ABSTRACT
Solvent-detergent treatment, although used routinely in plasma product processing to inactivate enveloped viruses, substantially reduces product yield from the human plasma resource. To improve yields in plasma product manufacturing, a new viral reduction process has been developed using the fatty acid caprylate. As licensure of plasma products warrants thorough evaluation of pathogen reduction capabilities, the present study examined susceptibility of enveloped viruses to inactivation by caprylate in protein solutions with varied pH and temperature. In the immunoglobin-rich solutions from Cohn Fraction II+III, human immunodeficiency virus, Type-1, bovine viral diarrhea virus (BVDV), and pseudorabies virus were inactivated by caprylate concentrations of >/=9 mM, >/=12 mM, and >/=9 mM, respectively. Compared to solvent-detergent treatment, BVDV inactivation in Fraction II+III solution was significantly faster (20-60 fold) using 16 mM caprylate. Caprylate-mediated inactivation of BVDV was not noticeably affected by temperature within the range chosen manufacturing the immunoglobulin product. In Fraction II+III solutions, IgG solubility was unaffected by =19 mM caprylate. In albumin solution from Cohn supernatant IV-1, 40 mM caprylate rapidly inactivated BVDV, demonstrating versatility in inactivating enveloped viruses potentially present in other protein solutions. Our data show that caprylate is a robust enveloped virus inactivating agent for immunoglobulins and albumin which may potentially be utilized for other proteins; viral inactivation was not adversely affected by protein content and the buffer composition conditions evaluated. Within the parameters examined, caprylate inactivation of enveloped viruses provided comparable activity or advantages relative to the current, standard solvent-detergent treatment.
Subject(s)
Caprylates/pharmacology , Detergents/pharmacology , Sterilization/methods , Virus Inactivation , Viruses/isolation & purification , Albumins/metabolism , Blood-Borne Pathogens , Chromatography, Gas , Chromatography, Ion Exchange , HIV-1/isolation & purification , Hydrogen-Ion Concentration , Immunoglobulin A/blood , Immunoglobulin A/metabolism , Immunoglobulin G/blood , Immunoglobulin M/blood , Kinetics , Lipids/chemistry , Nephelometry and Turbidimetry , Sodium Cholate/pharmacology , Solvents/pharmacology , Temperature , Time Factors , Virus Diseases/prevention & controlABSTRACT
The valine at position 82 (Val 82) in the active site of the human immunodeficiency virus (HIV) protease mutates in response to therapy with the protease inhibitor ritonavir. By using the X-ray crystal structure of the complex of HIV protease and ritonavir, the potent protease inhibitor ABT-378, which has a diminished interaction with Val 82, was designed. ABT-378 potently inhibited wild-type and mutant HIV protease (Ki = 1.3 to 3.6 pM), blocked the replication of laboratory and clinical strains of HIV type 1 (50% effective concentration [EC50], 0.006 to 0.017 microM), and maintained high potency against mutant HIV selected by ritonavir in vivo (EC50, =0. 06 microM). The metabolism of ABT-378 was strongly inhibited by ritonavir in vitro. Consequently, following concomitant oral administration of ABT-378 and ritonavir, the concentrations of ABT-378 in rat, dog, and monkey plasma exceeded the in vitro antiviral EC50 in the presence of human serum by >50-fold after 8 h. In healthy human volunteers, coadministration of a single 400-mg dose of ABT-378 with 50 mg of ritonavir enhanced the area under the concentration curve of ABT-378 in plasma by 77-fold over that observed after dosing with ABT-378 alone, and mean concentrations of ABT-378 exceeded the EC50 for >24 h. These results demonstrate the potential utility of ABT-378 as a therapeutic intervention against AIDS.
Subject(s)
Anti-HIV Agents/pharmacology , HIV Protease Inhibitors/pharmacology , Pyrimidinones/pharmacology , Animals , Anti-HIV Agents/metabolism , Anti-HIV Agents/pharmacokinetics , Area Under Curve , Crystallography, X-Ray , Dogs , Drug Interactions , Female , HIV Protease/chemistry , HIV Protease Inhibitors/metabolism , HIV Protease Inhibitors/pharmacokinetics , HIV-1/drug effects , Humans , In Vitro Techniques , Lopinavir , Macaca fascicularis , Male , Microsomes, Liver/metabolism , Models, Molecular , Pyrimidinones/metabolism , Pyrimidinones/pharmacokinetics , Rats , Rats, Sprague-Dawley , Ritonavir/chemistry , Ritonavir/pharmacologyABSTRACT
The 2-isopropyl thiazolyl group is a highly optimized P3 ligand for C2 symmetry-based HIV protease inhibitors, as exemplified in the drug ritonavir. Here we report that incorporation of this P3 ligand into a piperazine hydroxyethylamine series also yielded novel, highly potent inhibitors. In tissue culture assays, the presence of human serum was less deleterious to the activity of these inhibitors than to that of ritonavir. Furthermore, potent activity against ritonavir resistant HIV was observed.
Subject(s)
HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/pharmacology , Animals , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Blood , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme Inhibitors , HIV/drug effects , HIV Protease Inhibitors/chemical synthesis , Humans , Ligands , Microbial Sensitivity Tests , Microsomes, Liver/enzymology , Mixed Function Oxygenases/antagonists & inhibitors , Piperazines/chemical synthesis , Piperazines/chemistry , Piperazines/pharmacologyABSTRACT
Analysis of the HIV protease gene from the plasma of HIV-infected patients revealed substitutions at nine different codons selected in response to monotherapy with the protease inhibitor ritonavir. Mutants at valine-82, although insufficient to confer resistance, appeared first in most patients. Significant phenotypic resistance required multiple mutations in HIV protease, which emerged subsequently in an ordered, stepwise fashion. The appearance of resistance mutations was delayed in patients with higher plasma levels of ritonavir. Early mutants retained susceptibility to structurally diverse protease inhibitors, suggesting that dual protease inhibitor therapy might increase the duration of viral suppression.
Subject(s)
HIV Protease Inhibitors/pharmacology , HIV Protease/genetics , HIV/drug effects , Mutation , Thiazoles/pharmacology , Valine/analogs & derivatives , Codon , Genotype , HIV/enzymology , HIV/genetics , HIV Infections/blood , Humans , Phenotype , Ritonavir , Valine/genetics , Valine/pharmacologyABSTRACT
The Jurkat-tat cell line, carrying the transactivator (tat) gene of HIV-1 IIIB and thus constitutively expressing the tat protein, has the capacity to support replication of HIV isolates obtained from asymptomatic individuals, so called slow/low (s/l) type virus. A major characteristic of the s/l isolates in vitro is their inability to continuously replicate in cells of CD4+ established lines. In contrast, virus isolates designated rapid/high (r/h) obtained from patients in advanced stages of the HIV-infection do not show this restriction in replicative capacity. To analyze whether introduction of the tat protein into certain cell types or an over-expression of the tat protein would render cells permissive for s/l virus replication, the tat gene was transfected into cells of monocytoid and T cell origin. The resulting cell lines were then tested for their susceptibility to infection with s/l and r/h type HIV-1 isolates. The results conclusively show that mere constitutive expression of the tat protein in established CD4+ cell lines will not provide conditions allowing for continuous replication of s/l type virus. Thus, the Jurkat-tat cell line is a unique cell system for long-term propagation of this type of virus. In addition, it is a suitable system to study virus-host cell interactions and control of virus replication.