A phase I trial of an antisense inhibitor of hepatitis C virus (ISIS 14803), administered to chronic hepatitis C patients

Journal of Hepatology 44 (2006) 88–96 www.elsevier.com/locate/jhep

A phase I trial of an antisense inhibitor of hepatitis C virus (ISIS 14803), administered to chronic hepatitis C patients* John G. McHutchison1,2,*, Keyur Patel1,2, Paul Pockros2, Lisa Nyberg2, Stephen Pianko2, Rosie Z. Yu3, F. Andrew Dorr3, T. Jesse Kwoh3 1

The Duke Clinical Research Institute and Division of Gastroenterology, Duke University Medical Center, P.O. Box 17969, Durham, NC 27715, USA 2 Scripps Clinic, Division of Gastroenterology, La Jolla, CA, USA 3 Isis Pharmaceuticals, Carlsbad, CA, USA

Background/Aims: ISIS 14803 is a 20-unit antisense phosphorothioate oligodeoxynucleotide that binds to hepatitis C virus (HCV) RNA at the translation initiation region of the internal ribosome entry site (IRES) and inhibits protein expression in cell culture and mouse models. This Phase I, open-label, dose-escalation trial of ISIS 14803 was performed in chronic HCV patients. Methods: At least 7 days after receiving an initial single dose, twenty-eight patients received 0.5–3 mg/kg ISIS 14803 thrice weekly for 4 weeks by intravenous infusion or subcutaneous injection. Results: In most patients, the 4-week treatment did not reduce plasma HCV RNA. However, 3 patients receiving R2 mg/kg had transient HCV reductions of 1.2–1.7 log10 that persisted %32 days. These reductions were accompanied by asymptomatic, self-resolving elevations in serum alanine transaminase (ALT) levels to O10! the upper limit of normal. Two other patients had ALT flares without plasma HCV reduction. No clinical signs, symptoms of hepatic dysfunction, or laboratory changes in albumin or prothrombin time accompanied ALT elevations. Conclusions: ISIS 14803 treatment was associated with HCV reductions in only 3/28 patients. ALT flares in 5 patients also occurred. Further studies to evaluate ISIS 14803 treatment and the mechanisms of the ALT flares are now required. q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Hepatitis C virus; Treatment; Antisense therapy; New therapies; Nonresponders.

1. Introduction Approximately 170 million people worldwide are infected with hepatitis C virus (HCV), and infection persists in approximately 75–85% of cases, making hepatitis C infection an important cause of chronic liver disease Lauer Received 11 April 2005; received in revised form 6 September 2005; accepted 12 September 2005; available online 25 October 2005 * The authors who have taken part in this study have declared a relationship with the manufacturers of the drugs involved and they received funding from the drug companies involved to carry out their research. * Corresponding author. Duke Clinical Research Institute, P.O. Box 17969, Durham, NC 27715, USA. Tel.: C1 919-668-7193; fax: C1 919 684 716. E-mail address: [email protected] (J.G. McHutchison).

and Walker, (2001); NIH Consensus Development Conference Statement, (2002); Consensus, (2002); Alter et al., (1999); Lavanchy and McMahon, (2000). Liver cirrhosis and end-stage liver disease due to HCV is now the most common indication for liver transplantation in the United States. Moreover, the disease burden due to chronic infection is expected to increase over the next decade (Kim, (2002)). Treatment with peginterferon and ribavirin provides effective therapy in 50–60% of HCV patients Manns et al., (2001); Fried et al., (2002). However, this therapy is costly, prolonged, not suitable for many patients, and has significant side effects (NIH Consensus Development Conference Statement, (2002)). Thus, numerous efforts to develop new therapies are in progress (Pawlotsky et al., 2004).

0168-8278/$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2005.09.009

J.G. McHutchison et al. / Journal of Hepatology 44 (2006) 88–96

HCV genomic RNA is an attractive drug target because it is both the informational precursor for viral protein synthesis and an essential component of infectious virus (Penin et al., (2004)). Antisense therapeutics employ nucleic acids, usually oligonucleotides !25 nucleotides long, to disrupt protein translation through Watson–Crick basepairing of the antisense agent to the target RNA (Crooke, (1999); Crooke, (2001); Gerwitz et al., (1998); Scherer and Rossi, (2003)). Phosphorothioate oligodeoxynucleotides promote target RNA degradation via cellular RNase H enzymes (Crooke, 1999; Crooke, 2001; Gerwitz et al., 1998; Scherer and Rossi, 2003). Generally, the mechanisms that degrade RNA are the most potent (Crooke, 1999; Crooke, 2001; Scherer and Rossi, 2003). Several groups have identified antisense oligonucleotides that inhibit HCV RNA and polyprotein synthesis in in vitro, cell culture, and mouse models (Mizutani et al., 1995; Seki and Honda, 1995; Vidalin et al., 1996; Wakita and Wands, 1994; Alt et al., 1995; Lima et al., 1997; Brown-Driver et al., 1999; Hanecak et al., 1996; Zhang et al., 1999). One of these oligonucleotides, ISIS 14803, is a phosphorothioate oligodeoxynucleotide that is complementary to the HCV translation initiation region within the internal ribosome entry site (IRES) (Hanecak et al., 1996; Zhang et al., 1999). ISIS 14803 can reduce HCV RNA in immortalized human hepatocytes expressing a portion of the HCV genome (Hanecak et al., 1996). In addition to having the phosphorothioate modification, ISIS 14803 also contains 5-methyl-cytosine, instead of cytosine, which reduces the pro-inflammatory nature of this type of oligonucleotide (Zhang et al., 1999; Krieg, 2002; Levin et al., 2001). Here, we describe the first clinical study of this HCV-specific antisense approach. The study was performed in patients with chronic hepatitis C infection who had previously not responded to therapy or were unsuitable candidates for standard therapies.

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unwilling to take, or had a contraindication to conventional interferonbased therapy. Nonresponse to therapy was defined as retention of detectable serum or plasma HCV RNA after at least 3 months of interferon-based therapy. Patients with decompensated disease, hepatitis B virus (HBV) or HIV coinfection, or chronic liver disease from other causes were excluded from this study. Other exclusion criteria included comorbid illnesses including clinically significant cardiovascular, neurologic, autoimmune, or thyroid diseases. All patients had a pretreatment liver biopsy indicating chronic hepatitis, platelet count R1,00,000 cell/mm3, hemoglobin O11 g/dL, and prothrombin time, white blood cell count, bilirubin and creatinine within the normal range. The study was approved by the Scripps Clinic Institutional Review Board and performed in conformity with the Declaration of Helsinki. All patients provided written informed consent prior to enrollment.

2.3. Study evaluations Subjects were evaluated frequently for safety and adverse events. Safety endpoints included clinical symptoms, physical examination findings, and laboratory parameters. Serum chemistries, complete blood cell count with differential, urinalysis, and prothrombin and activated partial thromboplastin (aPTT) times were performed at baseline and weekly during therapy and the initial 3 weeks of the 8-week follow-up. In addition, complement split products (C3a, C5a, and Bb) were measured weekly before, during, and after administration of ISIS 14803. Plasma interferon-g (IFN-g), tumor necrosis factor-a (TNF-a), and interleukin-4 (IL-4) were measured before and after each patient’s first dose. Plasma HCV RNA was measured at a single central laboratory (National Genetics Institute, Los Angeles, CA) by quantitative polymerase chain reaction, with a detection limit of 100 copies/mL (SuperQuante, 2.5 copies/IU) (Tong et al., 1997; Pawlotsky et al., 2000). HCV genotyping was performed with Inno-LiPA HCV II (Innogenetics, Zwijnaarde, Belgium) as described previously (Stuyver et al., 1993). The plasma pharmacokinetics of ISIS 14803 was evaluated for both intravenous and subcutaneous administration. ISIS 14803 levels in plasma collected before, during, and following the single dose and the last dose of the treatment period were measured by capillary gel electrophoresis as previously described (Leeds et al., 1996). ISIS 14803 levels were similarly measured in urine collected for 24 h after these doses.

2.4. Statistical analysis This Phase I study was designed to evaluate the safety and tolerability of 4 ISIS 14803 doses given by two routes of administration. The study was not powered to detect significant changes in antiviral response rates between cohorts. Statistical comparisons between cohorts were not performed. Descriptive statistics (mean, SD, median) were employed to summarize selected findings.

2. Methods 2.1. Study design In this open-label, dose-escalation study, sequential cohorts of 3 patients were to be treated with 0.5, 1, 2, or 3 mg/kg of ISIS 14803. Patients were assigned to the cohorts open at the time of their enrollment. Initiation of each cohort was contingent upon completion of ISIS 14803 treatment in previous cohorts with an acceptable safety profile. Initially, doses were given by 2-h intravenous infusion. Each patient received an initial dose followed by a 7-day washout. Patients then were given thrice-weekly doses for 4 weeks and followed for 8 weeks post-treatment. After treatment was completed for the initial intravenous cohorts, separate sequential cohorts received treatment by subcutaneous injection.

2.2. Patient population Twenty-eight patients were enrolled and treated in this single-center study. All patients had chronic HCV infection as documented by the presence of detectable serum HCV antibodies and quantifiable plasma HCV RNA. Patients were enrolled if they had previously not responded to, were

3. Results 3.1. Study patients Twenty-eight chronic hepatitis C patients were enrolled. Twenty received ISIS 14803 intravenously, and 8 subcutaneously. The first patient was screened on March 13, 2000, and the last follow-up visit was July 17, 2002. Patients were middle-aged with elevated alanine transaminase (ALT) values, and most were male and had high plasma HCV RNA levels (Table 1). Twenty-four of the twenty-eight patients were virologic nonresponders to prior interferon-based therapy. Sequential 3-patient cohorts were treated intravenously at 0.5, 1, and 2 mg/kg ISIS 14803. Because of the unexpected ALT elevations in Patients 108 (598 U/L) and 109 (411 U/L), treated at 2 mg/kg, 2 subsequent patients

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Table 1 Patient characteristics at baseline Characteristic

Number

Patients treated Age (years) (median, range) Male/Female ALT, IU/L (meanGSD) HCV RNA (copies/mL) (median, range) HCV RNAO1!106 copies/mL HCV RNAO10!106 copies/mL Genotype 1 Cirrhosis Bridging fibrosis Portal fibrosis Prior therapy

28 47 (33–60) 18/10 69G41 7.7!106 (0.3–37!106) 26 10 24 2 3 18 24

reduction in 45 days. HCV was below pretreatment level for approximately 74 days and was O10-fold reduced for about 32 days. Patient 111’s HCV RNA reduction occurred after his seventh, and last, dose at 1 mg/kg. The nadir, a 1.2 log10 reduction, was reached 44 days after repeat dosing began. His HCV was below pretreatment for about 43 days and was O10fold reduced for approximately 15 days. Patient 208 had a pretreatment HCV RNA level of 4.3 million copies/mL, which initially increased to 39 million during treatment. The patient’s HCV RNA began to decline between 19 and 39 days after receiving the last dose and reached a nadir of 80,000 copies/mL, 1.7 log10 below pretreatment, at 81 days after starting repeat dosing. HCV was less than pretreatment levels O82 days.

ALT, alanine transaminase; HCV, hepatitis C virus.

3.3. ALT elevations (110 and 111) received 2 mg/kg for their single dose and then were treated at 1 mg/kg for their repeat dosing. Further, enrollment into the study was suspended when Patient 111 developed elevated ALT levels (830 U/L). A follow-up lasting several months indicated no clinical symptoms or sequelae amongst the 3 patients. Enrollment was resumed at the lower dose levels only after additional stopping rules were introduced and regulatory bodies were informed of the safety data. Sequential cohorts of 3 patients were then enrolled and treated intravenously at 1, 2, and 3 mg/kg. Patients received 3 mg/kg only after prior cohorts tolerated 1- and 2-mg/kg therapy without experiencing any further evidence of ALT flares. In parallel, sequential cohorts were treated subcutaneously with 0.5, 1, and 2 mg/kg. Enrollment was closed when dosing at the highest intravenous dose was completed. Additional subcutaneous dosing was not pursued to avoid subjecting patients to multiple injections at each dosing. 3.2. Plasma HCV RNA reductions Three of the 10 patients enrolled for treatment at 2 mg/kg (Patients 108, 111, and 208) had reductions in plasma HCV RNA of more than 1.0 log10 (Table 2, Fig. 1). None of the other patients enrolled for treatment at 0.5 (nZ6), 1 (nZ9), and 3 mg/kg (nZ3) had similar reductions. Patients 108 and 208 received all of their doses at 2 mg/kg, whereas Patient 111 only received 1 dose at that level, and his subsequent 7 doses were at 1 mg/kg. Treatment for Patients 108 and 111 were stopped early (after 11 and 8 doses, respectively) because their serum ALT was elevated O10!ULN. Patients 108 and 111 received ISIS 14803 intravenously, and Patient 208 by subcutaneous injection. These 3 patients had ALT elevations in association with reductions in HCV RNA (Fig. 1). The changes in viral concentrations varied between these 3 patients. Patient 108 had a pretreatment plasma HCV RNA level (average of Screen and Baseline values) of 12.4 million copies/mL. After initiation of thrice weekly dosing, HCV RNA levels progressively declined and reached a 1.4 log10

All 3 patients with HCV RNA reductions O1.0 log10 had elevated serum ALT. In addition, two other patients (Patient 105 treated at 1 mg/kg and Patient 109 treated at 2 mg/kg) also had ALT elevations (Fig. 1). For these 5 patients, peak ALT elevations ranged from 411 to 830 U/L, 8.6–18.4! ULN (upper limit of normal) (Tables 2 and 3). As with HCV RNA reductions, the time courses for ALT elevations varied. Peak ALT elevation occurred 18–25 days after initiation of thrice weekly dosing for Patients 105, 108, and 111, and 60–67 days for Patients 109 and 208 (Table 3). During these transient ALT elevations, the number of days during which ALT was R4-fold pretreatment levels ranged from 25 days to 84 days (Table 3). In 4 patients, their ALT elevations resolved to near baseline levels in 26–82 days following peak levels (Fig. 1). In contrast, Patient 109 had a second, lesser, ALT elevation, which peaked at 399 U/L, 8.3!ULN, 88 days after his first peak ALT elevation and 123 days after the last dose of ISIS 14803. Coincident with the resolution of this second peak, the patient’s plasma HCV RNA level declined 0.9 log10. Concurrent with ALT increases, elevations of aspartate transaminase (AST) (to 133–494 U/L, 2.8–11.9!ULN) and gamma-glutamyl transpeptidase (GGTP) (to 67–231 U/L, 2.3–7.9!ULN) occurred in all 5 patients. While alkaline phosphatase and bilirubin values remained within normal ranges, alkaline phosphatase increases (to 76–129 U/L, 1.6– 2.0 times pretreatment) were observed for Patients 108, 111, and 208, and a bilirubin increase to 1.6 mg/dL was observed for Patient 111. No significant changes were seen in other routine clinical laboratory tests including prothrombin time, albumin, and hematology and urinalysis parameters. Antinuclear and anti-smooth muscle antibody levels were also measured pre-study and during the ALT elevation in serum collected from Patients 105, 108, 109, and 111. No measurable antibody levels in these 4 patients suggested the induction of autoimmune liver injury (data not shown). Fatigue and headache were reported by 3 patients during their ALT flares, and nausea was by 2 patients. The association of these adverse events to the ALT flares is

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Table 2 HCV RNA and ALT changes observed during and after ISIS 14803 therapy Patient noa

Dose (mg/kg)

No doses

Plasma HCV RNA

ALT

!106 copies/mL

101 102 103 201 202 203 104 105 106 112 113 114 204 205 206 110k 111 107 108 109 115 116 117 207 208 118 119 120

0.5 0.5 0.5 0.5 0.5 0.5 1 1 1 1 1 1 1 1 1 2/1l 2/1l 2 2 2 2 2 2 2 2 3 3 3

13 13 13 13 13 12 13 13 13 13 13 13 13 13 13 8 8 13 11 13 13 13 13 12 11 13 11 13

Maximum observed Log10 reductionb

Week 0d

Week 4e

Week 7f

28.0 8.7 0.83 16.3 9.4 5.7 4.1 12.5 16.6 5.2 4.9 4.2 5.9 1.6 10.0 5.2 31.8 0.51 12.4 33.4 4.2 1.9 3.2 3.6 4.3 6.9 0.11 7.9

21.0 8.5 0.86 4.2 31.0 17.0 5.6 7.6 17.0 9.1 14.0 8.2 14.0 0.74 4.7 43.0m 10.0n 2.4 1.9o 25.0 5.1 2.7 9.8 7.4 22.0 3.6 0.10 24.0

31.0 5.4 0.66 3.5h 14.0 9.3 4.8 9.4 28.0 7.7 15.0 8.9 29.0 0.83 27.0 Na 1.9 1.2 0.46 29.0 2.8 3.8 4.0 3.5 34.0 3.4 0.06p 16.0

K0.4 K0.2 K0.1 K0.6 K0.1 K0.2 K0.1 K0.3 K0.2 0.0 K0.1 K0.1 K0.2 K0.3 K0.3 K0.1 K1.2 K0.1 K1.4 K0.6 K0.8 K0.6 K0.6 K0.2 K1.7 K0.7 K0.5 0.0

Peakc

U/L

Week 0g

Week 4e

Week 7f

!Wk 0

!ULN

123 114 65 113 32 107 91 70i 35 95 75 39 35i 42 154 88 74 29 51 50 65 30 36 160 43 47 68 34

80 129 52 145 19 86 93 397 77j 101 67 61 31 37 161 64m 830n 34j 598o 261 74 43 35 207 60 52 65 46

86 126 57 142h 18 69 126 102 67 120 69 67 28 38 158 na 424 32 189 346 58 46 49 185 144 56 48p 39

1.2 1.2 1.0 1.6 2.5 0.8 1.4 5.9 2.2 1.4 1.0 1.9 1.1 1.0 1.4 0.8 11.3 1.2 11.7 9.5 1.5 1.6 1.4 1.6 18.0 1.3 0.8 2.4

2.9 2.9 1.4 3.8 1.8 1.9 2.7 8.6 1.6 2.7 1.6 1.6 0.8 0.9 4.8 1.6 18.4 0.7 10.3 10.4 2.2 1.0 1.1 4.0 11.8 1.4 1.5 1.8

Days below are relative to the first day of thrice weekly dosing. ALT, alanine transaminase; HCV, hepatitis C virus: ULN, upper limit of normal. a Patients 101–120 were treated intravenously, and 201–208 were subcutaneously. The patients are grouped by dose level; the proportion of ISIS 14803 distributed to liver is equivalent between the two routes. b Maximal log10 reduction of HCV RNA observed at any time during the entire treatment period. c The peak ALT value observed for each patient is shown in these two columns as the ratio to the pretreatment or baseline values at week 0, and also as the ratio of this peak value to the upper limit of normal (ULN) for the ALT assay used in this study. d Geometric mean of Screen and Baseline values. e Day 25: day of last scheduled dose. f Days 42–44. g Arithmetic mean of Screen and Baseline values. h Day 35 (Day 42 not done). i Screen only (Baseline sample was hemolyzed). j Day 28 (Day 25 was hemolyzed). k Patient 110 stopped early because of serious adverse events (SAE): psychosis and substance abuse. l Patients 110 and 111 received single doses at 2 mg/kg and thrice-weekly doses at 1 mg/kg. m Day 21 (early termination visit) instead of Day 25 (last dose on Day 14). n Day 22 (unscheduled visit) instead of Day 25 because of early dosing termination (ALTO10!ULN, last dose on Day 14). o Day 28 (unscheduled visit) instead of Day 25 because of early dosing termination (ALTO10!ULN, last dose on Day 21). p Day 45.

unclear as their frequency and intensity were similar to those observed in patients without ALT elevations, who included patients treated at lower doses (e.g. 6 of 6 patients treated with 0.5 mg/kg reported fatigue). Other less frequent and minor adverse events concurrent with ALT flares were reported for individual patients.

Twenty-two days after a peak ALT elevation of 830 U/L, a liver biopsy was performed on Patient 111 when his ALT was 424 U/L, and plasma HCV RNA was at its nadir. Histology revealed an intense inflammatory cell infiltrate within the portal triads that was not present in the specimen collected 7 months prior to study entry. These biopsies were

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10

8

10

600

8

800

Pt 108

Pt 208 500

HCV

400

10

6

200 ALT

600 500 HCV

10 6

400 300

10

5

ALT

100

0

40

80

10 4

0 160

120

0

40

Days from Baseline

80

0 160

120

Days from Baseline

10 8

10

1000

8

500

Pt 111

Pt 105 400

ALT

HCV (copies/ml)

400

10

7

300

HCV

200

10 6 ALT

200

100 Doses

Doses

10 6 40

80

0 160

120

10

5

-20

0

20

Days from Baseline

10

ALT (U/L)

600 10 7

ALT (U/L)

HCV (copies/ml)

800 HCV

0

200 100

Doses

10 5

700

ALT (U/L)

300

HCV (copies/ml)

7

ALT (U/L)

HCV (copies/ml)

10 10

7

40

60

80

0 100

Days from Baseline

8

500

Pt 109

HCV

300 10

7

200

ALT (U/L)

HCV (copies/ml)

400

100

ALT Doses

10 6

0 0

40

80

120

160

200

240

280

Days from Baseline

Fig. 1. Plasma hepatitis C virus (HCV) RNA and alanine transaminase (ALT) levels during and after treatment with ISIS 14803. Patients were given their single dose of ISIS 14803 on Day 0. After a washout R7 days, patients were treated thrice weekly for up to 4 weeks. Patient 105 was treated at 1 mg/kg. Patients 108, 109, and 208 were treated at 2 mg/kg. Patient 111 received a single dose at 2 mg/kg and subsequent doses at 1 mg/kg. Patient 208 received the drug subcutaneously; all others did intravenously. Symbols: closed circles, plasma HCV RNA; open triangles, ALT; closed diamonds (on x-axis), administered doses.

reviewed by two separate experts who independently concluded that the histological findings were not suggestive of drug-induced hepatotoxicity. A liver biopsy was also performed on Patient 109 at 222 days after his first ALT peak and 134 days after his second. Besides moderate periportal hepatitis, there was no evidence of hepatotoxicity. 3.4. Safety Apart from the ALT flares, treatment with ISIS 14803 for 4 weeks was generally well-tolerated. Excluding

subcutaneous injection site reactions, the most frequent adverse events were headache (nZ25), fatigue (nZ15), pain (nZ9), abdominal pain (nZ9), arthralgia (nZ6), insomnia (nZ6), nausea (nZ6), dizziness (nZ5), myalgia (nZ5), dyspepsia (nZ5), diarrhea (nZ4), and constipation (nZ4). All were reported as mild to moderate in severity. All patients treated subcutaneously reported erythema and induration at some injection sites. Seven of 8 patients also reported pain at some subcutaneous injection sites. The local injection site reactions were judged by investigators and staff to be mild and did not require dose modifications or discontinuations.

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Table 3 Characteristics of ALT elevations Patienta

105 108 109 111 208 109e

Peak ALT (U/L)

411 598 469 830 765 399

Peak ALT (! Pre-Tx)

5.9 11.7 9.5 11.3 18.0 8.1

Day of eventb

Days elevated

ALTO4!PreTxc

Peak ALT

ALT!4!PreTxd

Last ISIS 14803 dose

11 18 18 11 56 120

18 25 60 22 67 148

36 44 78 95 136 232

25 21 25 14 25 25

25 26 60 84 80 112

a With the exception of Patient 108, the patients with ALT (alanine transaminase) flares were all prior nonresponders to interferon-based therapy. All were infected with HCV genotype 1 except Patient 208, who was infected with genotype 3a. There were no identifiable virologic or host characteristics that separated these patients as being different or explained the ALT flares. b Days from start of thrice weekly dosing. For Patients 105, 109, 111, and 208, thrice weekly dosing began 12 days after the single dose. Thrice weekly dosing for Patient 108 began 19 days after the single dose. c Day on which ALT greater than 4! pretreatment was first observed. Tx, treatment. d First day on which ALT declined below 4! pretreatment. e Second ALT flare.

No abnormalities in serum chemistry, hematology, and urinalysis were observed. As with other phosphorothioate oligodeoxynucleotides, transient aPTT prolongations were observed at the end of intravenous infusion (approximately 1.6-fold at 3 mg/kg) but not after subcutaneous injection (data not shown) (Levin et al., 2001; Dorr et al., 2001). No bleeding, bruising, or hemorrhage was associated with aPTT changes in this study or in studies of other oligonucleotides. Elevations in complement split products (C3a, C5a, and Bb) and specific cytokines (TNF-a, IFN-g, and IL-4) were not observed following intravenous or subcutaneous dosing (data not shown). 3.5. Pharmacokinetics The plasma pharmacokinetics of ISIS 14803 were very similar to those of other phosphorothioate oligodeoxynucleotides (Table 4) (Geary et al., 2001; Yu et al., 2001). For intravenous administration, maximum concentration (Cmax) occurred at or near the end of infusion, and drug levels declined rapidly thereafter, with distribution half-lives (t1/2) of 40–70 min. Both Cmax and area under the concentration– time curve (AUC) increased proportionally with dose and appeared to follow linear first-order pharmacokinetic principles. Cmax following subcutaneous administration was 4–13 times lower than that attained after intravenous infusion, but AUC was 45–111% of that for intravenous dosing. Animal studies have shown oligonucleotide distribution to liver is equivalent by the two routes of administration (Geary et al., 2001). As with intravenous administration, Cmax and AUC following subcutaneous injection increased proportionally with dose. Neither route led to differences in plasma pharmacokinetic parameters between Day 0 and Day 32, indicating no measurable plasma accumulation following alternate-day administration. Urinary excretion of ISIS 14803 during the 24 h

after dosing by either route was !3% of the administered dose (data not shown). Animals studies have shown oligonucleotide clearance from plasma represents distribution to tissue and not excretion (Geary et al., 2001).

4. Discussion This first clinical evaluation of an antisense inhibitor for HCV was primarily designed to evaluate the safety profile of ISIS 14803. The study design and dose selection were based on the high similarity of ISIS 14803 to other phosphorothioate oligonucleotides in preclinical toxicology and pharmacokinetic studies (Levin et al., 2001; Dorr et al., 2001). With the exception of serum transaminase elevations, primarily in ALT, the safety profile of ISIS 14803 was similar to other phosphorothioate oligodeoxynucleotides. The most frequent side effects were non-specific and included headache, fatigue, arthralgia, insomnia, and some gastrointestinal side effects. Aside from the transient aPTT prolongations following intravenous infusion, which are typical of this drug class, clinical laboratory abnormalities were observed only in the 5 ALT-flare patients. All patients receiving the drug subcutaneously experienced at least 1 local injection site reaction. These reactions were mainly mild or moderate erythema and induration at the injection site and did not affect dosing. In the 5 patients with ALT flares, ALT increased to 8.6–18.4!ULN. These ALT flares were asymptomatic, self-resolving, and not associated with changes in albumin or prothrombin time or hematology and urinalysis parameters. Similar ALT elevations were not seen in ISIS 14803 toxicology studies in mice and monkeys (unpublished results). ALT flares have also not been observed in clinical studies of other oligonucleotides of

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Table 4 Plasma pharmacokinetic parameters (MeanGSD) Study day Intravenous AUCa (h!mg/mL) Cmaxb (mg/mL) Clearancec (mL/h/kg) Vssd (mL/kg) T1/2e (h) Subcutaneous AUCa (h!mg/mL) Cmaxb (mg/mL) CL/fc (mL/h/kg) Vz/fd (mL/kg) T1/2 e(h) %BAVh a b c d e f g h

ISIS 14803 dose (mg/kg) 0.5

1

2

3

0 32 0 32 0 32 0 32 0 32

6.75G2.04 7.02G1.38 3.89G1.36 3.58G0.66 79.1G25.1 72.9G12.9 129G36.8 133G33.4 0.88G0.16 0.72G0.08

14.6G3.04 10.9G3.68 7.21G1.91 5.39G1.53 71.0G14.8 101G34.2 149G31.9 203G67.8 1.02G0.24 0.93G0.16

20.5G5.48 14.6G5.42 8.43G1.88 8.19G4.13 104G29.2 154G57.1 222G38.1 279G86.7 1.01G0.20 0.88G0.08

29.8G4.24 27.0G7.18 16.8G5.52 13.1G2.36 102G14.3 116G27.8 215G24.0 248G64.3 1.17G0.07 1.12G0.11

0 32 0 32 0 32 0 32 0 32 0 32

4.07f 3.18g 0.29f 0.36G0.09 123f 181g 1711f 1074g 9.66f 4.39g nc 45.3

10.8G6.29 7.86G3.06 1.58G0.42 1.50G0.94 112G50.3 141G53.1 397G118 535G322 2.63G0.57 2.63G1.50 74.0 72.1

18.9g 16.2g 2.02g 2.06g 106g 124g 960g 886g 6.29g 4.92g 92.2 111

Area under the concentration-time curve. nc, not calculated. Maximum plasma concentration. Plasma clearance. Volume of distribution. Plasma concentration half-life. nZ1. nZ2. Percent bioavailability (subcutaneous AUC divided by intravenous AUC!100).

the same chemistry, and such studies encompass more than 2000 patients with a variety of inflammatory, virologic, and oncologic diseases (Gerwitz et al., 1998; Dorr et al., 2001; Geary et al., 2001; Yu et al., 2001; Bennett, 1999; Field, 1999). A single liver biopsy performed during an ALT flare in this study did not reveal histological evidence of hepatotoxicity. However, HCV-infected hepatocytes could have increased sensitivity to oligonucleotide. Another potential source for the ALT elevations is the nonspecific pro-inflammatory property of this chemical class. Phosphorothioate oligodeoxynucleotides have been shown, primarily in rodents, to stimulate expression of pro-inflammatory cytokines and proliferation of B-cells (Krieg, 2002; Levin et al., 2001). However, we did not observe increases in serum TNF-a, IL-4, or IFN-g following ISIS 14803 dosing. Induction of autoimmune liver disease was also examined as a possible cause for the flares. Yet neither anti-nuclear nor smooth muscle antibodies were detected, and the histology of the single biopsy was not consistent with autoimmune hepatitis.

In this study, we also found that 3 patients of 13 treated at the two highest dose levels had significant (O1.0 log10) reductions in viral load. These reductions were temporary and tended to return to baseline over a variable time period following therapy. These reductions are intriguing because HCV levels are relatively stable over 3-month periods in untreated patients (Nguyen et al., 1996; Yoshimura et al., 1997). Yet the origin of these HCV reductions is unclear. Based on sequence analysis of virus specimens collected in this study, the IRES-quasispecies diversity in Patient 108 evolved during and following treatment, a change that has not been observed in interferon-treated patients (Soler et al., 2004). Thus, at least in Patient 108, the HCV changes may have been due to interactions between ISIS 14803 and HCV RNA. However, the post-treatment HCV reduction in Patient 208, and the occurrence of ALT flares with all 3 HCV reductions, suggests that other mechanisms may be involved. While the ALT flares in this study differ in several aspects from the immune clearance-mediated flares in antiviral therapy of chronic HBV, immune

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clearance may have a role in the ALT flares and HCV reductions seen with ISIS 14803 (Perrillo and Mason, 1994; Hoofnagle et al., 1988; Liaw et al., 1999). Studies examining the potential role of peripheral and intrahepatic lymphocytes in HCV-specific and non-specific immunological responses during ALT flare would be instructive. In conclusion, ISIS 14803 administration in this initial, Phase I, 4-week, safety study was accompanied by concerning ALT flares in a subgroup of patients. Transient plasma HCV RNA reductions greater than fluctuations generally seen in untreated patients were observed in 3 of 28 ISIS 14803-treated patients (Nguyen et al., 1996; Yoshimura et al., 1997). Further investigation into the relationship between the HCV reductions and ALT flares in ISIS 14803treated patients, and into which effect is secondary to the other, is needed.

Acknowledgements This work was supported in part by research grants from Isis Pharmaceuticals, Inc., Carlsbad, CA, and the General Clinical Research Center Grant at Scripps Clinic (MO1-RR00833). Jennifer King, PhD, of August Editorial, Inc., helped prepare the manuscript for publication.

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