by the institutional animal care and use committee, of OSU. Treatments. Animals were randomly assigned to each treatment group. Each animal received intravenous (iv) bolus injection at 15, 30, and 150 mg/kg dosing levels of the CPPPMO or PMO via an installed jugular vein catheter. In this study, the 150 mg/kg dose was chosen for toxicological evaluation. Blood samples were collected via the installed catheter at 0.2, 0.5, 1, 2, 4, 8, 12, and 24 h time points following a single dose for plasma PK evaluation. For the tissue uptake study, the animals were injected with a daily dose of 15 or 30 mg/kg for 5 days. The animals were euthanized 24 h or 5 days after the last injection by 100% carbon dioxide inhalation (in a plexiglass chamber). The liver, kidney, spleen, heart, brain, and lungs of

(1)

where C(t) is plasma concentration at time t; A and B are intercept terms; R is distribution rate constant; and β is elimination rate constant. Biological Stability of CPP in Plasma and Tissues. Stability of the CPP-PMO was examined in the rat plasma and lysates of the liver, heart, and kidney. The tissue lysates were prepared as previously described (32). The CPP-PMO was incubated in the lysates or plasma at 37 °C for 1, 6, and 24 h periods. The PMO were extracted and subsequently analyzed by matrixassisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using a method described previously (29). Safety Assessment. The safety of the CPP-PMO or PMO was evaluated on the basis of mortality as well as changes in physical appearance, behavior by cage-side observations, inlife body weight, and serum biochemistry profile. LD50 of the CPP-PMO was determined by iv administration of a single dose of the compound at the doses ranging from 1.2 to 400 mg/kg. The following serum components were monitored as part of the toxicological evaluation following administration of the CPP-PMO or PMO: serum alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase, total bilirubin, albumin, total protein, glucose, cholesterol, blood urea nitrogen (BUN), and serum creatinine. All samples were evaluated by the analytical laboratory at OSU college of Veterinary Medicine. Statistical Analysis. Data were analyzed using the statistical program GraphPad Prism v 4.01 (GraphPad Software, San Diego, CA). Statistical differences among compared groups were analyzed by one-way nonparametric ANOVA, and Tukey’s multiple comparison post-test analysis with a p value less than 0.05 was considered statistically significant. Comparison of two dosage levels was conducted using the Student’s t test. A two tailed p value less than 0.05 was considered statistically significant.

RESULTS Plasma Pharmacokinetics. Conjugation of PMO to the CPP improved kinetic behavior of the PMO. Pharmacokinetic (PK) analysis was based on plasma data following a single intravenous injection. The plasma concentration data was described by a two-compartment PK model describing both the distribution and elimination phases. The plasma concentration vs time curves and the PK parameters of the CPP-PMO and PMO are presented in Figure 2 and Table 1 respectively. The plasma PK for the CPP-PMO was characterized by rapid distribution from the vascular space to tissues with an initial distribution halflife (T1/2R) ranging from 0.40 to 1.56 h. The CPP-PMO showed a prolonged elimination half-life (T1/2β) ranging from 3.80 to 8.23 h, representing a 2-fold increase compared to the PMO at all three doses. Also, we observed a 4-fold increase in the

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Table 1. Single-Dose Plasma Pharmacokinetics Following IV Bolus Administration of the CPP-PMO and PMO in Ratsa dose (mg/kg)

AUC (µg-hr/mL)

15 30 150

11.48 ( 1.44 44.58 ( 2.46 74.79 ( 6.77

15 30 150

13.50 ( 2.61 56.47 ( 1.61c 163.40 ( 5.52b

CL (L/hr) PMO 0.32 ( 0.09 0.23 ( 0.02 0.47 ( 0.06 CPP-PMO 0.33 ( 0.01 0.27 ( 0.02 0.30 ( 0.04

Vd (L/kg)

T1/2R (hr)

T1/2β (hr)

3.31 ( 0.17 4.81 ( 0.44 11.20 ( 2.26

0.44 ( 0.13 0.76 ( 0.04 0.80 ( 0.05

2.24 ( 0.91 2.83 ( 0.20 3.80 ( 0.54

12.10 ( 0.65c 11.23 ( 0.96c 15.22 ( 2.22

0.40 ( 0.05 0.63 ( 0.02 1.56 ( 0.10b

5.04 ( 0.06d 5.83 ( 0.86d 8.23 ( 0.07c

a Blood samples were collected at different time points over a 24 h period. Data were fit to the following equation C(t) ) A e-Rt + B e-βt, where C(t) is the plasma concentration at time t; A and B are intercept terms; R is distribution rate constant; and β is elimination rate constant. Each data point represents the mean ( SD of triplicate values. Student’s t-test analysis: bp < 0.001, cp < 0.01, and dp < 0.05 CPP-PMO vs PMO treatment at equivalent dosing levels.

Figure 2. Plasma PMO concentration vs time profiles of (A) PMO and (B) CPP-PMO following a single iv bolus injection in rats at indicated dosing levels. Blood samples were collected at different time points over a 24 h period. The plasma PMO levels at the 12 and 24 h time points were below the lower limit of quantitation (LLOQ) of the detection method of 0.1 µg/mL. Each data point represents the mean ( SD of triplicate values. Table 2. Safety Assessment of the PMO and CPP-PMO Conjugatea dose (mg/kg) 15 30 150 15 30 150

∆wt (g)

BUN (mg/dL)

PMO 6.0 ( 6.93 24.50 ( 0.71 8.7 ( 5.03 16.00 ( 1.41 16.0 ( 8.72 21.50 ( 3.36 CPP-PMO 16.0 ( 7.21 19.67 ( 3.79 -19.0 ( 18.94 50.00 ( 36.77 -39.3 ( 24.44b 106.00 ( 39.85c

creatinine (mg/dL) 0.45 ( 0.07 0.20 ( 0.00 0.25 ( 0.07 0.37 ( 0.06 0.55 ( 0.21 2.03 ( 0.40d

a In-life body weight changes, serum BUN and creatinine data following iv bolus injection in rats. b Group received single daily injections for two consecutive days and animal body weight measured at the end of the 2 day period. The other groups received single daily injections for 5 days. c BUN (p < 0.05, one-way ANOVA, Tuckey’s multiple comparison test). d Serum creatinine (p < 0.001, one-way ANOVA, Tukey’s multiple comparison test). Reference range: BUN: 12-40 mg/dL, serum creatinine: 0.4-0.9 mg/ dL.

volume of distribution (Vd) (12.10-15.22 L/kg body weight) at the 15 and 30 mg/kg doses following conjugation of the CPP to the PMO. There was a small but statistically significant increase in the area under the plasma concentration vs time curve (AUC) at the 30 mg/kg dose and a larger increase in AUC for the 150 mg/kg dose.

Tissue Distribution. Conjugation of PMO to the CPP increased the tissue uptake and retention of the PMO. The liver, kidney, spleen, heart, brain, and lungs of the animals were harvested 24 h following five single daily injections of 15 and 30 mg/kg PMO or CPP-PMO, to assess the tissue distribution of the PMO. The tissue recovery at the 15 mg/kg dose following a 5 day washout period was also evaluated. The data is presented in Figure 3A-C. The CPP-PMO showed a broad tissue distribution with the kidney and liver as the primary sites of accumulation. Relatively lower concentrations were detected in spleen, lungs, heart, and brain. At equivalent dose, the tissue recovery data showed that conjugation of the CPP improved tissue uptake of the PMO in all of these tissues except brain, with the liver, spleen, and lungs showing greater increases at compared to PMO both dosing levels. In addition, tissue concentrations of the CPP-PMO treated samples were greater than the PMO treated samples 5 days postdose (Figure 3C), indicating that CPP conjugation prolongs the duration of tissue retention. Biological Stability of CPP-PMOs. The stability study found no evidence of PMO degradation, while the observed degradation of the CPP portion of CPP-PMO depended on time and tissue type. The degradation pattern varied among the tissue types and between tissue and plasma. The stability of the conjugate was examined in the plasma and the lysates of rat liver, heart, and kidney. The CPP-PMO was incubated with tissue lysates or plasma at 37 °C for 1, 6, and 24 h. The extracted materials were then analyzed by MALDI-TOF MS (29). The CPP portion of the conjugate degraded, more rapidly in plasma than in tissues (Figure 4). The intact CPP-PMO was observable in the plasma up to 6 h. A 24 h treatment resulted in complete degradation of the intact conjugate from (RXR)4XB-PMO to RXRXB-PMO. However, degradation of CPP was slower in tissue homogenates, with no apparent degradation at 6 h but much degradation at 24 h. Unlike the heart and kidney, some intact CPP was detectable in the liver at the 24 h time point. Therefore, the biological stability of the CPP was ranked in the order of liver > heart ) kidney > plasma. In the plasma, cleavage appeared to occur between adjacent L-arginine residues with the appearance of an m/z peak corresponding to RXR, which is consistent with the data observed in human serum (29). Comparatively, in the liver and kidney, degradation was observed to occur between adjacent L-arginine and 6-aminohexanoic acid residues with m/z peaks corresponding to XR(RXR) and XR(RXR)2. CPP degradation in the heart homogenate reflected a combination of the degradation patterns observed for plasma, liver, and kidney. Safety Assessment. Animals were observed for changes in behavior, appearance, or eating and drinking habits during the course of the study. Body weight measurements and serum chemistry were also used for the safety evaluation as shown in Table 2. The CPP-PMO conjugate did not cause detectable toxicity at the 15 mg/kg dose. It was well-tolerated, with no

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Figure 3. PMO concentration detected in tissue lysates. Rats were administrated with the PMO or the CPP-PMO at 15 mg/kg (A) and 30 mg/kg (B) with single daily iv injection for 5 days, and tissues were harvested 24 h after the last administration. Each data point represents mean ( SD of replicate values (N ) 5). (C) Rats were administrated with the PMO or the CPP-PMO at 15 mg/kg with single daily iv injection for 5 days, and the tissue were harvested 5 days after the last administration. The lower limit of quantitation (LLOQ) of the HPLC method was 0.075 µM. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test).

Figure 4. Biological stability of the peptide portion of the CPP-PMO conjugate in the rat tissue lysate or plasma. The mass spectrometry spectra of the samples are shown. Rat liver lysate (A), heart lysate (B), kidney lysate (C), and plasma (D) were treated with conjugate at 34 µM for 1, 6, and 24 h at 37 °C. The extracted materials were then analyzed by MALDI-TOF MS.

apparent adverse events. At 30 mg/kg, there was a decrease in body weight and a slight elevation in BUN levels but not creatinine. At 150 mg/kg, there was a greater decrease in body weight and a greater increase in BUN (p < 0.05) and serum creatinine (p < 0.001) levels. Animals appeared to be lethargic within minutes after the injection of 150 mg/kg of the conjugate. No changes were observed in albumin, total protein, glucose billirubin, cholesterol, electrolytes, as well as formed elements of blood (including red cells, neutrophils, and platelets) or serum enzymes such as LDH, or ALT (data not shown) at any dose level.

Free L-arginine was administered ad libitum in an attempt to restore BUN and serum creatinine to the normal plasma levels alongside treatment with CPP-PMO at a dose of 150 mg/kg. This was based on earlier reports demonstrating L-arginine to be effective against renal toxic or ischemic injury (33-39). L-Arginine supplementation significantly improved BUN (p < 0.01)) and serum creatinine (p < 0.05) clearance, as shown in Figure 5A and B, respectively. The data in Figure 6 show that the LD50 of the conjugate administered iv in rats was between 210 and 250 mg/kg. Death was observed at 3 h postdose at 400 mg/kg and was preceded

Cell-Penetrating Peptide−Morpholino Conjugate

Figure 5. Coadministration of L-arginine reduced serum BUN and creatinine levels. Mean serum creatinine and BUN levels vs time following iv administration of the CPP-PMO at a single 150 mg/kg dose and 0.5 g/animal L-arginine administered ad libitum. Serum creatinine and BUN following CPP-PMO + L-arginine treatment were significantly lower than the CPP-PMO alone, p < 0.05 and p < 0.01, respectively (one-way ANOVA, Tukey’s multiple comparison test).

Figure 6. Percent animal death vs log dose of the CPP-PMO following a single iv injection in rats.

by mild to severe lethargy, decreased food and water consumption, and ruffled fur.

DISCUSSION PMOs have been shown to have good biological stability (29), RNA binding affinity and specificity (2, 31), efficacy, and safety profiles (7, 13, 40). In spite of these qualities, delivery of PMOs into cells is limited. The arginine-rich cell-penentrating peptides, Tat, oligoarginine, and (RXR)4, have been shown to enhance the cytosolic and nuclear delivery of PMO (3, 23, 26-28). The (RXR)4 peptide has been found to be more efficacious (28) and more stable (29) than the Tat and oligoarginine peptides, and may have the potential to deliver therapeutic PMO. Therefore, evaluation of the PK, tissue distribution, biological stability, and safety of the (RXR)4-PMO conjugate is an important step for its therapeutic development. Conjugation of PMO to the (RXR)4 CPP improved kinetic behavior of the PMO as reflected by prolonged elimination halflife, increased AUC, and volume of distribution (Vd). Since the AUC and Vd are directly related to bioavailability and tissue uptake, respectively, it is expected that increases in these pharmacokinetic parameters will lead to increased tissue uptake. Indeed, the CPP-PMO demonstrated broad tissue distribution with increased uptake compared to PMO alone in all tissues except brain. This is in agreement with an earlier study which

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demonstrated enhanced tissue uptake of a Tat-protein-conjugated PMO compared to the same PMO in a nonconjugated form (41). Kidney and liver were the sites of the highest accumulation of CPP-PMO in our study, with relatively lower concentrations observed in the lungs, spleen, heart, and brain. The broad distribution of the PMO is therapeutically valuable, because it is targeted to c-myc which is ubiquitously expressed in nearly all replicating cells. c-myc is expressed in the process of liver regeneration following partial hepatectomy, polycystic kidney disease, and other cell proliferation disorders. Therefore, the observed accumulation of the CPP-PMO in tissues such as the liver and kidney may be therapeutically valuable. Once delivered to the tissues, CPP-PMO showed extensive retention time as shown by the tissue concentration of the compound detected 5 days after the last injection as shown in Figure 3C. This observation further goes to demonstrate a slow efflux of the compound from tissues back to the vascular space, which is vital in determining the dosing schedule in future clinical studies. Electrostatic interaction between the positively charged conjugate and cell surface proteoglycans is one of the factors that account for increased tissue uptake of CPP-PMO compared to PMO. The exact mechanism by which conjugated CPPs facilitate PMO delivery into the cytosolic and nuclear compartments of cells is not fully understood, although proteoglycaninvolved endocytosis has been suggested (28). The CPP had moderate biological stability with intact CPP detectable up to 6 h in plasma. This, along with the CPP-PMO having an estimated distribution half-life (T1/2R) range from 0.4 to 1.56 h, suggests that CPP-PMO will likely have sufficient time for distribution from plasma to peripheral tissues before the CPP component is completely degraded. Once the CPPPMO reached the tissues, it had a high degree of stability, with the majority of the conjugate still intact up to 6 h in all three tissue types examined. It is interesting that the rate and pattern of CPP degradation differed among the tissues. No detectable intact conjugate was found in heart and kidney after 24 h incubation, but the intact conjugate was still detectable in liver. Safety was assessed on the basis of changes in the animals’ physical appearance and activity, food and water consumption, in-life body weight, and serum biochemistry. The CPP-PMO conjugate was well-tolerated at 15 and 30 mg/kg doses, with no evidence of clinical abnormalities. This observation further emphasizes the therapeutic utility of this CPP-PMO conjugate, since the effective dose has been shown in previous studies to be several fold less than the lowest dose used in this study (13, 42). However, the 150 mg/kg dose caused adverse events such as lethargy and elevated BUN and serum creatinine levels. It must be noted that the 150 mg/kg dose was chosen to exclusively examine the toxicological profile of the CPP-PMO conjugate and therefore has no therapeutic relevance. BUN and creatinine are cleared from the plasma mainly by the kidneys, and a decrease in renal output leads to a simultaneous elevation of BUN and creatinine in plasma. We note that L-arginine is a known precursor for urea and creatinine biosynthesis in the liver and kidneys (43-46). It is therefore possible that the elevated BUN and serum creatinine levels could be due in part to the CPP degradants. However, the degree of elevation of serum creatinine and BUN levels appear to be significant and may not have been generated from the CPP portion of the CPPPMO. Therefore, we hypothesize that the observed simultaneous increase in serum creatinine and BUN concentrations is a result of reduced renal output. To test this hypothesis, L-arginine was administered ad libitum in an attempt to rescue the animals from the apparent peptideinduced renal failure. Like other cationic amino acids, L-arginine is transported into cells and tissues via the Na+-independent cationic amino acid transporter system y+, which is encoded

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by the Cat gene (47-52). We speculate that the delivery peptide which is arginine-rich utilizes the y+ system for transport into the kidney but with lower affinity compared to L-arginine. Therefore, L-arginine may be expected to displace the CPPPMO conjugate from the transporter, thereby reducing the delivery of the CPP-PMO to the kidney cells. Displacement of the CPP-PMO conjugate from the transporter by L-arginine may lead to reduced kidney exposure, and hence, the effect of the CPP-PMO on the kidney could be reduced. Also, L-arginine has been shown to increase renal glomerular filtration rate (GFR) either via nitric oxide (NO) production or stimulation of glucagon secretion (33-39). Indeed, our results showed improved renal clearance of creatinine and BUN following L-arginine supplementation. In conclusion, pharmacokinetic, tissue distribution, biological stability, and safety profiles of a CPP-PMO have been established by this study. CPP conjugation improved the kinetic behavior and tissue uptake of PMO. The CPP component has moderate stability in plasma and tissues. At e15 mg/kg, CPPPMO appeared to be nontoxic. Hence, the data from this study provide useful information when designing future CPP-PMO in ViVo efficacy studies.

ACKNOWLEDGMENT The authors thank the chemistry team at AVI BioPharma for the synthesis and purification and analysis of the PMO and CPP-PMO used in this study and the technical assistance provided by Oregon State University laboratory animal resource center (LARC) and Ariel Eberle. Also, we would like to thank Dave A. Stein for the critical reading of this manuscript.

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