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Poor solubility limiting significance of in-vitro studies with HIV protease inhibitors

Weiss, Johanna; Burhenne, Jürgen; Riedel, Klaus-Dieter; Haefeli, Walter Emil


Department of Internal Medicine VI, Clinical Pharmacology and Pharmacoepidemiology, University Hospital, Bergheimer Strasse 58, D- 69115 Heidelberg, Germany.

Sponsorship: This work was supported by grant 01EC9902 from the German Ministry for Education and Research (BMBF).

Received: 3 September 2001;

revised: 18 September 2001; accepted: 24 September 2001.

HIV protease inhibitors have effectively modified disease progression and mortality in HIV-infected patients when combined with nucleoside or non-nucleoside reverse transcriptase inhibitors [1,2]. However, their use is complicated by the frequent occurrence of adverse drug–drug interactions [3]. As high affinity substrates and inhibitors of both cytochrome P450 3A4 (CYP3A4) [2–6] and the ABC transporter P-glycoprotein [7–16], protease inhibitors may markedly alter the pharmacokinetics of numerous other substrates, thereby increasing drug exposure to other protease inhibitors [17,18] and non-HIV-therapeutic agents, and thus improving effectiveness but also promoting toxicity [3].

Because of the central role of CYP3A4 and P-glycoprotein in the pharmacokinetics of a substantial number of currently used drugs, models to study drug–drug interactions in vitro, and thus to predict significant interactions in vivo are a major focus of current drug development and safety. End-points of such in-vitro studies (Ki or IC50 values) serve as markers for the extent and risk of interactions. These values are calculated from the concentrations used in test systems and are obviously based on the known solubility of the compound under investigation. The rather poor solubility of protease inhibitors is known, but the data are conflicting [10,19–21], and only limited information is available for buffer systems commonly used in experiments with living cells or metabolism studies with microsomes.

We determined the maximum solubility of ritonavir, saquinavir mesylate and indinavir sulphate in Hank's balanced salt solution (HBSS) in the presence and absence of 1% of dimethylsulfoxide or ethanol. HBSS is a buffer system often used in vitro, and the two solvents are frequently added to increase the solubility of hydrophobic compounds in aqueous buffers.

Solubility tests were repeated once, each measured twice (quadruplicate determination). Indinavir sulphate, ritonavir or saquinavir mesylate (5.0 mg) were added to 10 ml HBSS or HBSS containing 1% dimethylsulfoxide or ethanol. After shaking, the suspensions were sonicated (1 h) and centrifuged (2000 g, 20 min). The clear supernatant was mixed with acetonitrile (1 : 1, v : v), pipetted into silanized autosampler vials and concentrations were determined using liquid chromatography–mass spectrometry. The liquid chromatography–mass spectrometry system was calibrated with solutions of each protease inhibitor (water : acetonitrile: 1 : 1, v : v) up to 100 μ M. All calibration curves were linear (correlation coefficients r ≥ 0.9989).

The lowest solubility was found with ritonavir, being approximately 5% of indinavir and approximately 9% of saquinavir solubility (Table 1). The influence of 1% organic solvent was negligible, except for ritonavir. The addition of 1% ethanol increased ritonavir solubility by 184%, whereas dimethylsulfoxide had no effect.

Table 1

Table 1

Our results agree well with protease inhibitor solubility data provided by the respective manufacturers (Table 1), and consistently show a limited solubility of this class of drugs in pH-adjusted solutions (buffers). Therefore, these data seriously question the reliability of in-vitro results in publications that claim to have used buffered solutions of ritonavir up to 10 or 20 μM [6,9,13,22], 50 μM [8], or even up to 100 μM [4,11,12,16,23,24], or of indinavir in concentrations of 50 μM [25], 100 μM [4,11,23], 163 μM [16], or even up to 10 000 μM [12], or of saquinavir in concentrations up to 1000 μM [4,12,23,24], without providing evidence for the dissolution of the compounds in their system. For other HIV protease inhibitors even fewer data are available. The solubility of nelfinavir in water is also low (6.8 mM at pH 2.6) [20] and, deduced from the above findings, is expected to be even lower in buffers. Nevertheless, investigators use this protease inhibitor in solutions up to 100 μM [11,12,23,24]. Likewise, the solubility of lopinavir in water is poor, the value indicated by the manufacturer (Abbott, Wiesbaden, Germany) is 70 μM and the solubility in buffers is unknown (Abbott, personal communication).

In accordance with unpublished information from the respective manufacturers, the current series of experiments confirms the limited solubility of HIV protease inhibitors in aqueous buffers used for in-vitro experiments. The common practice of using protease inhibitors at concentrations above the solubility limit is therefore likely to induce crystal formation, which may then result in an overestimation of the effective concentrations by orders of magnitude, and lead to incorrect Ki or IC50 values and meaningless or even wrong predictions of drug–drug interactions. Future reports on in-vitro studies with high protease inhibitor concentrations should therefore only be published if they provide evidence confirming solubility in the respective media.

As a gold standard for in-vitro studies with poorly soluble compounds, we suggest the determination of the actual concentrations in media to increase the reliability of in-vitro interaction studies, on the basis of valid concentration and not dose–response curves.

Johanna Weiss

Jürgen Burhenne

Klaus-Dieter Riedel

Walter Emil Haefeli

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The authors would like to thank Abbott (Wiesbaden, Germany), Roche (Basel, Switzerland), and MSD (Munich, Germany) for generously providing ritonavir, saquinavir mesylate, and indinavir sulphate, respectively, and for submitting their solubility data.

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