in rats, dogs and Cynomolgus monkeys

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Journal of Antimicrobial Chemotherapy

DOI: 10.1093/jac/dkg046

Page 1 of 7

. . . .

Absorption, pharmacokinetics and excretion of levovirin

in rats, dogs and Cynomolgus monkeys

**Chin-Chung Lin*, Trong Luu, David Lourenco, Li-Tain Yeh and Johnson Y. N. Lau**

Research and Development, Ribapharm, Inc., 3300 Hyland Avenue, Costa Mesa, CA 92626, USA Received 7 February 2002; returned 2 July 2002; revised 31 July 2002; accepted 20 August 2002

The absorption, pharmacokinetics and excretion of levovirin were studied in Sprague–Dawley rats (30 mg/kg) and Beagle dogs (30 mg/kg) following intravenous (iv) and oral administration of [3_{H]levovirin, and in Cynomolgus monkeys following iv and oral administration of [}14_C]levovirin. Oral absorption was 31.3% in rats, 67.3% in dogs and 17.5% in monkeys, and the bioavailability was 29.3% in rats, 51.3% in dogs and 18.4% in monkeys. After iv administration, the elimination half-life (t_1/2) was 1.47 h in rats, 3.70 h in dogs and 3.50 h in monkeys. The total body clearance was 8.24, 2.96 and 2.58 mL/min per kg, respectively, in rats, dogs and monkeys and the apparent volume of distribution was 0.79, 0.95 and 0.65 L/kg. No metabolite was detected in plasma or urine of rats, dogs or monkeys, indicating negligible metabolism of levovirin in these animals. Excretion of total radioactivity in urine after oral dosing accounted for 15.4% of the administered dose in rats, 49.9% in dogs and 21.4% in monkeys. Biliary excretion did not play a significant role in the elimination of levovirin.

Keywords: levovirin, hepatitis C, metabolism, pharmacokinetics

Introduction

Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide)

is a purine nucleoside analogue with broad-spectrum activity

against a variety of DNA and RNA virus infections.1,2_{It has}

been approved as monotherapy in paediatric patients as an aerosol in the treatment of respiratory syncytial virus (RSV) pneumonia with respiratory distress. The oral form is

ap-proved as combination therapy with either interferon-α-2b or

pegylated-interferon-α-2b (Rebetron and Pegatron,

Schering-Plough, Kenilworth, NJ, USA) in the treatment of patients with clinically compensated chronic hepatitis C virus (HCV) infection. Ribavirin directly inhibits the replication of many DNA and RNA viruses. More recently, a number of studies have suggested that ribavirin may exert its antiviral-activity

through an immunomodulatory mechanism.3–6_{In vitro and}

ex vivo studies suggested that ribavirin can promote

T-cell-mediated immunity against viral infection by augmenting anti-viral Th1 cytokine expression [interleukin-2 (IL-2), tumour necrosis factor] and suppressing Th2 cytokine levels (IL-4,

IL-5 and IL-10). In addition, Crotty et al.7_{showed that}

riba-virin could induce lethal mutagenesis to viral genome as a

result of RTP utilization by the viral RNA-dependent RNA polymerase and misincorporation of RMP into viral RNA. In clinical studies, ribavirin monotherapy lowered serum alan-ine transaminase (ALT) levels during treatment in patients with chronic HCV infection. The combination of ribavirin

and interferon-α is effective in inducing a sustained

viro-logical response in 41–54% of patients with chronic HCV

infection.8

Ribavirin has a known major side effect, namely haemo-lytic anaemia. Ribavirin is rapidly transported across cellular membranes, including those of red blood cells (RBCs). Since RBCs are anuclear, they have the capacity to phosphorylate nucleosides to their metabolites, but are devoid of phos-phatase activity to convert the phosphorylated metabolites back to ribavirin, which is capable of being transported out of the cells. This asymmetric activity in RBCs results in the intracellular accumulation of high levels of phosphorylated

ribavirin.9_{Ribavirin triphosphate is structurally similar to}

ATP and can competitively inhibit energy utilization, which results in a shorter life span for RBCs and haemolytic

anaemia.10–12

. . . .

*Corresponding author. Tel: +1-714-427-6236, ext. 2062; Fax: +1-714-641-7201; E-mail: cclin@ribapharm.com

at Pennsylvania State University on March 5, 2014

http://jac.oxfordjournals.org/

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Levovirin (1-β-L

-ribofuranosyl-1,2,4-triazole-3-carbox-amide) is a guanosine nucleoside analogue and the L

-enantio-mer (molecular mirror image) of ribavirin. Levovirin was discovered in the course of identifying compounds with similar or better immunomodulatory activities than ribavirin. The immunomodulatory effects of ribavirin are believed to be important for its activity in the combination treatment with

interferon-α for the treatment of chronic HCV infections. This

compound was selected for further development because of its immunomodulatory activities and its superior pre-clinical

toxicity profile compared with ribavirin.13_{Levovirin has been}

shown to stimulate host immune responses (enhanced Th1 and reduced Th2 cytokine expression). Levovirin is not rec-ognized by the host enzymes responsible for phosphorylation and has no measurable direct antiviral effect or the side effect

of haemolytic anaemia.6_{Like ribavirin, levovirin reduced}

serum ALT levels in a mouse hepatitis model.6_{In toxicology}

studies, levovirin did not appear to produce adverse effects, whereas ribavirin resulted in significant haemolytic anaemia

at the same dosage level.13_{In contrast to ribavirin, levovirin}

did not accumulate in RBCs and thus did not lead to

haemo-lytic anaemia.14_{Since levovirin has a much better safety}

pro-file than ribavirin and exerts similar immunomodulatory effects in a mouse efficacy model, it may provide a better therapeutic option for patients with chronic HCV infection.

The objectives of this study were to determine the absorp-tion, pharmacokinetics and excretion of levovirin in rats, dogs and monkeys.

Materials and methods

Compound

The compound [5-14_{C]levovirin (53 mCi/mmol) (Figure 1)}

was synthesized using [14_{C]barium carbonate as a precursor.}

[3_{H]Levovirin (14.6 Ci/mmol) was synthesized by}

hetero-geneous catalytic exchange with tritium gas. The labelled nucleosides were extensively purified by column chromatog-raphy and repeated recrystallization. The chemical identity

and purity were verified by mass spectrometry and proton magnetic resonance spectrometry, respectively. Radiopurity (>98%) was confirmed by HPLC coupled with radioflow detection.

Drug administration and sample collection in rats

Eight male Sprague–Dawley rats weighing ∼300 g were used

in the study. The protocol for the study was reviewed and approved by the Institutional Animal Care and Use Com-mittee at Premedica (Worcester, MA, USA). Following an overnight fast, four rats received an iv dose of 30 mg/kg of

[3_{H]levovirin via tail vein injection and four rats received an}

oral dose of 30 mg/kg of [3_{H]levovirin (}∼75 µCi in sterile

water) via oral gavage. Serial blood samples were collected directly into heparinized tubes at 0.1 h (iv only), 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48 and 72 h from four rats for each dose route and immediately centrifuged for 15 min to separate plasma and RBCs. In a separate study, urine and faecal samples at 0–6, 6–24, 24–48 and 48–72 h from four rats for each dose route were collected in metabolic cages for analysis.

Drug administration and sample collection in dogs Eight Beagle dogs weighing 9–12 kg were used in the study. The protocol for the study was reviewed and approved by the Institutional Animal Care and Use Committee at Premedica. Following an overnight fast, four dogs received an iv dose of

30 mg/kg of [3_{H]levovirin via a cephalic vein and four dogs}

received an oral dose of 30 mg/kg of [3_{H]levovirin via gavage}

followed by 5 mL of water to flush the dosing tube. Serial blood samples were collected directly into heparinized tubes at 0.1 h (iv only), 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48 and 72 h from four dogs in each dosing route and immediately centrifuged for 15 min to harvest plasma. Urine samples were collected from 0–6, 6–24, 24–48 and 48–72 h in plastic containers chilled by dry ice. Faeces were collected at 0–24, 24–48 and 48–72 h.

Drug administration and sample collection in Cynomolgus monkeys

Four male Cynomolgus monkeys weighing 4–5 kg were used in a two-way crossover study with a washout period of 2 weeks. The protocol for the study was reviewed and approved by the Institutional Animal Care and Use Committee at Pre-medica. Following an overnight fast, each monkey received

an iv bolus dose (10 mg/kg) of [14_{C]levovirin via a saphenous}

vein or an oral dose (10 mg/kg) of [14_{C]levovirin (}∼0.5 mCi in

sterile water) via oral gavage. Serial blood samples were collected directly into heparinized tubes at 5 min (iv only), 15 min (oral only) and 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144 and 168 h from each monkey and immediately centri-fuged for 15 min to separate plasma and RBCs. Urine and Figure 1. Chemical structures of levovirin and ribavirin.

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faecal samples at 0–6, 6–12, 12–24, 24–48, 48–72, 72–96, 96–120, 120–144 and 144–168 h were collected from each monkey for analysis.

Measurement of radioactivity

The radioactivity in plasma samples (0.5 mL) and urine samples (0.2 mL) was measured directly using Ultima Gold XR scintillation cocktail and a liquid scintillation counter (Model 1900TR; Packard Instrument Company, Meriden, CT, USA). Faeces and RBCs were combusted in a sample oxidizer (Model 306; Packard Instrument Company) and the

resulting 14_CO

2 was trapped in a mixture of Perma Fluor E+

and Carbo-Ssorb E followed by liquid scintillation counting. Scintillation counting data were automatically corrected for counting efficiency using a set of external standards and an instrument-based quench curve generated from a series of sealed quenched standards.

LC-MS/MS method for determination of levovirin in plasma

The LC-MS/MS (liquid chromatography/tandem mass spectrometry) method involved the addition of internal stand-ard (aciclovir), protein precipitation with acetonitrile, solvent evaporation, reconstitution of residue and Inertsil Silica column separation, followed by MS/MS detection. A Perkin-Elmer Sciex API 3000 in the MRM mode using positive electrospray ionization was used to monitor the 245 to 113 and 259 to 128 transitions for ribavirin and internal standard. The limit of quantification was 10 ng/mL with a relative

standard deviation (R.S.D.) of <3% and a deviation of the

mean from theoretical (DMT) of <4%. Linear regression of the concentration data (range 10–5000 ng/mL) yielded a correlation coefficient of >0.999. The overall precision was

<3%, <4% and <6% R.S.D. (n = 8), respectively, for the 30,

1500 and 3500 ng/mL QC samples. The accuracy, expressed as DMT, was not more than 9% for the intra-day and inter-day means.

HPLC procedure for studying the metabolic profiles of the drug in plasma and urine

Plasma and urine were mixed with an equal volume of acetonitrile. The mixtures were centrifuged and the super-natant was injected into the HPLC apparatus coupled with a

radioactivity detector (β-Ram Model 2; INU Systems, Inc.)

with Packard Ultima-flo AP as scintillant at a flow rate of 3 mL/min. The HPLC (Shimadzu Model SCL 10VP) was

equipped with an Amide-80 column (TSK, 4.6 mm × 110 cm).

The column was eluted with a solvent mixture consisting of 95% organic mobile phase (acetonitrile) and 10% aqueous phase (25 mM ammonium acetate) at a flow rate of 1.2 mL/min.

Immediately after injection, the solvent mixture was switched to 70% organic phase and 30% aqueous phase.

Pharmacokinetic analysis

Concentrations of radioactivity and levovirin in plasma and in RBCs were used to determine the pharmacokinetic para-meters using non-compartmental methods (Win Nonlin-2; Pharsight Corp., Mountain View, CA, USA). Maximum

con-centration (C_max) and time to maximum concentration (T_max)

were observed values. The area under the concentration–time curve to the last quantifiable sampling time (tf), AUC(tf), was computed using the linear trapezoidal rule. The area under the concentration–time curve to infinity, AUC(I), was calculated as the sum of AUC(tf) and the quotient of the last measurable

concentration (C_tf) and the elimination rate constant (K).

K was estimated as the negative slope of the regression of log

concentration versus time. Half-life (t_1/2) was calculated by

dividing 0.693 by K. Apparent total body clearance (CL) was calculated as the ratio of dose to AUC(I). Renal clearance was

calculated as the ratio of the cumulative urinary excretion (A_e)

to AUC(I). Volume of distribution (V_d) was calculated as the

ratio of total body clearance to K.

Results

Concentrations of levovirin and radioactivity in rat plasma

After iv administration (30 mg/kg), the disposition kinetics of levovirin were found to be biphasic (Figure 2) with an

elimination t_1/2 of 1.47 h, mean V_d of 0.79 L/kg and mean CL

of 8.24 mL/min per kg.

After oral administration (30 mg/kg), levovirin was rapidly

absorbed with a T_max of 1.5 h and a C_max of 2.30 mg/L. The

elimination t_1/2 was 4.46 h, which is longer than that obtained

Figure 2. Mean concentrations of levovirin and radioactivity in rat

plasma following iv and oral administration of [3_H]levovirin.

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after iv dosing. By comparing the AUC(I) obtained following oral dosing with that obtained after iv dosing, absolute bio-availability was calculated to be 29.7% (Table 1).

Radioactivity concentrations were measured in plasma following iv and oral administration. By comparing the plasma radioactivity AUC(tf) obtained after oral dosing with that obtained after iv dosing, absorption was estimated to be 31.3%.

Concentrations of levovirin and radioactivity in dog plasma

After iv administration (30 mg/kg), the disposition of levo-virin was found to be biphasic (Figure 3), with an elimination

t_1/2 of 3.70 h, mean V_d of 0.95 L/kg and mean CL of 2.96 mL/

min per kg.

After oral administration (30 mg/kg), levovirin was

absorbed with a T_max of 2.25 h and a C_max of 11.6 mg/L. The

elimination t_1/2 was 4.06 h, which is similar to that obtained

following iv dosing. By comparing AUC(I) after oral dosing with the AUC(I) obtained after iv dosing, absolute bioavail-ability was calculated to be 51.3% (Table 1).

Radioactivity concentrations were measured in plasma following iv and oral administration. By comparing plasma

radioactivity AUC(tf) obtained after oral dosing with that after iv dosing, absorption was estimated to be 67.3%. Concentrations of levovirin in monkey plasma

After iv administration (10 mg/kg), the disposition kinetics of levovirin were also found to be biphasic (Figure 4), with an Table 1. Pharmacokinetic parameters of levovirin in rats and dogs following iv or oral administration

of [3_H]levovirin

Species Analyte Parameter Unit Intravenous Oral

Rat levovirin Cmax mg/L 57.8 2.30

T_max h 0.1 1.5 AUC(I) mg·h/L 61.1 18.1 bioavailability % NA 29.7 t_1/2 h 1.47 4.46 CL mL/min/kg 8.24 NA CLr mL/min/kg 7.25 NA V_d L/kg 0.79 NA

radioactivity C_max mg-equivalent/L 45.7 45.7

T_max h 0.1 0.1

AUC(I) mg-equivalent·h/L 59.2 59.2

absorption % NA 31.3

Dog levovirin C_max mg/L 103.6 11.6

T_max h 0.1 2.25 AUC(I) mg·h/L 169.4 86.9 bioavailability % NA 51.3 t_1/2 h 3.70 4.06 CL mL/min/kg 2.96 NA CLr mL/min/kg 2.41 NA V_d L/kg 0.95 NA

radioactivity C_max mg-equivalent/L 75.0 10.9

T_max h 0.10 2.25

AUC(I) mg-equivalent·h/L 159.8 107.5

Figure 3. Plasma concentrations of radioactivity and levovirin in dogs

following iv or oral administration of [3_H]levovirin.

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elimination t_1/2 of 3.50 h, mean V_d of 0.65 L/kg and mean CL of 2.58 mL/h per kg.

After oral administration (10 mg/kg), levovirin was

absorbed with a T_max of 3 h and a C_max of 0.822 mg/L. The

elimination t_1/2 was 12.2 h. By comparing the AUC(I)

obtained after oral dosing with that obtained after iv dosing, absolute bioavailability was calculated to be 18.4% (Table 2). Radioactivity levels in monkey plasma and RBCs

After iv administration, the radioactivity AUC(tf) in RBCs (53.9 mg-equiv·h/L) was lower than that in plasma

(65.1 mg-equiv·h/L) (Table 2). Similar results were obtained after oral dosing, indicating that there was no preferential uptake of levovirin into RBCs after either iv or oral adminis-tration. By comparing the plasma radioactivity AUC(tf) obtained after oral dosing with that obtained after iv dosing, absorption was calculated to be 17.5%.

Urinary and faecal excretion of radioactivity in rats, dogs and monkeys

In rats, the cumulative excretion data for radioactivity in urine and faeces are shown in Table 3. Over a 96 h period, 88.6% of the iv dose and 15.4% of the oral dose were excreted in urine. In the same period, 2.08% of the iv dose and 75.8% of the oral dose were excreted in faeces. The data indicated that biliary excretion did not play a significant role in the elimination of levovirin in rats. By comparing the cumulative amount of

levovirin excreted in urine (A_e) with plasma AUC(I) for

levo-virin after iv dosing, the renal clearance (CLr) was estimated to be 7.25 mL/min/kg, which is slightly lower than CL after iv dosing (8.24 mL/min/kg).

The cumulative excretion data for radioactivity in urine and faeces in dogs are shown in Table 3. Over a 96 h period, 81.6% of the iv dose and 49.9% of the oral dose were excreted in urine. In the same period, 2.20% of the iv dose and 36.8% of the oral dose were excreted in faeces. These data indicate that biliary excretion did not play a significant role in the elimin-ation of levovirin in dogs. By comparing the cumulative amount of levovirin excreted in the urine with the plasma Figure 4. Plasma concentrations of levovirin and radioactivity in

monkeys following iv or oral administration of [14_C]levovirin.

Table 2. Pharmacokinetic parameters of levovirin in Cynomolgus monkeys following administration of [14_C]levovirin

Analyte Parameter Unit Intravenous Oral

Levovirin (plasma) C_max mg/L 28.9 0.822

T_max h 0.083 3 AUC(I) mg·h/L 65.3 12.0 AUC(tf) mg·h/L 65.1 10.6 bioavailability % NA 18.4 t_1/2 h 3.5 12.2 CL mL/min/kg 2.58 15.3 CLr mL/min/kg 2.16 NA V_d L/kg 0.645 NA

Radioactivity (plasma) C_max mg-equivalent/L 30.8 1.07

Tmax h 0 3

AUC(tf) mg-equivalent·h/L 64.7 11.3

Plasma levovirin/plasma radioactivity C_max 0.94 0.77

AUC(tf) 1.01 0.94

RBC radioactivity Cmax mg-equivalent/L 23.7 0.78

T_max h 0 3

AUC(tf) mg-equivalent·h/L 53.9 10.6

RBC radioactivity/plasma radioactivity AUC(tf) 0.83 0.94

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AUC(I) for levovirin after iv dosing, CLr was estimated to be 2.41 mL/min/kg, which is slightly lower than CL after iv dosing (2.96 mL/min/kg).

The cumulative excretion data for radioactivity in monkey urine and faeces are shown in Table 3. Over a 96 h period, 84.5% of the iv dose and 21.4% of the oral dose were excreted in urine. In the same period, 1.82% of the iv dose and 77.9% of the oral dose were excreted in faeces. These data indicate that biliary excretion did not play a significant role in the elimin-ation of levovirin in monkeys. The total recovery of

radio-activity in both urine and faeces accounted for ∼86.3% of the

iv dose and 99.3% of the oral dose. By comparing the cumu-lative amount of levovirin excreted in urine with the plasma AUC(I) for levovirin after iv dosing, CLr was estimated to be 2.16 mL/min/kg, which is slightly lower than CL after iv dosing (2.58 mL/min/kg).

Metabolic profiles in plasma and urine of rats, dogs and monkeys

Metabolic profiles were evaluated in (i) rat plasma at 2 and 8 h after oral dosing, (ii) rat urine 0–6 h and 6–24 h after oral dosing, (iii) dog plasma at 2 and 8 h after oral dosing, (iv) dog urine 0–6 h and 6–24 h after oral dosing, (v) monkey plasma at 1 h after iv dosing and 4 h after oral dosing, and (vi) monkey

urine 0–24 h after iv and oral dosing of [14_C]levovirin.

Unchanged levovirin was the only significant radioactive peak in the plasma and urine samples from rats, dogs and monkeys. These data indicate that there was no or negligible metabolism of levovirin in rats, dogs and monkeys.

Discussion

After iv or oral dosing of radiolabelled levovirin, plasma AUC(I) for radioactivity and plasma AUC(I) for unchanged levovirin were similar in rats, dogs and monkeys, indicating that there was no or negligible metabolism of levovirin in animals. This is in good agreement with the observed meta-bolic profiles in all animal species since unchanged levovirin was the only significant radioactive peak in plasma and urine. In contrast, ribavirin was extensively metabolized in both

rats and monkeys (C.-C. Lin, unpublished data). After oral

administration of [14_{C]ribavirin, plasma AUC for unchanged}

ribavirin accounted for only 8% and 18% of the plasma radio-activity AUC in rats and monkeys, respectively. It is

postu-lated that levovirin, the L-enantiomer of ribavirin containing

an L-ribose instead of the naturally occurring D-ribose, is not

a substrate for enzymes involved in the phosphorylation of ribavirin.

In monkeys, after oral dosing of [14_C]levovirin,

radio-activity AUC in plasma and RBCs were identical, indicating that no preferential uptake of levovirin occurred with RBCs. An earlier study in monkeys has demonstrated that the activity AUC in RBCs was 33-fold higher than the radio-activity AUC in plasma in monkeys after oral dosing with

[14_{C]ribavirin (C.-C. Lin, unpublished data). As discussed}

above, the RBC appears to be an important target of toxicity

for ribavirin.5,8_{Catlin et al.}9_{reported that after oral dosing}

with [14_{C]ribavirin in man, radioactivity was concentrated in}

RBCs with an RBC/plasma ratio of >100 on day 15 after dosing. It is clear that, unlike ribavirin, levovirin is not prefer-entially taken up by RBCs and hence haemolytic anaemia is minimal as confirmed in subsequent toxicology studies (C.-C. Lin, unpublished data).

After iv administration, V_d was similar in rats (0.79 L/kg),

dogs (0.95 L/kg) and monkeys (0.65 L/kg). Elimination t_1/2

in rats (1.47 h) was shorter than that in dogs (3.70 h) and monkeys (3.50 h). This is in good agreement with the find-ings that CL was higher in rats (8.24 mL/min/kg) than dogs (2.96 mL/min/kg) and monkeys (2.58 mL/min/kg). It is interesting to note that the CLr in rats (7.25 mL/min/kg), dogs (2.41 mL/min/kg) and monkeys (2.16 mL/min/kg) was similar to their respective CLs, suggesting that there was no or negligible metabolic clearance. This is in good agreement with the results generated from metabolic profile evaluation in that there was no or negligible metabolism of levovirin in the three species of animals tested.

It should be pointed out that CLr (2.16 mL/min/kg) and glomerular filtration rate (GFR) (1.96 mL/min/kg) were similar in monkeys, whereas CLr was smaller than GFR in rats (7.25 versus 10 mL/min/kg) and dogs (2.41 versus 4.29 mL/min/kg). These data indicated that active reabsorp-Table 3. Excretion of radioactivity in rats, dogs and monkeys following iv or oral administration of

[3_{H]levovirin or [}14_C]levovirin

Species Dosing route Time (h) Urine (% of dose) Faeces (% of dose) Total (% of dose)

Rat intravenous 0–96 88.6 2.08 90.7 oral 0–96 15.4 75.8 91.2 Dog intravenous 0–96 81.6 2.20 83.8 oral 0–96 49.9 36.8 86.7 Monkey intravenous 0–96 84.5 1.82 86.3 oral 0–96 21.4 77.9 99.3

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tion may play a role in dogs and to a lesser extent in rats, but not in monkeys. After iv administration, urinary excretion of radioactivity accounted for 88.6%, 81.6% and 84.5% of the dose in rats, dogs and monkeys, respectively, whereas faecal excretion of radioactivity accounted for 2.08%, 2.20% and 1.82% of the dose. These data indicate that urinary excretion is the predominant route of elimination, whereas biliary excretion did not play a significant role.

Based on the ratio of plasma radioactivity AUC obtained after oral dosing to that obtained after iv dosing, percentage absorption was estimated to be 31.3% in rats, 67.3% in dogs and 17.5% in monkeys. Based on the ratio of plasma levovirin AUC after oral dosing to that after iv dosing, percentage bio-availability was estimated to be 29.7% in rats, 51.3% in dogs and 18.4% in monkeys. The similarity between percentage absorption and percentage bioavailability in all animal species further confirmed that there was no or negligible metabolism of levovirin in rats, dogs and monkeys.

Plasma t_1/2 for levovirin after oral dosing (4.06 h) and

iv dosing (3.70 h) were similar in dogs, whereas t_1/2 after oral

dosing was much longer than that after iv dosing in monkeys (12.2 versus 3.50 h) and rats (4.46 versus 1.47 h). These data indicated that absorption rate was lower than the elimination rate (typical of a ‘flip-flop’ scenario) in monkeys and rats, but not in dogs. This slow absorption rate in monkeys and rats may be partially responsible for the low percentage absorp-tion of levovirin in monkeys (17.5%) and rats (31.3%) com-pared with dogs (67.3%). An earlier study demonstrated that ribavirin was well absorbed in rats (80.9%) and monkeys

(79.1%). Partsil et al.15_{reported that after oral administration,}

ribavirin was actively transported by gastrointestinal N1 sodium-dependent nucleoside transporters, which are local-ized in the proximal small bowel. In contrast, levovirin is not likely to be recognized by these nucleoside transporters and was absorbed by a passive gradient-dependent mechanism, as is typical for small molecules.

The present study indicated that levovirin, an L-enantiomer

of ribavirin, was orally absorbed in animals, underwent no or negligible metabolism, and was excreted primarily into urine as unchanged drug.

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