INTRODUCTION

• All currently marketed HIV protease inhibitors (PIs) are bound to plasma proteins (60 to >99%), primarily albumin and á 1 -acid glycoprotein (AGP).
• Protein-bound drug generally is considered to be too large to pass through most cellular membranes to exert pharmacological actions. 

In vitro experiments indicate that binding to plasma proteins indeed reduces the antiviral activity of PIs.1-4 Clinical data support these in vitro observations.5

• In vitro antiviral activity experiments are normally conducted in medium containing 10% fetal calf serum (FCS). In attempts to more realistically estimate the in vitro antiviral potency for protein-bound PIs, the 10% FCS medium has been modified by adding human serum (HS),1 purified AGP,1-4 or purified human serum albumin (HSA)1 . Use of these different systems can produce widely varied estimates of
  in vitro antiviral potency of PIs.
• HIV infection and hepatic impairment can alter the protein composition of plasma. The effect of these changes on the binding of PIs is unknown.
• Despite being highly protein bound, most HIV protease inhibitors exert antiviral activity in vivo. In order to understand the relevance of in vitro determinations to in vivo potency, we investigated various aspects of plasma protein binding of RTV and LPV. 

OBJECTIVES

• To assess if HIV infection and/or hepatic impairment affect the protein binding of LPV or RTV.
• To determine free fractions of LPV and RTV in various in vitro systems that have been used to determine the antiviral activity of PIs. 
• To determine the antiviral activity of LPV, RTV, and other PIs in two in vitro systems.

METHODS

• Sources of Plasma and Media
  – Healthy volunteers, who had fasted at least 8 hours, had taken no medication except aspirin
     during the preceding seven days and had taken no salicylates during the preceding 48 hours
  – Adult HIV-negative volunteers participating lopinavir/ritonavir pharmacokinetic studies
  – Adult HIV-infected subjects participating lopinavir/ritonavir or ritonavir Phase II studies
  – Fetal calf serum and RPMI 1640 medium were purchased from Gibco Chemical Co.
  – Human serum for mixed lymphocyte cultures was purchased from Sigma Chemical Co.
  
  
• Blood was collected into heparinized tubes and centrifuged to separate cells from plasma
  
  
• Ultrafiltration Method (Ex vivo HIV-positive samples)
  – Test medium spiked and incubated at 37°C for 15 minutes
  – 1 mL -> Centrifree ® Micropartition System (12-14kD cut off)
  – Centrifuged 1000-2000 x g in fixed angle rotor at 22°C
  – % Bound = 100 – (dpm/mL filtrate / dpm/mL test)  x 100%
  
  
• Equilibrium Dialysis ( In vitro and ex vivo HIV-negative samples)
  – Spectrum Equilibrium Dialysis System
  – 1 mL cells – Spectra/Por 2 membrane (12-14kD cut off)
  – Test medium with [ ¹⁴ C] Drug vs. dialysing medium (Test medium with serum/plasma component
      replaced with 0.02 M phosphate buffer, pH 7.4, containing 0.6% NaCl) at 37°C
  – 3 hour equilibration (drug stability tested by radio-HPLC)
  – % Bound = [(dpm/mL test – dpm/mL dialysing) / dpm/mL test] x 100% 

RESULTS AND DISCUSSION

• The binding of LPV (ca. 99%) and RTV (ca. 98%) ex vivo appeared to be similar in the plasma from both healthy volunteers (determined by equilibrium dialysis*) and HIV-infected subjects (determined by ultrafiltration*) (Table 1).
• The free fraction of LPV in healthy volunteers appeared to be relatively constant (0.8%) across the clinical plasma concentration range (Figure 1).
• Multiple dosing did not significantly alter the plasma protein binding of LPV and RTV in healthy volunteers dosed with LPV/r for 2 weeks (Table 1).

*In vitro experiments indicate that the LPV free fraction determined by ultrafiltration averages 1.7 X the value obtained by equilibrium dialysis (data not shown). Whether this difference also applies to ex vivo samples is unknown. Assuming this difference, the most conservative estimated free fraction of LPV at physiological concentrations in the plasma from HIV-infected subjects (ca. 0.7%) is within the error range of the various determinations. Ultrafiltration and equilibrium dialysis yielded similar free fractions of RTV over the clinically relevant concentration range (data not shown).

Figure 1. Free Fraction (%) of LPV in the Plasma of Healthy Volunteers After Multiple Dosing

Effect of Mild or Moderate Hepatic Impairment on the Protein Binding of RTV in HIV-infected Subjects

• RTV is significantly bound to albumin; however, no significant change was observed in plasma protein binding over a range of albumin concentrations in subjects with varying degrees of impairment in hepatic function (Table 2).
• The effect of hepatic impairment on the free fraction LPV is currently being evaluated.

The Free Fractions of LPV and RTV at Clinically Relevant Plasma Concentrations are Closely Approximated in 50% HS plus 10% FCS

• The free fraction of lopinavir in 10% FCS + 50% HS media and in whole human plasma was assessed in vitro.
• A total LPV concentration range of 0.1-10 µg/mL was assessed.
• At the IC 50 determined in the presence of 50% HS + 10% FCS (0.06 µg/mL), the estimated free fraction of LPV was ca. 0.65% (Figure 2 and Table 3).
• Similarly, at total mean plasma concentrations of LPV achieved by dosing LPV/r at 400/100 mg BID (5.5 to 9.6 µg/mL), the estimated free fraction was 0.6-0.8% in vitro (Table 1 and Table 3) and 1.1% ex vivo in HIV+ subjects (Table 1).

Figure 2. The Free Fraction of LPV in 50% HS + 10% FCS at the IC ₅₀ Approximates or Underestimates the Free Fraction in Patient Plasma

• In a similar manner to LPV, at the IC 50 of RTV determined in the presence of 50% HS + 10% FCS (0.96 µg/mL), the estimated free fraction of RTV was ca. 0.67% (Figure 3 and Table 4).
• At total mean plasma concentrations of RTV achieved by dosing at 600 mg BID (3.7 to 11.2 µg/mL), the estimated free fraction was 0.6 to 0.7% in vitro (Figure 3 and Table 4) and ca. 2.0% ex vivo (Table 1).

Figure 3. The Free Fraction of RTV in 50% HS + 10% FCS at the IC ₅₀ Approximates or Underestimates the Free Fraction in Patient Plasma

In Vitro Antiviral Potency of Protease Inhibitors Determined in 10% FCS + 50% HS

• The mean IC 50 values of various protease inhibitors against three wild-type laboratory HIV strains (IIIB, pNL4-3, and HXB2) were determined in side-by-side experiments using infected MT4 cells (Table 5).

Use of In Vitro Data for Development of Pharmacodynamic Models

• Given that the free fraction of LPV and RTV in human plasma is closely mimicked by media containing 10% FCS and 50% HS, the in vitro antiviral IC 50 of these drugs may be useful for the construction of pharmacodynamic models for estimating in vivo potency.
• The inhibitory quotient 6 (IQ) is one such model that characterizes the relationship between drug exposure (C trough ) and drug susceptibility (IC 50 ) (Figure 4).
• The inhibitory quotient has been shown to be predictive of virologic response to LPV/r 7 and RTV/IDV 8 regimens.

Figure 4. Inhibitory Quotient (IQ): Model for Understanding PI Pharmacodynamics

CONCLUSIONS

• The free fractions of LPV and of RTV did not appear to be affected by HIV infection.
• The free fraction of RTV did not appear to be affected by mild and moderate hepatic impairment. Data on LPV are pending.
• The free fraction of RTV and LPV in 10% fetal calf serum + 50% human serum closely resembles the free fraction of each in 100% human plasma at clinically relevant concentrations.
• Therefore, IC 50 values determined in 10% fetal calf serum + 50% human serum should provide a reasonable estimate for IC 50 values in 100% human serum.
• The free fractions of protease inhibitors in the media of various in vitro assays used to assess antiviral potency should be investigated to provide insight into the possible clinical relevance of data from such assays.

REFERENCES

1. Molla, A., Vasavanonda, S., Kumar, G., Sham, H. L., et al. Human serum attenuates the activity of protease inhibitors toward wild-type and mutant human immunodeficiency virus. Virology 250: 255-262 (1998).
2. Lazdins J. K., Mestan, J., Goutte, G., Walker, M. R., et al. In vitro effect of a1-acid glycoprotein on the anti-human immunodeficiency virus (HIV) activity of the protease inhibitor CGP 61755: A comparative study with other relevant HIV protease inhibitors. J Infect. Dis. 175: 1063-1071 (1997).
3. Bilello, J. A., Bilello, P. A., Prichard, M., Robins, T., and Drusano, G. L. Reduction of the in vitro activity of A77003, an inhibitor of human immunodeficiency virus protease by human serum α 1-acid glycoprotein. J Infect. Dis. 171: 546-551 (1995).
4. Bilello, J. A., Bilello, P. A., Stellrecht, K., Leonard, J, et al. Human serum α 1-acid glycoprotein reduces uptake, intracellular concentration, and antiviral activity of A80987, an inhibitor of the human immunodeficiency virus type-1 protease. Antimicrob. Agents Chemother. 40: 1491-1497 (1996).
5. Getman, D. P., DeCrescenzo, G. A., Heintz, R. M., Reed, K. L., et al. Discovery of a novel class of potent HIV-1 protease inhibitors containing the (R)-(hydroxyethyl)urea isostere. J. Med. Chem. 36:288-291 (1993).
6. Ellner, P. D., Neu, H. C. The inhibitory quotient. A method for interpreting minimum inhibitory concentration data. JAMA. 246:1575-8 (1981).
7. Hsu, A., Granneman, G. R., Kempf, D. J., Isaacson, J., et al. The C trough inhibitory quotient predicts virologic response to lopinavir/ritonavir (ABT-378/ritonavir) therapy in treatment experienced patients. Fifth International Congress on Drug Therapy in HIV Infection, Oct. 22-26, Glasgow, UK (2000).
8. Kempf, D., Hsu, A., Jiang, P., Rode, R. et al. Response to ritonavir (RTV) intensification in indinavir (IDV) recipients is highly correlated with virtual inhibitory quotient. 8th Conference on Retroviruses and Opportunistic Infections, Feb. 4-8, Chicago, IL (2001)

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Poster 753
  Protein Binding of Lopinavir (LPV) and Ritonavir (RTV): In Vitro and Ex Vivo Data from HIV-infected Patients and Healthy Volunteers

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