DRUG DEVELOPMENTÂ AND USE
The journey of Saquinavir from the lab to effective HIV treatment
The first of its kind, Saquinavir has been in uncharted territory from the beginning. Today, it continues to be essential in the treatment of HIV.
STRUCTURAL EVOLUTION OF SAQUINOVIR
SAQUINOVIR WAS DEVELOPED FROM THE INITIAL SUBSTRATE SEQUENCE
Saquinavir was developed by using standard medicinal chemistry approaches to a key substrate of HIV-1 protease. Within the viral proteome, HIV protease cleaves an unusual amino acid pair site between either tyrosine or phenylalanine and proline. Most mammalian proteases are unable to cleave between these amino acid residues. Based on the sequence of amino acids 165-169 in the pol polyprotein (Leu-Asn-Phe-Pro-Ile) around the Tyr/Phe-Pro cleavage site, Roche scientists synthesized a series of inhibitor molecules that corresponded to the transition state isosteres at this cleavage site. This included a stable hydroxyl-containing isostere that replaced the amide bond and mimicked the tetrahedral intermediate that results from enzymatic hydrolysis as seen in Figure 1 below.
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For this series of compounds containing the transition state isosteres, it was determined that the stereochemistry of the hydroxyl group was critical for potent enzyme inhibition. To optimize the series, Roche scientists determined the smallest sequence that preserved strong enzyme binding. The N and C termini and amino acid side chains of the lead compounds were also optimized. Finally, saquinavir was identified as a development candidate.[1]
Figure 1. Evolution of Saquinavir adapted from Ghosh and Chapsal (2013). The transition state mimic is highlighted in gold and red. The final product, Saquinavir itself, is revealed as the most potent, and was thus chosen as the target to move forward with.
HIV-infected cell lines treated with saquinavir showed profound protease inhibition and antiviral activity. The inhibitor’s potency as measured by the inhibition constant, Ki, was found to be 0.23 nM in vitro. The minimum concentration for 50% inhibition of viral replication, ID50, measured by p24 viral antigen production was determined to be 12-25 nM in the same cell lines.[2] In fact, saquinavir remains the most potent FDA-approved HIV protease inhibitor when measured in vitro. However, the in vivo potency of saquinavir is severely diminished due to its bioavailability being less than 4%. After receiving FDA approval, Roche discovered that to increase the in vivo potency of saquinavir, saquinavir can be administered together with a low dose of the protease inhibitor ritonavir. Ritonavir has a strong inhibitory effect on the cytochrome P450 liver isoenzyme CYP3A4 that mediates more than 90% of the hepatic metabolism saquinavir, resulting in higher blood plasma concentrations, or Area Under the Curve, of saquinavir. Saquinavir’s AUC increased by 17-23 times when administered with 400 mg and 200 mg of ritonavir respectively compared to lone saquinavir administration.[3] Using ritonavir as a booster allowed saquinavir to be administered in a twice-daily dosing regimen and reduced its capsule burden.
ORAL DELIVERY OF SAQUINAVIR
Roche patented saquinavir in 1989.[4] Roche developed two formulations under the trademarks INVIRASE (saquinavir base stabilized by mesylate in a hard-gel capsule) and FORTOVASE (a microemulsified soft gel capsule formulation that improved bioavailability).[5] FORTOVASE delivers roughly eightfold more active drug than INVIRASE at the standard dosage without ritonavir boosting.
SAQUINAVIR IN CLINICAL TRIALS
Clinical trials that followed conclusively showed reduced viral loads, improved CD4+ cell counts, and stunted disease progression of HIV/AIDS. In a randomized, double-blind clinical study (NV14256) in ZDV-experienced, HIV-infected patients, INVIRASE in combination with HIVID was shown to decrease the cumulative incidence of clinical disease progression to AIDS-defining events or death. In another randomized study (ACTG229/NV14255), patients with advanced HIV infection with a history of prolonged ZDV treatment and who were given INVIRASE 600 mg tid + ZDV + HIVID experienced greater increases in CD4+ cell counts compared to those who received HIVID + ZDV. FORTOVASE 1000 mg bid coadministered with ritonavir 100 mg bid was studied in a heterogenous population of 148 HIV-infected patients. At baseline, 42 patients were treatment naïve and 106 patients were treatment experienced. Of these patients, 52 had an HIV RNA level less than 400 copies/mL. Results showed that 91 of 148, or 61% of subjects, achieved and/or sustained an HIV RNA level of less than 400 copies/mL after 48 weeks of treatment.[6] Based on strong in vitro, in vivo, and clinical evidence of HIV viral replication inhibition, Roche’s INVIRASE became the sixth antiretroviral indicated for the treatment of HIV/AIDs and the first FDA-approved HIV-1 protease inhibitor in December 1995. This approval was received via the FDA’s Accelerated Approval program. It was a first-in-class drug that marked the genesis of the highly active antiretroviral therapy (HAART) protocol. In November 1997, FORTOVASE also gained FDA approval. Saquinavir is now used clinically as a first-line protease inhibitor for HIV-1 infection in adults in combination with ritonavir and other antiretroviral agents. It is also used as a salvage therapy in patients with viral resistance to a regimen of other protease inhibitors.
References
[1] Lyle, T. (2007) Ribonucleic Acid Viruses: Antivirals for Human Immunodeficiency Virus. Comprehensive Medicinal Chemistry II 7, 329–371.
[2]Ghosh, A. K., and Chapsal, B. D. (2013) Design of the anti-HIV protease inhibitor darunavir. Introduction to Biological and Small Molecule Drug Research and Development 355–384.Â
[3]Aronson, J. K. (2016) Saquinavir, in Meyler's side effects of drugs: the international encyclopedia of adverse drug reactions and interactions 16th ed., pp 306–309. Elsevier Amsterdam, Netherlands.Â
[4]Martin, J. A., and Redshaw, S. (1993, March 23) Amino Acid Derivatives.
[5]Bailey, C. A., Ferdinando, J. C., and Shah, N. (1999, December 28) Pharmaceutical compositions containing proteinase inhibitors.Â
[6]Roche Laboratories. (2004) INVIRASE (saquinavir mesylate) capsules and tablets. Access Data FDA. U.S. Food and Drug Administration.