Synthesis and in vitro biological evaluation of mannose-containing prodrugs derived from clinically used HIV-protease inhibitors with improved transepithelial transport

In an approach to improve the pharmacological properties, safety and pharmacokinetic profiles, and their penetration into HIV reservoirs or sanctuaries, and consequently, the therapeutic potential of the current protease inhibitors (PIs) used in clinics, we investigated the synthesis of various mannose-substituted saquinavir, nelfinavir, and indinavir prodrugs, their in vitro stability with respect to hydrolysis, anti-HIV activity, cytotoxicity, and permeation through a monolayer of Caco-2 cells used as a model of the intestinal barrier. Mannose-derived conjugates were prepared in two steps, in good yields, by condensing an acid derivative of a protected mannose with the PIs, followed by deprotection of the sugar protecting group. With respect to hydrolysis, these PI prodrugs are chemically stable with half-life times in the 50-60 h range that are compatible with an in vivo utilization aimed at improving the absorption/penetration or accumulation of the prodrug in specific cells/tissues and liberation of the active free drug inside HIV-infected cells. These stabilities correlate closely with the low in vitro anti-HIV activity measured for those prodrugs wherein the coupling of mannose to the PIs was performed through the peptidomimetic PI's hydroxyl. Importantly, mannose conjugation to the PIs was further found to improve the absorptive transepithelial transport of saquinavir and indinavir but not of nelfinavir across Caco-2 cell monolayers, by contrast to glucose conjugation which had the opposite effect. The mannose-linked prodrugs of saquinavir and indinavir display therefore a most promising therapeutic potential provided that bioavailability, penetration into the HIV infected macrophages, and HIV-reservoirs of these PIs are improved.     

Determination of protease inhibitors using liquid chromatography-tandem mass spectrometry

A method for the analysis of six protease inhibitors and one metabolite has been developed and validated. Amprenavir, ritonavir, saquinavir, lopinavir, indinavir, nelfinavir, and an active metabolite of nelfinavir (M8) are quantitated using reversed-phase liquid chromatography coupled to tandem mass spectrometry, equipped with an electrospray ionization source (ESI-LC-MS-MS). The validation data presented here shows that the method allows the rugged analysis of these species from one aliquot. The evolution of complex drug interactions assessments and the clinical use of therapeutic drug monitoring for these antiretrovirals will be a potential immediate application of this method.     

Human Immunodeficiency Virus Type 1 Protease Genotypes and In Vitro Protease Inhibitor Susceptibilities of Isolates from Individuals Who Were Switched to Other Protease Inhibitors after Long-Term Saquinavir Treatment

An understanding of the mechanisms of virologic cross-resistance between human immunodeficiency virus type 1 protease inhibitors is important for the establishment of effective treatment strategies for patients who no longer respond to their initial protease inhibitor. Protease gene sequencing results from patients treated with saquinavir showed significant increases in the frequency of the G48V protease mutation in patients receiving higher doses of the drug. In addition, all six patients who developed the G48V mutation during saquinavir therapy developed the V82A mutation either on continued saquinavir or after a switch to nelfinavir or indinavir. In vitro susceptibility assays showed that all 13 isolates with reduced susceptibilities to two or more protease inhibitors had either the G48V or L90M mutation, along with an average of six other protease mutations. Reduced susceptibility to nelfinavir was found in 14 isolates, but only 1 possessed the D30N mutation. These results suggest that mutations selected in vivo by initial saquinavir therapy may provide more cross-resistance to the other protease inhibitors than has been previously reported.     

Effectiveness of commercial inhibitors against subtype F HIV-1 protease

Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K_i and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.     

Effectiveness of commercial inhibitors against subtype F HIV-1 protease

Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K(i) and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.     

Novel anthracycline-spacer-beta-glucuronide,-beta-glucoside, and -beta-galactoside prodrugs for application in selective chemotherapy.

A series of anthracycline prodrugs containing an immolative spacer was synthesized for application in selective chemotherapy. The prodrugs having the general structure anthracycline-spacer-beta-glycoside were designed to be activated by beta-glucuronidase or beta-galactosidase. Prodrugs with -chloro, -bromo or -n-hexyl substituents on the spacer were synthesized as well as prodrugs containing a -beta-glucuronyl, -beta-glucosyl or -beta-galactosyl carbamate specifier. The key step in the synthesis of all prodrugs is the highly beta-diastereoselective addition reaction of the anomeric hydroxyl of a glycosyl donor to a spacer isocyanate resulting in the respective beta-glycosyl carbamate pro-moieties. The resulting protected pro-moieties were coupled to an anthracycline. Prodrugs were evaluated with respect to activation rate by the appropriate enzyme and additionally, their IC50 values were determined. Optimal prodrugs in this study were at least 100- to 200-fold less toxic than their corresponding drug in vitro and were activated to the parent drug in a half-life time of approximately 2 h.     

Rapid enzymatic test for phenotypic HIV protease drug resistance

A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid one-step procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIV-infected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for high-throughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (Ki). The Michaelis constant (Km) and the inhibition constant (Ki) of recombinant wild-type protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIV-positive patients scored from 'sensitive' to 'highly resistant' according to mutations detected by genotypic analysis. The measured Ki values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The non-infectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays.     

Peptide mimetic HIV protease inhibitors are ligands for the orphan receptor SXR

The orphan nuclear receptor SXR coordinately regulates drug clearance in response to a wide variety of xenobiotic compounds. This signaling system protects the body from exposure to toxic compounds; however, it can also pose a severe barrier to drug therapy. We now demonstrate that the human immunodeficiency virus (HIV) protease inhibitor ritonavir binds SXR and activates its target genes. This represents an example of a commonly used therapeutic agent that effectively activates SXR. We also show that other protease inhibitors are weaker (saquinavir) or unable to activate SXR (nelfinavir, indinavir) thus defining analogs that fail to induce SXR-regulated clearance pathways. Interestingly, HIV protease inhibitors are distinct from previously known SXR ligands in that they are peptide mimetic compounds. This expands the ligand specificity of SXR to include this unique chemical class whose pharmaceutical significance is expanding. Finally, we show that SXR ligands activate expression of multiple resistance protein 2, a critical regulator of bile flow and biliary drug excretion. These findings have important implications for the role of SXR in regulating drug clearance and hepatic disorders associated with impaired bile flow.     

Simultaneous determination of HIV protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir in human plasma by high-performance liquid chromatography-tandem mass spectrometry

We report a precise and accurate method for simultaneous quantification of protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir in plasma. An internal standard was added to samples prior to protein precipitation with acetonitrile followed by addition of ammonium formate buffer. Analysis was by HPLC-MS/MS. Calibration curves were validated over concentration ranges encompassing both subtherapeutic and potentially 'toxic' drug concentrations. Inter- and intra-assay variation were below 11% and PI recovery was above 87%. The bioanalytical method described is successfully applied to measure PI concentrations obtained from clinical pharmacokinetic studies and routine therapeutic drug monitoring (TDM).     

Active-site mutations in the South african human immunodeficiency virus type 1 subtype C protease have a significant impact on clinical inhibitor binding: kinetic and thermodynamic study

Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Study of active-site mutations (the V82A single mutation and the V82F I84V double mutation) in the less-studied South African HIV type 1 subtype C (C-SA) protease indicated that neither mutation had a significant impact on the proteolytic functioning of the protease. However, the binding affinities of, and inhibition by, saquinavir, ritonavir, indinavir, and nelfinavir were weaker for each variant than for the wild-type protease, with the double mutant exhibiting the most dramatic change. Therefore, our results show that the C-SA V82F I84V double mutation decreased the binding affinities of protease inhibitors to levels significantly lower than that required for effective inhibition.     

Non-active site changes elicit broad-based cross-resistance of the HIV-1 protease to inhibitors.

Three high level, cross-resistant variants of the HIV-1 protease have been analyzed for their ability to bind four protease inhibitors approved by the Food and Drug Administration (saquinavir, ritonavir, indinavir, and nelfinavir) as AIDS therapeutics. The loss in binding energy (DeltaDeltaG(b)) going from the wild-type enzyme to mutant enzymes ranges from 2.5 to 4.4 kcal/mol, 40-65% of which is attributed to amino acid substitutions away from the active site of the protease and not in direct contact with the inhibitor. The data suggest that non-active site changes are collectively a major contributor toward engendering resistance against the protease inhibitor and cannot be ignored when considering cross-resistance issues of drugs against the HIV-1 protease.     

Select HIV protease inhibitors alter bone and fat metabolism ex vivo

Human immunodeficiency virus (HIV) therapies have been associated with alterations in fat metabolism and bone mineral density. This study examined the effects of HIV protease inhibitors (PIs) on bone resorption, bone formation, and adipocyte differentiation using ex vivo cultured osteoclasts, osteoblasts, and adipocytes, respectively. Osteoclast activity, measured using a rat neonatal calvaria assay, increased in the presence of nelfinavir (NFV; 47.2%, p = 0.001), indinavir (34.6%, p = 0.001), saquinavir (24.3%, p = 0.001), or ritonavir (18%, p < 0.01). In contrast, lopinavir (LPV) and amprenavir did not increase osteoclast activity. In human mesenchymal stem cells (hMSCs), the PIs LPV and NFV decreased osteoblast alkaline phosphatase enzyme activity and gene expression significantly (p < 0.05). LPV and NFV diminished calcium deposition and osteoprotegrin expression (p < 0.05), whereas the other PIs investigated did not. Adipogenesis of hMSCs was strongly inhibited by saquinavir and NFV (>50%, p < 0.001) and moderately inhibited by ritonavir and LPV (>40%, p < 0.01). Expression of diacylglycerol transferase, a marker of adipocyte differentiation, decreased in hMSCs treated with NFV. Amprenavir and indinavir did not affect adipogenesis or lipolysis. These results suggest that bone and fat formation in hMSCs of bone marrow may be coordinately down-regulated by some but not all PIs.