INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Hypericin (4,5,7,4',5',7'-hexa-hydroxy 2,2' dimethyl-mesonaphthodianthron) is a natural compound found in the stems and petals of members of the genus Hypericum, including the common St. Johns wort plant Hypericum perforatum (32). It has shown in vitro activity against a variety of viruses, including murine Friend leukemia virus (22), Rauscher leukemia virus (30), equine infectious anemia virus (14), murine immunodeficiency virus (16), murine cytomegalovirus (P. L. Barnard, J. H. Huffmann, and S. G. Wood, Prog. Abstr. 30th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1093, 1990), influenza virus (30), vesiculostomatitis virus (18), Sendai virus (18), herpes viruses (30), and duck hepatitis B virus (23). Although hypericin has in vitro antiviral activity without light activation, this activity is enhanced by exposure to light (11, 16, 30). Furthermore, hypericin was effective against Friend leukemia virus and herpes simplex virus type 1 in mice (16, 30).

Recently, bovine diarrhea virus (BVDV) was found to be completely inactivated by hypericin in vitro in the presence of light (26). BVDV is a pestivirus that has structural similarities to the hepatitis C virus (HCV) (17, 24). Infection with HCV establishes a persistent infection in up to 90% of cases (1-3, 7). Four million Americans and 100 million people worldwide are chronically infected with HCV (4). Cirrhosis and hepatocellular carcinoma are long-term sequelae of this infection (27-29, 31). Although hypericin's activity against BVDV in vitro was not studied in the absence of light, it was decided to proceed with an exploratory clinical study of its activity against HCV. It was felt that there was no practical, consistent method of generating a stable, photo-activated form of hypericin for administration to patients.

We conducted a phase I dose escalation study to determine the safety and antiviral activity of hypericin in patients with chronic HCV infection.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Study population. We enrolled patients in the study if they met the following criteria: an age of 18 to 70 years, evidence of virologically active HCV infection as determined by a positive PCR HCV RNA assay within 60 days prior to study entry, a hemoglobin concentration greater than 11 g/dl, a total white blood cell count greater than 3,000/mm³, an absolute neutrophil count greater than 1,500/mm³ a platelet count greater than 180,000/mm³ cubic millimeter, a serum bilirubin concentration of less than 1.3 mg/dl, a serum alanine aminotransferase (ALT) concentration less than five times the upper limit of normal, a serum albumin concentration greater than 38 g per liter, a serum creatinine concentration less than 1.6 mg/dl, a prothrombin time less than 2 s above control, no more than 30 mg/dl urinary protein or 5 to 25 erythrocytes/µl urinary blood, and negative anti-DNA and anti-smooth muscle antibody assays.

Treatment regimens. The first 12 patients enrolled in the study were to receive an 8-week course of 0.05 mg of hypericin per kg of body weight in liquid form orally once a day (the study medication was kindly provided by VimRx Pharmaceuticals, Wilmington, Del.). The next 12 patients were to receive an 8-week course of 0.1 mg of hypericin per kg orally once a day. The drug was taken in the morning without regard to food. The subjects were instructed to return the empty drug vials to the research nurses after they were used. This was to monitor adherence to the drug regimen. Patients were also instructed to keep a medication log. In addition, dosing in the clinic at the time of pharmacokinetic blood sampling was observed by the research nurses.

Pharmacologic studies. Hypericin was reconstituted from a lyophilized 40-mg vial to a concentration of 1 mg/ml in 1.9% benzyl alcohol-4.5% dextrose and given orally at doses of 0.05 and 0.10 mg/kg. Blood was drawn into heparinized tubes at predetermined times as follows: week 0, 0 and 6 h; week 1, 0 and 6 h; week 2, 0 h; and week 4, 0 h. At week 8, detailed pharmacokinetic samples were taken at 0, 6, 9, 12, 24, and 48 h. The plasma was centrifuged at 1,000 × g for 10 min, aliquoted in cryovials, and stored at 20°C.

Criteria for response. The primary end point of the study was a change in plasma HCV RNA level of more than 1.0 log₁₀ from baseline to week 8.

Evaluation of patients and follow-up. After the screening and baseline evaluations, the patients were seen weekly for the first 2 weeks and then every 2 weeks until week 10 (2 weeks after the end of study treatment). At each of these visits, an interval clinical history was obtained, a physical examination was performed, and blood specimens were obtained for complete blood cell counts, serum chemistries, and hepatic and renal function. Two plasma samples for the measurement of HCV RNA were obtained at baseline; the first one was taken within 7 days before starting study treatment, and the second was taken on the day of starting treatment. Follow-up samples were obtained every 2 weeks until week 10. In addition, blood specimens were obtained for measuring hypericin levels at baseline.

Statistics. Response was defined as a reduction in HCV RNA levels of at least 1.0 log₁₀. The study was designed to have 80% power and a type I error rate of 0.05 to detect at least a 10% response rate (2 or more responders out of 10 subjects in each group) if the true response rate were 50%. To allow for a 20% dropout rate, 12 subjects were accrued to each dose arm of the study.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Study population. Between January 1997 and October 1997, 19 patients were enrolled; 12 received the 0.05-mg/kg daily dose of hypericin and seven received 0.10 mg/kg/day. The baseline characteristics of the study subjects are shown in Table 1.

Virologic data. The median plasma HCV RNA levels at baseline were 7.3 log₁₀ RNA molecules/ml for the 0.05-mg/kg dosage group and 7.6 log₁₀ RNA molecules/ml for the 0.1-mg/kg dosage group (Table 1). No subjects had a change of plasma HCV RNA levels of more than 1.0 log₁₀ (Table 2). With the 11 evaluable subjects in the 0.05-mg/kg dose cohort and 7 in the 0.10-mg/kg dose cohort, the study had 89 and 77% power, respectively, to detect a response rate of more than 10%. There were no significant changes from baseline to week 8 in plasma HCV RNA levels with either dose of study treatment (Table 2; Fig. 1). At the end of the 8-week study treatment period, the median plasma HCV RNA level was 7.27 log₁₀ RNA molecules/ml for the 0.05-mg/kg/day dosage group and 7.35 log₁₀ RNA molecules/ml for the 0.10-mg/kg/day dosage group.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Median plasma HCV RNA levels in patient cohorts treated with hypericin (either 0.05 or 0.10 mg/kg/day) for 56 days. Inf and Inf, positive and negative infinity, respectively; error bars, 95% confidence intervals.

Safety data. Seven of 12 subjects treated with the 0.05-mg/kg dosage and all 7 treated with the 0.1-mg/kg dosage had a photosensitivity reaction(s) while taking hypericin. There were four different types of photosensitivity reactions identifiedparesthesias, dermatitis, darkened coloration of exposed skin, and pruritic nodules (Table 3). All of the photosensitivity reactions were judged to be probably related to hypericin.

Other clinical data. Most subjects who entered the study had no symptoms of chronic illness. Five subjects complained of chronic fatigue at baseline: two in the 0.05-mg/kg dosage group and three in the 0.1-mg/kg dosage group. Four subjects, two in each dosage group, had intermittent right upper quadrant abdominal pain at baseline. The degree of fatigue improved during study treatment for only one of the five subjects who initially reported this symptom. In the other four subjects, the severity of the symptom remained approximately the same. In the four subjects with abdominal pain, no significant change occurred while on study treatment for three of them. Only one subject in the 0.1-mg/kg group reported a slight decrease in abdominal pain. All but two subjects had mild-to-moderate elevations of serum ALT levels at baseline (median, 70 U/liter; range, 39 to 158 U/liter). There were no significant changes in these values with either dose of hypericin given (Table 4).

Pharmacology. Blood specimens were analyzed initially in terms of the peak and trough levels. Averages of peak levels were derived from the day 14 and day 56 samples, while the trough averages were from the day 7, day 14, and day 28 samples (Table 5). A second set of trough values (minimum drug concentration if serum [C_min] at the end of the treatment cycle) which were derived from the measurements at day 56 and day 57 are also summarized. These data show good consistency over these different time points and do not indicate any effects of chronic dosing over 58 days. The mean values of the minimum drug concentration in serum (C_min) and the maximum drug concentration in serum (C_max) are dose proportional. Figure 2 shows a comparison of the mean ± standard deviation (SD) values from plasma decays obtained from those subjects receiving doses of 0.05 and 0.10 mg/kg who had detailed pharmacokinetic samplings at the end of the dosing cycle. These plots show dose proportionality, and the pharmacokinetic estimates from those subjects who had evaluable data are summarized in Table 6. The plasma hypericin decays were found to have a mean ±SD elimination half-life of 36.1 ± 22.6 and 33.8 ± 18.8 h, respectively, for the 0.01- and 0.05-mg/kg doses. The mean area under the curve (AUC) determinations for the two hypericin doses were, respectively, 1.5 and 3.1 µg/ml × hr, which again showed dose proportionality. The time to C_max, 4.4 ± 2.5 h, along with C_max values from the earlier study monitoring (Table 5) comparable with those at the end of the study (Table 6) showed enough overlap and confirmed that the monitoring for C_max at 6 h was adequate. The volume of distribution and clearance were greater at the 0.10-mg/kg dose. This possibly reflects a secondary tissue absorption that is more apparent at the higher concentration.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Summary of mean values ± SD (error bars) for hypericin decay plots from patients receiving hypericin at 0.05 (n = 7) and 0.1 (n = 4) mg/kg who agreed to undergo detailed pharmacokinetic sampling. The decays were fitted with one-compartment model with a first-order absorption (nonlinear fit) and showed dose proportionality for the AUC determinations (1.5 versus 3.1 µg/ml × h, respectively, for 0.05- and 0.1-mg/kg dose levels).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In the doses studied hypericin demonstrated no detectable anti-HCV activity. No subjects treated with an 8-week course of hypericin (either 0.05 or 0.10 mg/kg/day) had a change in plasma level of HCV RNA of more than 1.0 log₁₀, the natural variability of the laboratory assay. Twelve subjects receiving the 0.05-mg/kg/day dose and seven receiving the 0.10-mg/kg/day dose were studied. These results provide significant evidence that hypericin in these doses is not effective in lowering plasma HCV levels in HCV-infected patients. In addition, hypericin had no effect on improving elevated serum liver enzyme levels in the patients studied.

Although immune responses to the infecting organism are likely to play a role in disease pathogenesis (13), elimination of detectable virus from the blood is closely associated with the successful treatment of chronic HCV infection (15, 20). In all clinical trials involving interferon preparations, long-term histologic and clinical outcome in a patient was determined by the sustained (>6 months after treatment ended) suppression of detectable HCV activity in plasma (15, 20). Thus, the lack of an effect of hypericin on plasma HCV levels strongly suggests that hypericin as a single agent has no role in the treatment of chronic hepatitis C.

Five of 12 subjects receiving the 0.05-mg/kg/day dosing schedule and almost all (6 of 7) subjects receiving the 0.10-mg/kg/day dosing schedule developed uncomfortable phototoxicity. This necessitated treatment discontinuation in only two subjects in the 0.05-mg/kg/day treatment group and none in the 0.10-mg/kg/day treatment group. Nevertheless, it is likely that the phototoxic reactions would be even more severe at higher doses. Without even a hint of activity at the doses studied, it would be difficult to justify evaluating higher doses of hypericin for hepatitis C. Similar phototoxicity was reported recently in a study of hypericin given intravenously in doses of 0.25 and 0.50 mg/kg given two or three times weekly for HIV infection (8). No antiviral activity was detected in that study either (8). No other serious adverse events associated with hypericin use occurred during our study.

Hypericin is being evaluated for other antiviral and antineoplastic activities (6, 8-10, 33). The plant from which it is derived, St. Johns wort, has wide use for the treatment of depression (5). Thus, the safety and pharmacokinetic data reported here have usefulness for the application of hypericin to these indications.

The pharmacokinetic data from the two dose levels of hypericin examined in this study were consistent with respect to dose proportionality in the steady-state levels observed as well as for the pharmacokinetic parameter estimates yielding the AUC calculations. These data are also comparable in terms of the long elimination half-life (mean values of 36.1 and 33.8 h, respectively, for doses of 0.05 and 0.10 mg/kg/day) with the data derived from the phase I studies of hypericin in HIV-infected adults, in which an elimination half-life of 35.3 ± 9 h was observed (McAuliff et al., 1st Natl. Conf. Hum. Retrovir. Relat. Infect). A study with plant-derived hypericin preparations administered to healthy volunteers at hypericin levels 5- to 10-fold lower than those used in the present study also showed an elimination rate of 43 h (12). The AUC calculation from the 0.05-mg/kg dosing group of 3.1 ± 1.3 µg/ml × h is in good agreement with the 22% bioavailability that was determined from the phase I HIV-infected-adult study, in which a theoretical extrapolation from the intravenous AUC determined from the 0.5-mg/kg dose level of 53.7 µg/ml × h corrected for the ~20% bioavailability gives an expected AUC for 0.05-mg/kg dosing of 2.39 µg/ml × hr. There was no discernible drug accumulation from the daily dosing, as evidenced over time.

If one uses a multidosing simulation of the single-dosing pharmacokinetic parameter estimates (McAuliffe et al., 1st Natl. Conf. Hum. Retrovir. Relat. Infect.), steady-state levels should be reached by 10 days with a twofold peak/trough ratio. This was observed in our study, in which there was good consistency for the day 14, 28, and 56 trough levels as well as for the peak level measurements obtained at study days 14 and 56 (data not shown). While the daily dosing employed in this study appears to take close to 2 weeks to achieve steady state, it still appears to be preferable not to dose more frequently given the considerable toxicity with daily dosing at the 0.5-mg/kg level in this study as well as in the phase I HIV study (8).

It is worthwhile to comment on the lack of any detectable hypericin metabolites, even in any of the detailed pharmacokinetic samples taken on day 42 of the study. Our findings extend the observations of Kerb and coworkers (12), who found little difference between the multidose and the single dose AUC from 0 h to infinity. They also found no change in the plot of trough levels over their 14 days of continuous dosing. It is possible that the recent suggested perturbations imparted by St. Johns wort on the pharmacokinetics of cyclosporin and protease inhibitors (25) could be related to the use of additional plant-derived components and the concentrations used. A recent report by Markowitz and coworkers (21) found little perturbation of cytochrome P-450 and 3A4 activity in healthy volunteers taking St. Johns wort preparations (H. perforatum) at recommended doses for depression.

In sum, hypericin given orally in tolerable doses had no detectable anti-HCV activity in patients with chronic HCV infection. It has considerable phototoxicity when administered in doses of 0.05 mg/kg/day or more. Nevertheless, hypericin is being evaluated for other antimicrobial, antineoplastic, and antidepressant indications, and the pharmacokinetic and safety data reported here can be used to guide further study of this compound.