Assessment of drug-drug interactions between daclatasvir and methadone or buprenorphine-naloxone.

Hepatitis C virus (HCV) infection is common among people who inject drugs, including those managed with maintenance opioids. Pharmacokinetic interactions between opioids and emerging oral HCV antivirals merit evaluation. Daclatasvir is a potent pangenotypic inhibitor of the HCV NS5A replication complex recently approved for HCV treatment in Europe and Japan in combination with other antivirals. The effect of steady-state daclatasvir (60 mg daily) on stable plasma exposure to oral opioids was assessed in non-HCV-infected subjects receiving methadone (40 to 120 mg; n = 14) or buprenorphine plus naloxone (8 to 24 mg plus 2 to 6 mg; n = 11). No relevant interaction was inferred if the 90% confidence interval (CI) of the geometric mean ratio (GMR) of opioid area under the plasma concentration-time curve over the dosing interval (AUCτ) or maximum concentration in plasma (C max) with versus without daclatasvir was within literature-derived ranges of 0.7 to 1.43 (R- and S-methadone) or 0.5 to 2.0 (buprenorphine and norbuprenorphine). Dose-normalized AUCτ for R-methadone (GMR, 1.08; 90% CI, 0.94 to 1.24), S-methadone (1.13; 0.99 to 1.30), and buprenorphine (GMR, 1.37; 90% CI, 1.24 to 1.52) were within the no-effect range. The norbuprenorphine AUCτ was slightly elevated in the primary analysis (GMR, 1.62; 90% CI, 1.30 to 2.02) but within the no-effect range in a supplementary analysis of all evaluable subjects. Dose-normalized C max for both methadone enantiomers, buprenorphine and norbuprenorphine, were within the no-effect range. Standardized assessments of opioid pharmacodynamics were unchanged throughout daclatasvir administration with methadone or buprenorphine. Daclatasvir pharmacokinetics were similar to historical data. Coadministration of daclatasvir and opioids was generally well tolerated. In conclusion, these data suggest that daclatasvir can be administered with buprenorphine or methadone without dose adjustments.

Sofosbuvir and daclatasvir combination therapy in a liver transplant recipient with severe recurrent cholestatic hepatitis C.

Recurrent HCV infection following liver transplantation can lead to accelerated allograft injury that is difficult to treat with interferon. The aim of this study is to describe the first ever use of an interferon-free, all oral regimen in a liver transplant recipient with severe recurrent HCV. A 54-year-old male with HCV genotype 1b developed severe cholestatic HCV at 6 months posttransplant with ascites, AST 503 IU/mL, alkaline phosphatase of 298 IU/mL, HCV RNA of 12 000 000 IU/mL, and histological cholestasis with pericellular fibrosis. Sofosbuvir, an HCV polymerase inhibitor (400 mg/day), and daclatasvir, an HCV NS5A replication complex inhibitor (60 mg/day), were co-administered for 24 weeks. Within 4 weeks of initiating treatment, serum HCV RNA levels became undetectable and liver biochemistries normalized with concomitant resolution of ascites. The patient achieved a sustained virological response with undetectable HCV RNA at 9 months posttreatment. During and following treatment, the daily dose and blood level of tacrolimus remained stable and unchanged. The rapid and sustained suppression of HCV replication in this liver transplant recipient provides great promise for the use of combination oral antiviral regimens in other immunosuppressed and interferon refractory HCV patients.

Synthesis, structure proof, and biological activity of epothilone cyclopropanes.

[structure--see text] A semisynthetic route to epothilone cyclopropanes from epothilones A and B is described. Of significance, the deoxygenation of the 12, 13-epoxide to give the corresponding olefin was achieved with high efficiency. The title compounds (8, 9) were active in both tubulin polymerization and cytotoxicity assays, which is in direct contrast to a previously published report. These results provide further evidence that the role of the 12,13-epoxide of epothilones is largely conformational and argue against some of the current pharmacophore models.

Generation of replication-competent hepatitis B virus nucleocapsids in insect cells.

The double-stranded DNA genome of human hepatitis B virus (HBV) and related hepadnaviruses is reverse transcribed from a pregenomic RNA by a viral polymerase (Pol) harboring both priming and RNA- and DNA-dependent elongation activities. Although hepadnavirus replication occurs inside viral nucleocapsids, or cores, biochemical systems for analyzing this reaction are currently limited to unencapsidated Pols expressed in heterologous systems. Here, we describe cis and trans classes of replicative HBV cores, produced in the recombinant baculovirus system via coexpression of HBV core and Pol proteins from either a single RNA (i.e., in cis) or two distinct RNAs (in trans). Upon isolation from insect cells, cis and trans cores contained Pol-linked HBV minus-strand DNA with 5' ends mapping to the authentic elongation origin DR1 and also plus-strand DNA species. Only trans cores, however, were highly active for the de novo priming and reverse transcription of authentic HBV minus strands in in vitro endogenous polymerase assays. This reaction strictly required HBV Pol but not the epsilon stem-loop element, although the presence of one epsilon, or better, two epsilons, enhanced minus-strand synthesis up to 10-fold. Compared to unencapsidated Pol enzymes, encapsidated Pol appeared to be (i) highly processive, able to extend minus-strand DNAs of 400 nucleotides from DR1 in vitro, and (ii) more active for HBV plus-strand synthesis. These observations suggest possible contributions to the replication process from the HBV core protein. These novel core reagents should facilitate the analysis of HBV replication in its natural environment, the interior of the capsid, and also fuel the development of new anti-HBV drug screens.

A functional interaction of ICP8, the herpes simplex virus single-stranded DNA-binding protein, and the helicase-primase complex that is dependent on the presence of the UL8 subunit.

The herpes simplex virus type 1 (HSV) single-stranded DNA-binding protein (SSB, ICP8) stimulates the viral DNA polymerase (Pol) on an oligonucleotide-primed single-stranded DNA template. This stimulation is non-specific since other SSBs also increase Pol activity. However, only ICP8 was stimulatory when Pol activity was dependent upon priming by the viral helicase-primase complex. ICP8 also specifically stimulated the primer synthesis and ATPase activities of the helicase-primase. The mechanism of stimulation was different from that of Pol; helicase-primase stimulation required much lower amounts of ICP8 than the amount that saturates the DNA and optimally stimulates Pol. Furthermore, ICP8 did not act by removing secondary structure as stimulation also occurred on homopolymer templates. While the UL8 component of the helicase-primase is not required for enzymatic activities by a subassembly of the UL5 and UL52 proteins, only the holoenzyme (UL5/8/52) was stimulated by ICP8. These results identify a unique, functional interaction between the ICP8 SSB and the helicase-primase complex, mediated by the UL8 subunit.

Establishment of an in vitro assay to characterize hepatitis C virus NS3-4A protease trans-processing activity.

An in vitro cleavage system was established to measure HCV NS3 protease trans-processing activity. This system utilizes purified NS3-4A protein from baculovirus, purified substrates expressed by in vitro transcription and translation and defined buffer components. The 41-residue substrates, 5A/5B and 4A/4B, were processed efficiently in trans by wild-type NS3 but not by a catalytically inactive mutant protease; radiolabel sequencing confirmed that NS3-mediated cleavage occurred at the correct cysteine/serine sites, thereby authenticating this system. Two striking features of this in vitro assay are: (1) analogous 4B/5A and 3/4A substrates cannot be processed in trans under the same conditions, and (2) in vitro cleavage of the 5A/5B and 4A/4B sites is highly dependent on the presence of NS4A, which we show is not the case in vivo.

Characterization of monoclonal antibodies recognizing amino- and carboxy-terminal epitopes of the herpes simplex virus UL42 protein.

A panel of monoclonal antibodies (MAbs) directed against the herpes simplex virus type 1 (HSV-1) DNA polymerase (Pol) accessory protein, UL42, was developed and characterized. Thirteen different MAbs were isolated which exhibited varied affinities for the protein. All MAbs reacted with UL42 in ELISA, Western blot and immunoprecipitation analyses. Competitive ELISA was used to show that 6 different epitopes within UL42 were recognized by the MAbs. Immunoprecipitation of amino- and carboxy-terminal truncations of UL42 mapped the epitopes to regions containing amino acids 1-10, 10-108, 338-402, 402-460, and 460-477. All but one of these epitopes were outside the minimal active portion of the protein previously mapped to amino acids 20-315. None of these MAbs, alone or in combination, specifically neutralized the ability of UL42 to stimulate Pol activity in vitro. These results are consistent with structure-function studies that showed that N- and C-terminal regions of the UL42 protein, those recognized by the MAbs, are not involved in UL42 function in vitro.

Sequence-dependent primer synthesis by the herpes simplex virus helicase-primase complex.

The herpes simplex virus helicase-primase complex, a heterotrimer of the UL5, UL8, and UL52 proteins, displays a single predominant site of primer synthesis on phi X174 virion DNA (Tenney, D. J., Hurlburt, W. W., Micheletti, P. M., Bifano, M., and Hamatake, R. K. (1994) J. Biol. Chem. 269, 5030-5035). This site was mapped and found to be deoxycytosine-rich, directing the synthesis of a primer initiating with several guanine residues. The size and sequence requirements for primer synthesis were determined using oligonucleotides containing variations of the predominant template. Although the efficiency of primer synthesis on oligonucleotides was influenced by template size, it was absolutely dependent on nucleotide sequence. Conversely, the ATPase activity on oligonucleotide templates was dependent on template size rather than nucleotide sequence. Furthermore, only oligonucleotides containing primase templates were inhibitory in a coupled primase-polymerase assay using phi X174 DNA as template, suggesting that primer synthesis or primase turnover is rate-limiting. Additionally, stimulation of helicase-primase by the UL8 component and that by the ICP8 protein were shown to differ mechanistically using different templates: the UL8 component stimulated the rate of primer synthesis on phi X174 virion DNA and oligonucleotide templates, while ICP8 stimulation occurred only on phi X174 virion DNA.

The UL8 component of the herpes simplex virus helicase-primase complex stimulates primer synthesis by a subassembly of the UL5 and UL52 components.

The herpes simplex virus type 1 (HSV) UL5, UL8, and UL52 proteins form a helicase-primase complex in infected cells. Several laboratories have demonstrated that helicase and nucleoside triphosphatase activities of the heterotrimer (UL5/8/52) are indistinguishable from that of a subassembly of UL5 and UL52 (UL5/52). Although the UL5/52 subassembly functions in coupled primase-polymerase assays on homopolymeric templates, its activity on natural DNA templates has been reported to require UL8. To determine the role of UL8 in primase assays, the activity of the UL5/52 subassembly was compared to that of the heterotrimer reconstituted by adding UL8 to UL5/52. We detected significant activity of the UL5/52 subassembly in coupled primase-polymerase and oligoribonucleotide primer synthesis assays on phi X174 and M13 virion DNAs. However the addition of UL8 to UL5/52 stimulated this activity in a dose-dependent manner. We demonstrate that stimulation occurred at the level of primer synthesis. UL8 did not affect the amount or size of primers annealed to template, their utilization by DNA polymerase, or the use of specific initiation sites within the template. In kinetic studies, the rate of primer synthesis was increased by UL8 but the Km for phi X174 DNA template was unchanged. These results suggest that a function of the UL8 component of the HSV helicase-primase complex is to increase the efficiency of primer synthesis by UL5/52.

The herpes simplex virus type 1 DNA polymerase accessory protein, UL42, contains a functional protease-resistant domain.

Herpes simplex virus type 1 encodes its own DNA polymerase (Pol), the product of the UL30 gene, and a polymerase accessory subunit, the product of the UL42 gene, both of which are required for viral DNA replication. Pol and the UL42 protein associate to form a heterodimeric complex (Pol/UL42) which is more active and has a higher processivity than the Pol catalytic subunit alone. The Pol/UL42 complex has been reconstituted by mixing together highly purified Pol and UL42 subunits obtained from recombinant baculovirus-infected cells. We have used polymerase activity on poly(dA):oligo(dT20), a template that the Pol subunit utilizes with low efficiency, to measure the formation of the Pol/UL42 complex. Our data indicate that the association constant for the Pol/UL42 complex is 1 x 10(8) M-1. Proteolytic digestions of UL42 were performed to determine whether structural domains of UL42 could be disclosed by differential amino acid accessibilities. The ability of these protease-resistant domains to form a functional complex with Pol was determined by measuring their ability to stimulate Pol activity on poly(dA):oligo(dT20). We have found that trypsin digestion of UL42 in the presence of DNA generates protease-resistant fragments of 28K and 8K which co-elute from a MonoQ column and are able to stimulate Pol activity on poly(dA):oligo(dT20). Complex formation of the 28K and 8K tryptic fragments with Pol was also shown by their co-immunoprecipitation with antibody to Pol. It was determined that the 28K fragment of UL42 comprised amino acids 1 to 245 or 1 to 254 of UL42, whereas the 8K fragment started at amino acid 255. Thus, controlled proteolysis of UL42 revealed two closely contiguous structural domains that retained the ability to complex with Pol and stimulate Pol activity.

Deletions of the carboxy terminus of herpes simplex virus type 1 UL42 define a conserved amino-terminal functional domain.

The herpes simplex virus type 1 UL42 protein was synthesized in reticulocyte lysates and assayed for activity in vitro. Three functional assays were used to examine the properties of in vitro-synthesized UL42: (i) coimmunoprecipitation to detect stable complex formation with purified herpes simplex virus type 1 DNA polymerase (Pol), (ii) a simple gel-based assay for DNA binding, and (iii) a sensitive assay for the stimulation of Pol activity. UL42 synthesized in reticulocyte lysates formed a stable coimmunoprecipitable complex with Pol, bound to double-stranded DNA, and stimulated the activity of Pol in vitro. Carboxy-terminal truncations of the UL42 protein were synthesized from restriction enzyme-digested UL42 gene templates and gene templates made by polymerase chain reaction and assayed for in vitro activity. Truncations of the 488-amino-acid (aa) UL42 protein to aa 315 did not abolish its ability to bind to Pol and DNA or to stimulate Pol activity. Proteins terminating at aas 314 and 313 showed reduced levels of binding to Pol, but these and shorter proteins were unable to bind to DNA or to stimulate Pol activity. These results suggest that all three of the biochemical functions of UL42 colocalize entirely within the N-terminal 315 aas of the UL42 protein. Amino acid sequence alignment of alpha herpesvirus UL42 homologs revealed that the N-terminal functional domain corresponds to the most highly conserved region of the protein, while the dispensable C terminus is not conserved. Conservative aa changes at the C terminus of the 315-aa truncated protein were used to show that conserved residues were important for activity. These results suggest that 173 aa of UL42 can be deleted without a loss of activity and that DNA-binding and Pol-binding activities are correlated with the ability of UL42 to stimulate Pol activity.

Mutations in the C terminus of herpes simplex virus type 1 DNA polymerase can affect binding and stimulation by its accessory protein UL42 without affecting basal polymerase activity.

We have analyzed the effects of mutations in the herpes simplex virus type 1 DNA polymerase (Pol) C-terminal UL42 binding domain on the activity of Pol and its ability to form complexes with and be stimulated by UL42 in vitro. Wild-type Pol expressed in Saccharomyces cerevisiae was both bound and stimulated by UL42 in vitro. C-terminal truncations of 19 and 40 amino acids (aa) did not affect the ability of Pol to be stimulated by UL42 in vitro. This stimulation as well as basal Pol activity in the presence of UL42 was inhibited by polyclonal anti-UL42 antiserum, thus indicating a physical interaction between Pol and UL42. Removal of the C-terminal 59 aa of Pol and internal deletions of 72 aa within the Pol C terminus eliminated stimulation by UL42. None of the truncations or deletions within Pol affected basal polymerase activity. In contrast with their ability to be stimulated by UL42, only wild-type Pol and Pol lacking the C-terminal 19 aa bound UL42 in a coimmunoprecipitation assay. These results demonstrate that a functional UL42 binding domain of Pol is separable from sequences necessary for basal polymerase activity and that the C-terminal 40 aa of Pol appear to contain a region which modulates the stability of the Pol-UL42 interaction.

Hypothalamic-pituitary somatotropic function in prepubertal hypothyroid rats: effect of growth hormone replacement therapy.

The effect of thyroid hormone deficiency and growth hormone (GH) treatment on hypothalamic GH-releasing hormone (GHRH)/somatostatin (SS) concentrations, GHRH/SS mRNA levels, and plasma GH and somatomedin-C (IGF-I) concentrations were studied in 28- and 35-day-old rats made hypothyroid by giving dams propylthiouracil in the drinking water since the day of parturition. Hypothyroid rats, at both 28 and 35 days of life, had decreased hypothalamic GHRH content and increased GHRH mRNA levels, unaltered SS content and SS mRNA levels, and reduced plasma GH and IGF-I concentrations. Treatment of hypothyroid rats with GH for 14 days completely restored hypothalamic GHRH content and reversed the increase in GHRH mRNA, but did not alter plasma IGF-I concentrations. These data indicate that, in hypothyroid rats, the changes in hypothalamic GHRH content and gene expression are due to the GH deficiency ensuing from the hypothyroid state. Failure of the GH treatment to increase plasma IGF-I indicates that the feedback regulation on GHRH neurons is operated by circulating GH and/or perhaps tissue but not plasma IGF-I concentrations. Presence of low plasma IGF-I concentrations would be directly related to thyroid hormone deficiency.

Changes in the hypothalamic-pituitary somatotropic function of infant hypothyroid rats.

The effects of the perturbation of the pituitary-thyroid axis induced during development on the functional activity of the growth hormone (GH) regulatory neuronal systems, GH-releasing hormone (GHRH), and somatostatin (SS) were studied in 14- and 21-day-old rats made hypothyroid by giving dams propylthiouracil in the drinking water since the day of parturition. Infant hypothyroid rats, both at 14 and 21 days of life, had elevated plasma thyroid-stimulating hormone levels and decreased pituitary and plasma GH levels. Simultaneous determination of hypothalamic GHRH/SS-like immunoreactivity (LI) and GHRH/SS mRNA levels did not reveal any difference in 14-day-old hypothyroid rats when compared with age-matched controls. In contrast, 21-day-old hypothyroid rats had decreased GHRH-LI content and a striking rise in GHRH mRNA levels, whereas SS-LI content and SS gene expression remained unaltered. These data indicate that in infant hypothyroid rats, changes in the functional activity of the GHRH neuronal system occur later than changes in GH secretion and are probably dependent on the GH deficiency. The functional activity of SS neurons was apparently unaltered in these hypothyroid rats, pointing to a lesser sensitivity of this system to the perturbation of the pituitary-thyroid axis.