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 . 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 .
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.
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 , or even up to 100 μM [4,11,12,16,23,24], or of indinavir in concentrations of 50 μM , 100 μM [4,11,23], 163 μM , or even up to 10 000 μM , 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)  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.
Walter Emil Haefeli
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.
1. Lee LM, Karon JM, Selik R, Neal JJ, Fleming PL. Survival after AIDS diagnosis in adolescents and adults during the treatment era, United States, 1984–1997. JAMA 2001, 285: 1308–1315.
2. Flexner C. HIV-protease inhibitors. N Engl J Med 1998, 338: 1281–1292.
3. Piscitelli S, Gallicano K. Interactions among drugs for HIV and opportunistic infections. N Engl J Med 2001, 344: 984–996.
4. Eagling VA, Back DJ, Barry MG. Differential inhibition of cytochrome P450 isoforms by the protease inhibitors, ritonavir, saquinavir and indinavir. Br J Clin Pharmacol 1997, 44: 190–194.
5. von Moltke LL, Greenblatt DJ, Grassi JM. et al
. Protease inhibitors as inhibitors of human cytochromes P450: high risk associated with ritonavir. J Clin Pharmacol 1998, 38: 106–111.
6. Koudriakova T, Iatsimirskaia E, Utkin I. et al
. Metabolism of the human immunodeficiency virus protease inhibitors indinavir and ritonavir by human intestinal microsomes and expressed cytochrome P4503A4/3A5: mechanism-based inactivation of cytochrome P4503A by ritonavir. Drug Metab Dispos 1998, 26: 552–561.
7. Kim RB, Fromm MF, Wandel C. et al
. The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors. J Clin Invest 1998, 101: 289–294.
8. Huisman MT, Smit JW, Wiltshire HR, Hoetelmans RMW, Beihnen, JH, Schinkel AH. P-glycoprotein limits oral availability, brain, and fetal penetration of saquinavir even with high doses of ritonavir. Mol Pharmacol 2001, 59: 806–813.
9. Alsenz J, Steffen H, Alex R. Active apical secretory efflux of the HIV protease inhibitors saquinavir and ritonavir in Caco-2 cell monolayers. Pharmacol Res 1998, 15: 423–428.
10. Aungst BJ. P-glycoprotein, secretory transport, and other barriers to the oral delivery of anti-HIV drugs. Adv Drug Del Rev 1999, 39: 105–116.
11. Profit L, Eagling VA, Back DJ. Modulation of P-glycoprotein function in human lymphocytes and Caco-2 cell monolayers by HIV-1 protease inhibitors. AIDS 1999, 13: 1623–1627.
12. Washington CB, Duran GE, Man MC, Sikic BI, Blaschke TF. Interaction of anti-HIV protease inhibitors with the multidrug transporter p-glycoprotein (P-gp) in human cultured cells. J Acquir Immun Defic Syndr 1998, 19: 203–209.
13. Gutmann H, Fricker G, Drewe J, Török M, Miller DS. Interactions of HIV protease inhibitors with ATP-dependent drug export proteins. Mol Pharmacol 1999, 56: 383–389.
14. Srinivas RV, Middlemas D, Flynn P, Fridland A. Human immunodeficiency virus protease inhibitors serve as substrates for multidrug transporter proteins MDR1 and MRP1 but retain antiviral efficacy in cell lines expressing these transporters. Antimicrob Agents Chemother 1998, 42: 3157–3162.
15. Lee CG, Gottesman MM, Cardarelli CO. et al
. HIV-1 protease inhibitors are substrates for the MDR1 multidrug transporter. Biochemistry 1998, 37: 3594–3601.
16. van der Sandt ICJ, Vos C, Nabulski L, Blom-Roosemalen MCM, Voorwinden HH, de Boer AG, Breimer DD. Assessment of active transport of HIV protease inhibitors in various cell lines and the in vitro blood–brain barrier. AIDS 2001, 15: 483–491.
17. Shibata N, Matsumura Y, Okamoto H, Kawaguchi Y, Ohtani A, Yoshikawa Y, Takada K. Pharmacokinetic interactions between HIV-1 protease inhibitors in rats: study on combinations of two kinds of HIV-1 protease inhibitors. J Pharm Pharmacol 2000, 52: 1239–1246.
18. Hsu A, Granneman GR, Cao G. et al
. Pharmacokinetic interactions between two human immunodeficiency virus protease inhibitors, ritonavir and saquinavir. Clin Pharmacol Ther 1998, 63: 453–464.
19. Vella S, Floridia M. Saquinavir clinical pharmacology and efficacy. Clin Pharmacokinet 1998, 34: 189–201.
20. Longer M, Shetty B, Zamansky I, Tyle P. Preformulation studies of a novel protease inhibitor, AG1343. J Pharm Sci 1995, 84: 1090–1093.
21. Kempf DJ, Marsh KC, Denissen JF. et al
. ABT-538 is a potent inhibitor of human immunodeficiency virus protease and has high oral bioavailability in humans. Proc Natl Acad Sci U S A 1995, 92: 2484–2488.
22. Jones K, Hoggard PG, Sales SD, Khoo S, Davey R, Back DJ. Differences in the intracellular accumulation of HIV protease inhibitors in vitro
and the effect of active transport. AIDS 2001, 15: 675–681.
23. Hesse LM, von Moltke LL, Shader RI, Greenblatt DJ. Ritonavir, efavirenz, and nelfinavir inhibit CYP2B6 activity in vitro
: potential drug interactions with bupropion. Drug Metab Dispos 2001, 29: 100–102.
24. Shiraki N, Hamada A, Yasuda K, Fujii J, Arimori K, Nakano M. Inhibitory effect of human immunodeficiency virus protease inhibitors on multidrug resistance transporter P-glycoprotein. Biol Pharm Bull 2000, 23: 1528–1531.
25. Hochman JH, Chiba M, Nishime J, Yamazaki M, Lin JH. Influence of P-glycoprotein on the transport and metabolism of indinavir in Caco-2 cells expressing cytochrome P-450 3A4. J Pharmacol Exp Ther 2000, 292: 310–318.