A phase I/II study of the safety and activity of a microsphere formulation of KNI-272 in patients with HIV-1 infection

D. R. Churchilla, P. M. Sladea, M. Youleb, B. G. Gazzardb and J. N. Webera,*

a Department of Genitourinary Medicine and Communicable Diseases, Imperial College School of Medicine at St Mary's, Norfolk Place, London W2 1PG; b Department of HIV Medicine, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Eighteen patients with symptomatic HIV disease were enrolled into a phase I/II study of a microsphere formulation of the HIV protease inhibitor KNI-272, with doses escalated up to a maximum dose of 60 mg/kg/day. One patient developed reversible elevation in hepatic transaminase. The plasma half-life of the drug was very short, varying between 0.25 and 1.1 h. No consistent effect on plasma HIV RNA levels or CD4+ lymphocyte counts was seen.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
KNI-272 is a tripeptide HIV protease inhibitor containing a unique unnatural amino acid (allophenylnorstatine). This compound has shown potent antiretroviral activity in vitro, with IC50 values of 0.02–0.1 µM against a range of virus strains in ATH8 cells.1 However, further in vitro experiments indicated a high degree of protein binding of the drug in plasma (98–99%), suggesting that high plasma levels of the drug would need to be achieved to produce a potent antiretroviral effect in vivo.2

In a phase I/II study in HIV-infected patients,3 an initial dose of 4 mg/kg four times daily was well tolerated, but significant reversible elevations in hepatic transaminases were seen in two of five patients in whom the dose was increased to 6.6 mg/kg four times daily. In patients enrolled subsequently into this study, the dose of KNI-272 was increased gradually to 10 mg/kg four times daily without major abnormalities in liver function. At higher doses, there was some evidence of an antiretroviral effect.3

The objective of the current study was to assess the safety, tolerability and efficacy on surrogate markers of KNI-272 in patients with HIV infection, using a novel microsphere formulation and higher doses of the drug.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Eighteen patients were enrolled at two study sites. To be eligible for the study, patients had to have symptomatic HIV disease or AIDS, CD4+ lymphocyte counts of 50–500 cells/mm3 and plasma HIV RNA >20000 copies/mL (Roche Amplicor v.1.0, Nutley, NJ, USA) and no previous history of treatment with protease inhibitors. Patients with major haematological, renal or hepatic dysfunction, evidence of significant cardiac disease, active opportunistic infection or Kaposi's sarcoma likely to require chemotherapy were excluded.

A dose-titration schedule was used to try to minimize the possibility of liver toxicity. Over 4 weeks the dose of the drug was increased from 6 to 24 mg/kg/day in 6 mg/kg/day increments. Each capsule of KNI-272 contained 75 mg of drug, and the number of capsules to be taken per dose was calculated to the nearest whole capsule. The drug was taken in five doses per day (the first four doses were one-sixth of the total daily dosage, and the last dose was one-third of the daily dosage). Patients who tolerated the target dose could elect to have their dose escalated further to a maximum dose of 36 mg/kg/day for the remainder of the study period. Initial dosing levels were later revised to 15 mg/kg/day, increasing to 60 mg/kg/day (or maximal tolerated dose if lower) in 15 mg/kg/day increments.

A subset of subjects underwent pharmacokinetic studies at day –1 with a single dose of 4 mg/kg, with half of the cohort fed with a standard meal and half fasted. Additional pharmacokinetic data were collected in the same cohort at day 25 when the target dose of 24 mg/kg/day had been reached.

Efficacy was measured by changes in CD4+ lymphocyte counts and plasma HIV RNA (Roche Amplicor 1.0, lower limit of detection 500 copies/mL).

For pharmacokinetic studies, blood was collected in heparinized tubes and plasma was immediately separated and stored at –20°C. Analysis of plasma for KNI-272 levels was carried out by high-performance liquid chromatography, using a validated technique with a lower limit of detection of 10 ng/mL (Huntingdon Life Sciences, Cambridge, UK).

The study was approved by the Local Research Ethics Committees at St Mary's and at the Chelsea and Westminster hospitals. Written informed consent was obtained from all subjects before participation in the trial.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Baseline characteristics are summarized in Table IGo. Eighteen patients were recruited to the study, 17 of whom discontinued study medication prematurely (i.e. before completion of at least 3 weeks at 60 mg/kg/day). Eleven patients withdrew for personal reasons, five withdrew because of an adverse event and one because of a falling CD4+ lymphocyte count.


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Table I. Baseline demographics of patients: all 18 patients were male
 
Adverse events

One patient developed grade 3 elevation of alanine transaminase, and withdrew from the study. This was the only serious adverse event that was judged to be probably related to the study drug.

Pharmacokinetic results

Single-dose pharmacokinetic data are shown in Table IIGo. For later pharmacokinetic studies, patients were dosed in the fed state. After 25 days of treatment (at which time patients were taking 24 mg/kg/day) there was no change in Cmax, AUC0–4 or Tmax following a dose of the drug at 4 mg/kg compared with results from patients given a single dose.


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Table II. Results of pharmacokinetic studies after a single dose of 4 mg/kg KNI-272
 
Virological efficacy

There was no clear pattern in viral load response to treatment with KNI-272 at different final doses. Mean plasma HIV RNA decreased by 0.2 log10 from baseline to day 25 in patients on 24 mg/kg/day, but there was an increase of 0.06 log10 from baseline after 21 days of treatment at this dose. Twenty-one days after dosing at 36 and 60 mg/kg/day, changes from baseline were –0.1 and +0.23 log10, respectively.

For eight patients, full pharmacokinetic profiles and viral load data from day 25 of treatment at 24 mg/kg/day were available for an analysis that related AUC0–4 to change in plasma HIV RNA. This showed a weak association between exposure to KNI-272 and change in plasma HIV RNA (data not shown).

CD4+ lymphocyte count response

There was no consistent trend of change in CD4+ counts at any dose level (Table IIIGo).


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Table III. Changes in CD4+ lymphocyte counts (cells/mm3) from baseline at last sample during each dose period of drug
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite the recent dramatic improvements in the treatment of HIV infection,4,5 there is a need for the continued development of new antiretroviral drugs, particularly agents with toxicity and resistance profiles that do not overlap with currently available agents. In vitro studies of KNI-272 suggested that the drug had many favourable properties, including high potency and low toxicity. Animal work confirmed that serious toxicity with the drug was seen largely only at very high doses, and early phase I/II studies in HIV-infected subjects suggested some evidence of antiretroviral efficacy, although plasma levels of the drug were only transiently above the in vitro IC90.

Despite concerns about hepatic toxicity from other studies of KNI-272 in HIV-infected subjects, only one of the 18 patients in this study developed abnormalities in liver function tests. Since we found the drug to have a short plasma half-life, and saw no change in pharmacokinetics following administration of multiple doses nor evidence of increasing toxicity with higher doses, the dose escalation that we used to try to limit toxicity was probably unnecessary.

The formulation of KNI-272 used in this study was demonstrated to have little measurable antiretroviral activity, and had no clear effect on CD4+ lymphocyte counts. Although the drug was rapidly absorbed, the plasma half-life of the drug was very short, and there was no accumulation of the drug with repeated dosing. Since very little of the drug is excreted unchanged in the urine, it is likely that rapid metabolism of the drug accounts for the short half-life. The unfavourable pharmacokinetics of the drug might be overcome if it were given together with drugs that would inhibit metabolism of KNI-272 by cytochrome P450. For example, co-administration of KNI-272 with ritonavir, indinavir, nelfinavir or delavirdine to rats led to increases in AUC for KNI-272 of up to 22-fold.6 It remains to be seen, however, whether a similar effect is seen in human subjects, and whether the increased drug exposure that would result would be associated with any additional toxicity.


    Notes
 
* Corresponding author. Tel: +44-20-7886-1539; Fax: +44-20-7886-6645. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Kageyama, S., Mimoto, T., Murakawa, Y., Nomizu, M., Ford, H., Shirasaka, T. et al. (1993). In vitro anti-human immunodeficiency virus (HIV) activities of transition state mimetic HIV protease inhibitors containing allophenylnorstatine. Antimicrobial Agents and Chemotherapy 37, 810–7.[Abstract]

2 . Kageyama, S., Anderson, B. D., Hoesterey, B. L., Hayashi, H., Kiso, Y., Flora, K. P. et al. (1994). Protein binding of human immunodeficiency virus protease inhibitor KNI-272 and alteration of its in vitro antiretroviral activity in the presence of high concentrations of proteins. Antimicrobial Agents and Chemotherapy 38, 1107–11.[Abstract]

3 . Humphrey, R. W., Wyvill, K. M., Nguyen, B. Y., Shay, L. E., Kohler, D. R., Steinberg, S. M. et al. (1999). A phase I trial of the pharmacokinetics, toxicity, and activity of KNI-272, an inhibitor of HIV-1 protease, in patients with AIDS or symptomatic HIV infection. Antiviral Research 41, 21–33.[ISI][Medline]

4 . Hammer, S. M., Squires, K. E., Hughes, M. D., Grimes, J. M., Demeter, L. M., Currier, J. S. et al. (1997). A controlled trial of two nucleoside analogues plus indinavir in persons with human immunodeficiency virus infection and CD4 cell counts of 200 per cubic millimeter or less. AIDS Clinical Trials Group 320 Study Team. New England Journal of Medicine 337, 725–33.[Abstract/Free Full Text]

5 . Palella, F. J., Delaney, K. M., Moorman, A. C., Loveless, M. O., Fuhrer, J., Satten, G. A. et al. (1998). Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. New England Journal of Medicine 338, 853–60.[Abstract/Free Full Text]

6 . Sato, H., Shintani, M. S., Mimoto, T. M., Terashima, K. T., Hayashi, H. H. & Mitsuya, H. M. (1998). In vitro antiviral activity and pharmacokinetic (PK) profiles of KNI-272 when combined with other protease inhibitors (PIs). XIIth World AIDS Conference, Geneva, 1998. Abstract 12339.

Received 10 January 2000; returned 3 May 2000; revised 25 September 2000; accepted 17 October 2000





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