Interferon alpha receptor 2a correlated with proinflammatory cytokines in the serum of patients chronically infected hepatitis C virus

HEPATOLOGY Vol. 34, No. 4, Pt. 2, 2 0 0 1

219A

AASLD ABSTRACTS

181

182

SAFETY STUDIES OF LEVOVIRIN, A SECOND GENERATION RIBAVIRIN CANDIDATE, SHOWED EXCELLENT SAFETY PROFILE. Suzana Corr i t o r i , J o h n s o n Y Lau, C h i n - C h u n g Lin, I C N Pharmaceuticals, Costa Mesa, CA

COMPARATIVE ABSORPTION, METABOLISM AND EXCRETION STUDIES OF RIBAVIRIN VERSUS VIRAMIDINE, A LIVER-TARGETED RIBAVIRIN PRODRUG. C h i n - C h u n g Lie, K e n n y Luu, David Lourenco, Jingfan H u a n g , J a n e W Fang, J o h n s o n Y Lau, I C N Pharmaceuticals, Costa Mesa, CA

Background: Ribavirin forms part of the c u r r e n t c o m b i n a t i o n t h e r a p y for c h r o n i c hepatitis C b u t its RBC toxicity represents a challenge in clinical m a n a g e m e n t . Levovirin, the L - e n a n t i o m e r of ribavirn, has b e e n s h o w n to possess the i m m u n o m o d u l a t o r y f u n c t i o n of ribavirin in e n h a n c i n g T h l o v e r T h 2 activity in favor of an antiviral state b u t does trot possess the direct anfiviral activity. Aim: To evaluate the safety profile of levovirin f r o m animal general toxicity, genetic toxicity, and safety p h a r m a c o l o g y perspectives. Study design/ Methods: Single dose toxicity was evaluated in rats at oral doses of 0 , 3 0 0 , 600, 1200, 2400, 4 8 0 0 a n d 9600 m g / k g a n d in dogs at oral doses of 0 , 2 0 0 , 600 a n d 1800 mg/kg. 28-days toxicity s t u d y w a s e v a l u a t e d in rats at oral doses of 0 , 3 0 0 , 600 a n d 2400 mg/kg, and in dogs at oral doses of 0~ 1 0 0 , 5 0 0 a n d 1000 mg/kg. Genetic toxicity was evaluated w i t h (1) A m e s test (Salmonella t y p h i m u r i u m ) , (2) m o u s e l y m p h o m a assay, a n d (3) m o u s e m i c r o n u c l e u s test. Safety p h a r m a cology evaluation i n c l u d e d n e u r o p h a r m a c o l o g i c a l effects in mice, cardiovascular effects in dogs, renal effects in rats, respiratory effects in guinea pigs a n d gastrointestinal p r o p u l s i o n effects in mice. Results: No toxicity was observed in b o t h rats a n d dogs in single dose toxicity in the entire dose range. In 28-day toxicity studies (rats a n d dogs), there w e r e no mortality, no c h a n g e in b o d y w e i g h t a n d food c o n s u m p t i o n , a n d no pathological gross findings n o r m i c r o scopic lesions. In vitro Salmonella t y p h i m n r i u m reverse m u t a t i o n assay confirmed that levovirin (up to 5 0 0 0 / x g / m L ) did n o t cause a n increase in m e a n n u m b e r of revertants colonies in either the p r e s e n c e or absence of m i c r o s o m a l e n z y m e s f r o m A r o c o l o r - i n d u c e d rat liver. Mouse l y m p h o m a cell TK +/- f o r w a r d gene m u t a t i o n assay indicated that levovirin (up to 5000 /zg/mL) was n o t m u t a g e n i c . Mouse b o n e m a r r o w erythropoietic cell m i c r o n u c l e u s test also indicated that levovirin (2000 m g / k g ) did n o t increase the m i c r o n u c l e i in m o u s e p o l y c h r o m a t i c erythrocytes. T h e r e was no adverse effect in n e u r o p h a r m a e o l o g y profile in m i c e (up to the dose of 1000 m g / k g ) , cardiovascular/ h e m o d y n a m i c p a r a m e t e r s in dog (up to dose of 500 m g / k g ) , a i r w a y resistance/ lung c o m p l i a n c e in g u i n e a pigs (up to dose of 350 m g / k g ) , and gastrointestinal motility in m i c e (up to dose of 1000 m g / k g ) . T h e r e was a u r i n e v o l u m e d e p e n d e n t t e n d e n c y t o w a r d increased e x c r e t i o n of Na +, K + a n d C1- at the highest dose (1000 m g / k g ) , b u t w i t h o u t toxicological significance. Conclusions: (1) L e v o v i r i n did n o t s h o w a n y toxicity in single dose, 28-day dosing, g e n e t i c / m u t a g e n i c i t y testing, a n d a n i m a l safety p h a r m a c o l o g y studies. (2) In contrast to ribavirin ( w h i c h s h o w e d significant toxicity in all tests), levovirin has an excellent safety profile, e n d o r s i n g its potential as a s e c o n d g e n e r a t i o n ribavirin f r o m a safety perspective.

Background: Viramidine is a liver-targeting ribavirin prodrug with reduced exposure to RBC. It is converted mainly in liver to ribavirin to exert its activity. Aim: To compare the absorption, metabolism and excretion of ribavirin and viramidine in rats and monkeys to further define the pharmacokinetic characteristics of these compounds. Method: Absorption, metabolism and excretion were evaluated after 1V and oral dosing of I4C-fibavirin, and J4C-viramidine in rats (30 mg/kg) and cynomolgus monkeys ( 10 mg/kg). Plasma, RBC,urine and fecal samples (up to 168 hr) were collected and analyzed for radioactivity-using sample oxidizer followed by liquid scintillation spectrometry. Plasma samples were also analyzed for ribavirin and viramidine by LC-MSBvlS method with a LOQ of 10 ng/mL, a precision with a CV of < 6% and an accuracy with a deviation of mean from theoretical values of < 9%. Concentrations of radioactivity and ribavirin in plasma were used to determine various pharmacokinetic parameters using noncompartmental methods (Win Nonlin-2). Results: Major pharmacokmetic parameters are summarized in the table below. Ribavirin was well absorbed in both rats and monkeys following oral dosing. It was more extensively metaboIized in rats than monkeys. Unchanged ribavirin accounted for 7% of plasma radioactivity iu rats and 33% in monkeys. As a result, ribavirin had a shorter T1/2, a larger CL, and thus a lower bioavailability in rats than monkeys. This species difference in phannacokinetics was also observed for viramidine in rats and monkeys. Viramidine was readily absorbed in rats and monkeys following oral dosing. It was rapidly converted into ribavirin, followed by further metabolic degradation. This accounted for shorter Tta and larger CL of viramidine than ribavirin. However oral dosing of either viramidine or ribavirin yielded similar plasma ribavirin AUC in both rats and nmnkeys. Both viramidine and ribavirm were excreted primarily into urine as metabolRes. Conclusions: (I) Viramidine, a liver-targeting ribavirin prodrug, is metabolized extensively into ribavirin. (2) Viramidine has an acceptable pharmacokinetic profile. Compound Dosed Species (Dose) tt~ (PC) C! (IV) (mU __.__ (m~'kg) _(hr) v~C*Rt~vtrio(R) Pal: 9,9 2800 (30) Monkey 130 224 Rat (30) Monkey

Compound Dosed Species (Beset (rnglkg) 14C-Rihavirtn (R)

~c-giramidine N) _

Oral

Oral

i14oo

80

2z 59

29400

88

1.8

13103

~2852

65

10

23

1459

19952

~

16

(,0;,

~C-Virarnidiee (V)

Yes (IV)

Oral AUC

Oral Urinary le~ (%of Dose)

(ttrx uglml) R

V

2,48

0

83 67

Rat

14C 31,9

bbnkey

50,8

16.9

0

Rat I30)

43,6

3.27

0,20

59

Monkey I10)

30,2

12.2

t,25

46

(30)

183

184

A NOVEL RIBOZYME-BASED METHOD FOR THE DIRECT DETECTION OF H E P A T I T I S C VIRUS RNA. Scott Seiwert, N a r e n d r a Vaish, Lori A n d r e w s , S h a w n Zinnen, L e o n i d Beigelman, L a w r e n c e Blatt, R i b o z y m e Pharmaceuticals, Boulder, CO

INTERFERON ALPItA RECEPTOR 2A CORRELATED W I T H PROINFLAMMATORY CYTOKINES IN THE SERUM OF PATIENTS CHRONICALLY INFECTED HEPATITIS C VIRUS. Koji Ishii, Mie Shinohara, Naoko T a k a m n r a , T o m o k o Ishikawa, K a z u h i r o Shin, Masao Hagiwara, Takashi Ikehara, Yasunobu Nishio, Soichiro Hata, Takashi Kawafune, Yasukiyo Sumino, T o h o University, School of Medicine, T o k y o Japan; W a t a r u Y a m a m u r o , Internal Medicine, Saiseikai-kanagawaken Hospital, Y o k o h a m a J a p a n

T h e direct detection of Hepatitis C viral RNA by RT-PCR a n d other m e t h o d ologies has b e c o m e an i m p o r t a n t c o m p o n e n t in the diagnosis a n d t r e a t m e n t of chronic Hepatitis C. O n e issue w i t h c u r r e n t l y available m e t h o d s is the high rate of assay variability a n d h i g h assay cost. In addition, the c u r r e n t i m m u n o - b a s e d p r i m a r y screens for H C V do not detect infected individuals w h o h a v e yet to s e r o c o n v e r t or those that are i m m u n o c o m p r o m i s e d . Further, the use of RTPCR to diagnose H C V infection in patients w h o h a v e n o t yet s e r o c o n v e r t e d has b e e n cost prohibitive. Here, w e describe a novel, generalizable a n d low cost alternative a p p r o a c h for detection of low copy nucleic acids (including b o t h DNA a n d RNA) u s i n g H C V as a m o d e l system. O u r t e c h n o l o g y is based o n Halfzymes TM, r i b o z y m e s that r e q u i r e a target nucleic acid to s u p p l y nucleotide s e q u e n c e s for r i b o z y m e catalysis. H a l f z y m e molecules are inexpensive biosensers that can b o t h detect a n d signal in a single step. This simplicity should r e d u c e the variability a n d cost of c u r r e n t l y available H C V detection platforms. To target detection of the H C V g e n o m e , w e designed H a l f z y m e molecules that b i n d a n d are activated by a highly c o n s e r v e d RNA s e q u e n c e p r e s e n t in the 5' UTR in stem- loop IIIb. In the p r e s e n c e of a m o d e l oligonucleotide, the H C V H a l f z y m e was activated to cleave a labeled target indicating detection a n d signaling in a single step. In the absence of oligonncleotide target no r i b o z y m e activity was detected. Q u a n t i t a t i o n of these data indicate that this system has a signal to noise ratio o v e r 2 X 106, several orders of m a g n i t u d e greater t h a n that displayed by RT-PCR m e t h o d s . T h e native H C V 5' U T R is also able to p r o m o t e halfzyme activation if first d i s r u p t e d b y p r e - t r e a t m e n t w i t h RNase H a n d a specific D N A oligonucleotide. Titration e x p e r i m e n t s d e m o n s t r a t e that H a l f z y m e activity faithfully reports target a b u n d a n c e in a quantitative fashion. A l t h o u g h n o t fully characterized, w e estimate that the limit of detection for H a l f z y m e t e c h n o l o g y to be c o m p a r a b l e to amplification based m e t h o d s s u c h as PCR. T h u s these initial proof-of-principle e x p e r i m e n t s d e m o n s t r a t e the feasibility of a r i b o z y m e - b a s e d a p p r o a c h to the detection of H C V RNA a n d low copy n u m b e r nucleic acids.

BACKGROUNS:Type 1 interferon (IFN) receptor consists of two chains (IFNAR i and 2), and IFNAR-2 takes a soluble, short, or long form (IFNAR 2a, 2b, 2c, respectively). Although it became possible to measure 1FNAR-2ain the set-am, the clinical significance of IFNAR-2a has not been well known in patients chronicafiy infected with hepatitis C virus (HCV). Recently, proinflammatory eytokines and viral infection were reported to lip-regulate IFNAR-2 expression (Int J Mol Med, 2000;6:621-7) or type 2 IFN receptor (J lmmuno11992;149: t 67I-5). The goal of the present study was to clarify relation between serum IFNAR-2a and other liver tests including serum cytokine levels in patients chronically infected with HCV (CHC). PATIENTSAND METHODS: The study consisted of 82 patients with CHC. The patients consisted of 53 men and 29 women ranging frmn 21 to 80 years old (mean age, 55 +/- 13 years). All patients were positive for HCV (anti-HCV) antibody and for HCV-RNA in the serum. Anti-HCV antibody was assayed by a third-generation enzyme-linked imnmnosolbent assay (ELISA).The amount of serum HCV-RNA was quantified using the Amplicor Monitor Version 2 test (Nippon Roche Inc., Tokyo, japan). According to serological genotyping assay as reported by Tanaka et al. (Hepatology I994; 19: 1347-1353), serum types of HCV were d vided into 3 serotypes; typel (genotype ia, lb) was detected in 50 patients, and type 2 (genotype 2a, 2b) was in 20 patients, unclassified was in 12 patients. Patients who were known to be homosexual or to have intravenous drug addictions, these positive for hepatitis B surface antigens (HBsAg) or antinuelear antibodies, and those with metabolic liver dystunction were excIuded. The object was divided into 4 groups; 14 patients with continuously normaI ALT in the serum (can" er), 49 pat ents with chronic hepatitis (CH), 9 patients with liver cirrhosis (LC), and 10 patients with hepatocellular carcinoma (HCC). On each occasion, alanine amtnotransferase (ALT) in the serum, and platelets in peripheral blood were measured according to standard procedures. Serum levels of IFNAR-2a were measured using an IFNAR-2a assay k t (Otuka Co., Ltd., Tokyo, Japan). Serum levels of interleukin (IL)-I beta (IL-1 beta), 1L-6, IL-10, IL-18, and TNF-alpha were measured using each ELISAkit. RESULTS:Serum levels of IFNAR-2a in 82 patients with CHC widely varied from 1.4 to 9.21 ng/mL (mean +/- SD, 3.4 +/- t.5 ng/mL). Serum IFNAR-2a levels were significandy (p<0.05, by Seheffe's test) more increased in patients with LC (4.7 +/- 2.1 ng/mk) and HCC (4.7 +/- 2.0 ng/mL) than in patients with CH (3.3 +/- 1.1 ng/mL) and calxier (2.5 +/- 0.9 ng/mL). There were insignificant differences in serum levels of IFNAR-2a between patients with carrier and those with CH, or patients with LC and those with HCC. There were insignificant differences in the serum levels of IFNAR-2a among patients infected with serotype 1, 2, and unclassified HCV. Serum levels of HCV-RNA did not correlate with any other parameters. Serum levels of IFNAR-2a significantly correlated with ALT (r=0.34, P<0.0I, by Spearman's correlation coefficient) with lL-6 (r=0.32. P<0.01) with TNFalpha (r=0.35, P<0.01), and with pIatelets in peripheral blood (r=-0.54, P<0.01). Serum ALT slightly co,xelated with TNF-alpha (r=0.25, P<0.05) and with platetets in peripheral blood (r=0.27 P<0.05). Serum levels of 1L-1beta significant y correlated with IL 6 (r=0.36, P<0.01) with 1L-18 (r=0.43, P<0.01), and with TNF-alpha (r-0.42, P<0.01). Serum levels of IL-6 significantly correlated with IL-18 (r--0.33, P<0.0t) and with TNP-alpha (r=0.48, P<0.01). CONCLUSIONS:It was revealed that serum IFNAR-2a levels increase if patients with CHC develop to LC or HCC. In addition, proinflammatory cytokines such as TNF-alpha and IL-6, which are both produced by monocytes, might be related with serum levels of IFNAR-2a, because correlation coefficients among IFNAR 2a, IL6, and TNF-alpha were relatively high.