INTRODUCTION

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(+)-Calanolide A, (+)-[10R,11S,12S]-10,11-trans-dihydro- 12-hydroxy-6,6,10,11-tetramethyl-4-propyl-2H,6H-benzo[1,2-b: 3,4-b':5,6-b"]tripyran-2-one, is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) with potent activity against the human immunodeficiency virus type 1 (HIV-1) (7, 10). The compound was first isolated from a tropical tree (Calophyllum lanigerum) in Malaysia (10). Due to low availability of naturally occurring (+)-calanolide A, a total synthesis of this polycyclic coumarin was developed to provide material for preclinical and clinical research (6, 10).

Previous in vitro studies have demonstrated the protective activity of (+)-calanolide A to both established cell lines and primary human cells against a wide variety of HIV-1 isolates including syncytium-inducing (SI) and non-syncytium-inducing (NSI) viruses, T-tropic, and monocyte-macrophage tropic viruses (1, 2, 4, 7, 10). The activity (i.e., the 50% effective concentration) of the compound ranged from 0.02 to 0.5 µM. No activity was detected against HIV-2 or simian immunodeficiency virus. Direct cytotoxicity (i.e., the 50% infective concentration) of (+)-calanolide A was apparent at concentrations approximately 100 to 200 times greater than the anti-HIV-1-activity concentration in all cell lines tested (3, 5, 10). Kinetic analyses indicated that (+)-calanolide A inhibited HIV-1 reverse transcriptase (RT) by a complex mechanism involving two possible binding sites, a property that has not been observed for any other NNRTI (5, 10). Evidence suggests that one of the (+)-calanolide A binding sites is near both the pyrophosphate binding site and the active site of the RT enzyme (5, 10).

In vitro synergy has been demonstrated between (+)-calanolide A and a number of other antiretroviral agents, including NRTI's, NNRTI's, and protease inhibitors (8, 10; R. W. Buckheit, Jr., J. Russell, V. F. Boltz, L. A. Pallansch, Z.-Q. Xu, and M. T. Flavin, Abstr. Conf. Rec. 12th World AIDS Conf., abstr. 12366, p.86, 1998). (+)-Calanolide A remains fully active against virus isolates with zidovudine (AZT) and 3TC resistance-engendering mutations (1, 2, 8, 10). The compound has enhanced activity against virus isolates with the Y181C mutation, which confers resistance to other NNRTIs and against viruses that have both AZT resistance and the Y181C mutation. Even though (+)-calanolide A exhibits reduced activity against HIV-1 with the K103N mutation, it remains fully active against virus isolates that express both the K103N and the Y181C mutations. This resistance profile is a unique feature of the compound, since the Y181C and K103N mutations are two of the most commonly observed mutations in laboratory and clinical virus isolates from patients receiving other NNRTIs, including nevirapine, delavirdine, and efavirenz (9). In vitro, (+)-calanolide A predominantly selects for a unique drug-resistant virus having a mutation at amino acid residue 139 (T139I). This virus remains susceptible to all other anti-HIV agents tested, including other NNRTIs (3).

The toxicity of (+)-calanolide A in a number of animal species, including mice, rats, and dogs, has been studied (P. Frank, M. T. Flavin, J. Roca-Acin, and Z.-Q. Xu, Abstr. 4th Conf. Retrovir. Opportunistic Infect. [CROI], abstr. 225, p. 106, 1997). (+)-Calanolide A was well tolerated at oral doses of up to 150 mg/kg in rats and 100 mg/kg in dogs. Toxicities associated with the oral administration of (+)-calanolide A for up to 28 days in animals were gastric irritation and subsequent gastric hyperplasia and edema in the rat and salivation in the dog. Emesis in the dog was the dose-limiting side effect, but a 50% lethal dose could not be attained. In vitro and in vivo assays for mutagenicity have been negative, and (+)-calanolide A does not produce teratologic effects when administered to rats during gestation (Frank et al., 4th CROI).

In vitro studies indicate that metabolism is qualitatively similar in rats, dogs, monkeys, and humans, with four to seven main metabolites produced (S. D. Patil, A. K. Thilagar, P. Frank, and Z.-Q. Xu, PharmSci Suppl. 1:S-41, abstr. 1125, 1998). CYP3A4 is the primary isoform of P450 that metabolizes (+)-calanolide A, although CYP2C may be involved as a minor isoform (S. D. Patil, A. K. Thilagar, P. Frank, and Z.-Q. Xu, PharmSci Suppl. 1:S-41, abstr. 1126, 1998). Animal studies have shown that compound-related radioactivity distributes into both the brain and the lymph after oral administration, while after intravenous administration the radioactivity accumulates in the brain (Frank et al., 4th CROI). These studies indicate that (+)-calanolide A crosses the blood-brain barrier and may be preferentially distributed in the lymphatic system. (Frank et al., 4th CROI). Indeed, in rat studies, the oral administration of radiolabeled (+)-calanolide A results in a mean ratio of lymph to serum radioactivity of 2.8:1 after 6 h. (+)-Calanolide A binds extensively (>97%) to human and animal plasma proteins and to human ₁-acid glycoprotein (Frank et al., 4th CROI).

The favorable safety profile and pharmacokinetics of (+)-calanolide A, coupled with its unique in vitro resistance pattern, has led to further clinical development. The first phase I study, described in this report, was conducted primarily to evaluate the safety and secondarily to evaluate the pharmacokinetics of single escalating doses of (+)-calanolide A in humans. In addition, the effect of dosing in a fasted or fed state was explored in the second and third cohorts in order to obtain information valuable in the design of future clinical trials. Because of a concern in regard to the rapid emergence of viral resistance with many monotherapy antiretroviral therapies in HIV-positive patients, this preliminary study was conducted in healthy, HIV-negative subjects.

	MATERIALS AND METHODS

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Study population. Enrollment in the study was limited to healthy, HIV-negative subjects of any race, 18 years of age or older, weighing within 20% of their ideal body weight (Society of Actuaries and Association of Life Insurance Medical Directors of America; also called the "Met-Life Tables"). Subjects were excluded from enrollment if they had a history of hemophilia, sickle cell disease, or other known blood dyscrasias or if they had intractable diarrhea or severe malabsorption. Subjects were also excluded if they had any chemical or hematological findings that were more than 10% outside the laboratory normal range (modified Adult ACTG toxicity grading scale) within 30 days prior to receiving the study dose of (+)-calanolide A. Treatment within 30 days of dosing, or required use during the study, of biological response modifiers, antimetabolites, systemic corticosteroids, antibiotics, or antivirals was prohibited. Subjects previously treated with dexfenfluramine, phentermine, or fenfluramine were excluded from participation. Negative hepatitis panel, serum pregnancy test, and HIV-1 enzyme-linked immunosorbent assay results were required prior to enrollment. Subjects were excluded if pregnant, lactating, or unwilling to employ adequate birth control. Qualified subjects must not have had any history of intolerance to any components of the (+)-calanolide A formulation. Participation in any other clinical trial involving drug therapy within the previous 6 months was prohibited. Written informed consent was obtained from all participants, and the study was both reviewed and approved by the Western Institutional Review Board (Olympia, Wash.).

Study medication and dosing. (+)-Calanolide A was supplied as translucent, soft gelatin capsules containing 100 mg of (+)-calanolide A formulated in an oil-based vehicle. Study medication was supplied in blister-packs of 10 capsules each.

Pharmacokinetic sampling. Blood samples for pharmacokinetic analysis were obtained via an indwelling catheter or via direct venipuncture. Patency of the indwelling catheter was maintained by saline flush, and heparin flush was not permitted. Blood samples were collected before dosing and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 24 h postdosing. Cohort 4 subjects had additional samples drawn at 32, 36, and 48 h postdose. The exact times of doses, meals, and pharmacokinetic blood collections were recorded.

Assay for plasma samples. Concentrations of (+)-calanolide A in plasma were determined using a validated high-performance liquid chromatographic (HPLC) method with fluorescent detection (11). Briefly, the synthetic intermediate (±)-12-oxocalanolide A prepared in 100% acetonitrile (4 µg/ml) was used as an internal standard, and 50 µl of this internal standard solution was mixed with the plasma sample to be analyzed. The sample mixtures (1.0 ml) were loaded onto a Varian Bond Elut column (Harbor City, Calif.) (6 ml containing 1.0 g of C₁₈ packing material). Each column was preconditioned with 5 ml of acetonitrile followed by 5 ml of HPLC-grade water flowing through the column by gravity. The column was washed with water (acidified with acetic acid) and eluted with 5 ml of acetonitrile under vacuum, and the eluents were collected and dried under N₂ at 50 to 60°C. The residue was reconstituted in 300 µl of acetonitrile, mixed by vortexing, sonicated, and filtered (0.2 µm [pore size]; 13-mm PTFE Gelman filter attached to a 1-ml disposable syringe) into an autoinjector vial for HPLC analysis.

Safety evaluation. The safety of single escalating doses of (+)-calanolide A was evaluated based on adverse experience reports, measurements of vital signs (heart rate, body temperature, respiratory rate, and blood pressure), clinical laboratory values, and the results of physical examination. Successive cohorts were not dosed until all subjects in the previous cohort had completed their week 1 follow-up visits with no unacceptable toxicities. For this study, toxicity was considered unacceptable if, within any cohort, the following occurred: (i) any subject experienced a life-threatening grade 4 toxicity that was reasonably attributable to study treatment or (ii) any three subjects experienced non-life-threatening grade 3 or 4 toxicities that were reasonably attributable to study medication or did not resolve within a reasonable time after dosing. Periodic clinical trial monitoring was conducted according to protocol and regulatory requirements.

Statistical analysis. The primary outcome measure in the study was the safety of escalating single doses of (+)-calanolide A, as measured by the appearance of adverse signs and/or symptoms. The primary planned comparisons between dosing cohorts evaluated the profile of changes in all safety measures. The study was not designed to detect statistically significant differences in safety measures but rather was designed to identify a safe dosing regimen for use in future studies.

	RESULTS

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(+)-Calanolide A was administered to 47 healthy HIV-negative subjects in four successive single-dose cohorts of 200, 400, 600, and 800 mg. All cohorts were balanced by gender, and the demographics are detailed in Table 1. The subjects in cohorts 2 (400-mg dose) and 3 (600-mg dose) were randomly assigned to receive the drug either with food or fasting.

Safety evaluation. In the four dosing cohorts, a total of 110 adverse events were reported, 101 of which were considered to be mild (grade 1). Forty-two treated subjects reported at least one adverse event, but only seven subjects experienced any event of greater than grade 1 severity. Table 2 summarizes the most frequent and/or most severe adverse events reported in this study. Of the events listed in Table 2, only dizziness, taste perversion (oily aftertaste), headache, eructation, and dyspepsia were considered to be probably related to study drug.

Pharmacokinetic evaluation. Pharmacokinetic parameters were highly variable among subjects. Table 4 and Fig. 1 and 2 summarize the overall profile. Intrasubject variability and limited elimination phase sampling precluded calculation of the half-life (t_1/2) for all subjects in the first three cohorts. The data from cohort 4 (800-mg dose) indicated a t_1/2 of about 20 h (Table 4). Concentrations of (+)-calanolide A were measurable beyond 24 h for all but three test subjects in this cohort. Both C_max and AUC values increased with dose (Fig. 2). An increase in C_max was accompanied by a respective increase in the AUC. Dose escalation across cohorts increased 100% from cohort 1 to cohort 2, 50% from cohort 2 to cohort 3, and 33% from cohort 3 to cohort 4. As determined from mean AUC values, the drug exposure increased 340, 129, and 159% across the three cohorts, while mean C_max values increased 379, 126, and 130%.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Mean (+)-calanolide A concentration-time curve following oral administration of single-escalating doses to healthy, HIV-negative subjects.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   (A) Examination of mean Cmax versus (+)-calanolide A dose following oral administration. (B) Examination of mean AUC-versus-(+)-calanolide A dose following oral administration.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Analysis of mean AUC versus (+)-calanolide A dose for male and female healthy, HIV-negative individuals following single oral doses. The dose represented in the figure is based on the body weight of the individual subject. Linear regression was performed for both male and female subjects with the results of regression analysis indicated in the figure.

	DISCUSSION

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This was the first study designed to evaluate the safety and pharmacokinetics of (+)-calanolide A in humans. In order to evaluate multiple doses of (+)-calanolide A, these phase I studies were performed in an escalating dose fashion, with careful monitoring of multiple safety parameters at each dosing increase. During the course of this study, no acute serious or life-threatening adverse experiences were seen. In fact, almost all previously described adverse experiences were of minimal clinical significance and without consequence.

Because the (+)-calanolide A ring structure has some resemblance to anticlotting clotting agents, such as coumarin and warfarin, special attention was paid to hematological findings in both previous animal studies and these human studies. No evidence of a clotting deficiency was observed in rats or dogs after 28 days of administration of (+)-calanolide A (Frank et al., 4th CROI). In this initial phase I human trial, no evidence of aberrations in PT or PTT values was observed.

(+)-Calanolide A produced venous irritation after intravenous administration to rats and dogs. In addition, some signs of gastric mucosal irritation were observed after its oral administration to animals. Consequently, signs and symptoms of nausea, upset stomach, and oral irritation have been closely monitored in this phase I study. No evidence of any serious gastrointestinal intolerance or oral irritation was seen in any of the dosing cohorts.

Single-doses of (+)-calanolide A appeared to be well tolerated in healthy, HIV-negative individuals. With the possible exception of dizziness, no adverse event appeared to be dose related. The dizziness reported by 51% of the subjects was transient and mild; in many cases, it appeared to be temporally related to phlebotomy or to the subjects having taken the drug while fasting. However, since (+)-calanolide A can readily cross the blood-brain barrier in animals, it is not possible to rule out causality in relationship to the observed transient dizziness.

A single serious adverse event occurred during this study. This event involved an unexplained increase in a subject's lipase value 24 h postdose. This male subject's lipase value increased to 1,046 IU/liter, approximately 3.5 times the upper limit of normal. While this event qualified as a grade 3 adverse experience according to a modified Adult ACTG laboratory test toxicity grading scale, the subject was completely asymptomatic, and the event completely resolved within 1 week with no complications.

A great deal of intrasubject variability was seen in the pharmacokinetic parameters examined. While this was seen across all dosing cohorts, certain relative significance and possible trends can be extrapolated from these data. Since the possibility of gender differences in pharmacokinetics had been suggested by animal studies, the study was designed to be gender balanced in order that any differences could be examined.

Elimination profiles of (+)-calanolide A were not fully characterized as a result of inadequate sampling times postdosing. Only three subjects of the 32 examined in the first three cohorts had undetectable (+)-calanolide A levels in plasma at the 24-h postdosing sampling point. This lack of data regarding the complete elimination of (+)-calanolide A precluded the calculation of the elimination rate constant, which resulted in the inability to calculate t_1/2 values as well as the AUC_0- for most subjects in the first three cohorts. In addition, all 12 subjects receiving 800 mg of (+)-calanolide A had detectable drug concentrations in plasma 48 h postdosing. In future studies, additional time points may help to better assess the elimination phase of the parent drug and aid in calculating a more precise half-life.

Due to the large degree of variability, inadequate sampling duration postdosing, and small sample size, it was impossible to calculate a mean half-life for the 200-, 400-, and 600-mg cohorts. The calculated half-life seen in the 800-mg dosing cohort was approximately 20 h. This rather long half-life may allow for a decreased frequency of dosing. The half-life observed in female subjects was approximately 3 h longer than that seen in male subjects. This may be due to a difference in the metabolic rate of (+)-calanolide A elimination or may simply be a result of the increased dose (in milligrams per kilogram) taken by female subjects due to their decreased weight.

The C_max and AUC data demonstrate an increase in each parameter with increasing dose (Fig. 2). This increase appears to become more linear at the higher doses of (+)-calanolide A, with a disproportionate increase observed between the 200- and 400-mg dosing regimens. This may indicate a possible saturable metabolism upon dose escalation, or it may simply be the result of increased variability in the detection of the low plasma concentrations seen in subjects receiving the 200-mg dose. In animal studies, this nonlinearity was not observed, and this may have been due to the relatively higher doses (150 mg/kg) of (+)-calanolide A administered to the animals, resulting in plasma levels significantly greater than those seen in these initial human studies. Once again, female subjects across all cohorts demonstrated an increased AUC and increased C_max compared to male subjects. However, when the dose was expressed as milligrams per kilogram, the AUC and C_max profiles of (+)-calanolide A were virtually identical in males and females. Males appeared to display increased apparent clearance rates compared with females; however, these differences were less pronounced at higher dosages.

The mean AUC data appears to indicate that in the 600-mg dosing cohort there is little difference in (+)-calanolide A absorption between male and female subjects. This discrepancy can be explained by the existence of a single female study subject whose body weight was considerably higher than the other female subjects. This subject reduced the mean value of the AUC significantly, resulting in what appears to be a reduction in gender difference in this cohort.

The effect of food on the pharmacokinetic profile of (+)-calanolide A was initially examined in this study. Briefly, the food effect seemed to be most evident in the T_max values obtained. The presence of food appeared to delay the onset of T_max across all groups examined. In addition, fed subjects appeared to display increased clearance compared to fasting subjects. A general conclusion, however, could not be reached with regard to the C_max and AUC results obtained. These parameters appeared to be much more variable between fed and fasted subjects. Obviously, the small sample size played a role in this high degree of variability, and more -defined studies will be required to properly delineate the effect of food on the pharmacokinetic profile of (+)-calanolide A.

In general, the levels of (+)-calanolide A in plasma seen in human subjects receiving single doses were higher than would have been predicted from animal studies. The highest AUC observed previously in any animal study was 30,455 ng · h/ml documented in rats receiving a dose of 150 mg/kg. In this first human study, the highest AUC observed was 26,569 ng · h/ml in a subject receiving 800 mg of (+)-calanolide A (11.1 mg/kg). These data support the assertion that humans may achieve significantly higher plasma levels of (+)-calanolide A with a given dose than have been seen in any animal model.

Taken together, the above data demonstrate that the favorable safety profile, long half-life, and increased plasma concentrations may allow for twice-daily dosing of this novel anti-HIV agent. Further clinical development is ongoing for this compound.