Preclinical characterization of the novel HCV NS3 protease inhibitor GS-9256.

BACKGROUND: GS-9256 is an inhibitor of HCV NS3 protease with a macrocyclic structure and novel phosphinic acid pharmacophore. METHODS: Key preclinical properties of GS-9256 including in vitro antiviral activity, cross-resistance and pharmacokinetic properties were investigated in non-human species. RESULTS: In genotype (GT) 1b Huh-luc cells with a replicon encoding luciferase, GS-9256 had a mean 50% effective concentration (EC50) value of 20.0 nM, with minimal cytotoxicity. Antiviral activity was similar in a number of additional GT1b and GT1a replicon cell lines. Similar potency was observed in chimeric replicons encoding the NS3 protease of GT1 clinical isolates. GS-9256 was less active in GT2a replicon cells (14.2-fold increase in EC50). Additive to synergistic in vitro antiviral activity was observed when GS-9256 was combined with other agents including interferon-α, ribavirin, NS5B polymerase inhibitors GS-6620 and tegobuvir, as well as the NS5A inhibitor ledipasvir. GS-9256 retained wild-type activity against all tested NS5B and NS5A inhibitor resistance mutations. GS-9256 was metabolically stable in microsomes and hepatocytes of tested species, including rodents, dogs and humans. GS-9256 had high bioavailability in mice (near 100%) and moderate bioavailability in rats (14%), dogs (21%) and monkeys (14%). Elimination half-lives were approximately 2 h in mice, 0.6 h in rats, 5 h in dogs and 4 h in monkey. A study in bile duct-cannulated rats indicated that the major route of elimination is through biliary excretion of unmetabolized GS-9256. CONCLUSIONS: GS-9256 showed a favourable preclinical profile supportive of clinical development for the treatment of chronic HCV infection in GT1 patients.

[Effects of Huanglian ointment on wound healing of mice with full-thickness skin defect and the related mechanism].

Objective: To observe the effects of Huanglian ointment on wound healing of mice with full-thickness skin defect, and to explore the related mechanism. Methods: Thirty male C57BL/6J mice were divided into Huanglian ointment group and vehicle group according to the random number table after round wounds of full-thickness skin defect with diameter of 7.5 mm were inflicted on the back of each mouse, with 15 mice in each group. Wounds of mice in Huanglian ointment group and vehicle group were treated with Huanglian ointment and vehicle respectively from post injury day (PID) 1 on, 2 times each day. Five mice from each group were selected to observe wound changes on PID 0, 3, 7, 10, and 14, and wound healing rates were calculated. Five mice out of the 10 mice that hadn't been used for general observation in each group were sacrificed on PID 3 and 7 respectively, and 5 mice after being used for general observation in each group were sacrificed on PID 14. Wound and skin tissue within 2 mm from the edge of wound was collected. Histologic scoring was conducted based on the histomorphological observation with HE staining. The expression of double positive cells of alpha smooth muscle actin (α-SMA) and Ki-67 (myofibroblast) in tissue of wounds of mice was observed by immunofluorescence staining. Protein expressions of transforming growth factor beta (TGF-ß) and collagen in tissue of wounds of mice were determined by enzyme-linked immunosorbent assay. Data were processed with analysis of variance for repeated measurement, analysis of variance of factorial design, t test of two independent samples, one-way analysis of variance, and Bonferronni test or correction. Results: (1) Wounds of mice in two groups were red and swollen on PID 0, while they were neither red nor swollen with scabs on PID 3 and 7. On PID 10, woundsof mice in Huanglian ointment group contracted obviously, while the contracted wounds of mice in vehicle group were smaller than those in Huanglian ointment group. On PID 14, wounds of most mice in Huanglian ointment group were healed, while wounds of some mice in vehicle group failed to heal. Wound healing rates of mice in two groups were close on PID 3 and 7 (with t values respectively 0.64 and 1.90, P values above 0.05). Wound healing rates of mice in Huanglian ointment group on PID 10 and 14 were (76±7)% and (93±5)% respectively, significantly higher than those of vehicle group [(48±9)% and (68±11)%, with t values respectively 7.44 and 3.89, P values below 0.01]. Wound healing rates of mice in two groups on PID 7, 10, and 14 were significantly higher than those on the previous time points of the same group (with P values below 0.01). (2) Histologic scores of wounds of mice in two groups were close on PID 3 (t=-0.76, P>0.05). Histologic scores of wounds of mice in Huanglian ointment group on PID 7 and 14 were (7.0±1.6) and (11.6±2.1) points respectively, significantly higher than those of vehicle group [(4.2±1.3) and (7.2±1.3) points, with t values respectively 1.96 and 2.50, P<0.05 or P<0.01]. Histologic scores of wounds of mice in two groups on PID 7 and 14 were significantly higher than those on the previous time points of the same group (with P values below 0.01). (3) Percentages of double positive cells of α-SMA and Ki-67 in tissue of wounds of mice in Huanglian ointment group on PID 3 and 7 were (35±12)% and (62±10)% respectively, significantly higher than those of vehicle group [(17±12)% and (34±6)%, with t values respectively -2.48 and -5.25, P<0.05 or P<0.01]. The percentage of double positive cells of α-SMA and Ki-67 in tissue of wounds of mice in Huanglian ointment group on PID 14 was (25±5)%, significantly lower than that of vehicle group [(44±17)%, t=2.50, P<0.05]. The percentage of double positive cells of α-SMA and Ki-67 in tissue of wounds of mice on PID 7 was significantly higher than that on PID 3 or 14 in Huanglian ointment group (with P values below 0.01). Percentages of double positive cells of α-SMA and Ki-67 in tissue of wounds of mice on PID 7 and 14 were significantly higher than those on the previous time points in vehicle group (with P values below 0.05). (4) Protein expressions of TGF-ß in tissue of wounds of mice in Huanglian ointment group on PID 3 and 7 were (396±45) and (722±96) pg/mL respectively, significantly higher than those of vehicle group [(290±42) and (382±62) pg/mL, with t values respectively -8.17 and -6.65, P values below 0.01]. Protein expressions of TGF-ß in tissue of wounds of mice in two groups were close on PID 14 (t=1.60, P>0.05). The protein expression of TGF-ß in tissue of wounds of mice in Huanglian ointment group on PID 7 was significantly higher than that on PID 3 or 14 (with P values below 0.01). Protein expressions of TGF-ß in tissue of wounds of mice in vehicle group on PID 7 and 14 were significantly higher than those on the previous time points (with P values below 0.05). Protein expressions of collagen in tissue of wounds of mice in two groups were close on PID 3 (t=1.99, P>0.05). Protein expressions of collagen in tissue of wounds of mice in Huanglian ointment on PID 7 and 14 were (47±10) and (70±14) ng/mL respectively, significantly higher than those of vehicle group [(34±10) and (42±12) ng/mL, with t values respectively 3.15 and 3.52, P<0.05 or P<0.01]. Protein expressions of collagen in tissue of wounds of mice in two groups on PID 7 and 14 were significantly higher than those on the previous time points of the same group (P<0.05 or P<0.01). Conclusions: Huanglian ointment can promote wound healing of full-thickness skin defect of mice through increasing production of myofibroblasts and protein expressions of TGF-ß and collagen.

Preclinical characterization of the novel hepatitis C virus NS3 protease inhibitor GS-9451.

GS-9451 is a selective hepatitis C virus (HCV) NS3 protease inhibitor in development for the treatment of genotype 1 (GT1) HCV infection. Key preclinical properties of GS-9451, including in vitro antiviral activity, selectivity, cross-resistance, and combination activity, as well as pharmacokinetic properties, were determined. In multiple GT1a and GT1b replicon cell lines, GS-9451 had mean 50% effective concentrations (EC50s) of 13 and 5.4 nM, respectively, with minimal cytotoxicity; similar potency was observed in chimeric replicons encoding the NS3 protease gene of GT1 clinical isolates. GS-9451 was less active in GT2a replicon cells (EC50 = 316 nM). Additive to synergistic in vitro antiviral activity was observed when GS-9451 was combined with other agents, including alpha interferon, ribavirin, and the polymerase inhibitors GS-6620 and tegobuvir (GS-9190), as well as the NS5A inhibitor ledipasvir (GS-5885). GS-9451 retained wild-type activity against multiple classes of NS5B and NS5A inhibitor resistance mutations. GS-9451 was stable in hepatic microsomes and hepatocytes from human and three other tested species. Systemic clearance was low in dogs and monkeys but high in rats. GS-9451 showed good oral bioavailability in all three species tested. In rats, GS-9451 levels were ∼40-fold higher in liver than plasma after intravenous dosing, and elimination of GS-9451 was primarily through biliary excretion. Together, these results are consistent with the antiviral activity observed in a recent phase 1b study. The results of in vitro cross-resistance and combination antiviral assays support the ongoing development of GS-9451 in combination with other agents for the treatment of chronic HCV infection.

Neurogenesis and identification of developing layers in the visual cortex of the wallaby (Macropus eugenii).

Birthdates of the neurons that comprise the layers of the mature visual cortex in the wallaby (Macropus eugenii) have been determined with the aid of tritiated thymidine autoradiography. The laminar positions of cells, identified by their birthdates, have then been followed at early stages during development and compared with previously published data on the distribution of thalamocortical afferents and corticothalamic projecting cells (Sheng et al. [1991] J. Comp. Neurol. 307:17-38). Neurons are born in a deep to superficial sequence typical of other mammals. The loosely packed zone of cells, which develops at the base of the thin compact zone of cells at the superficial margin of the cortical plate early in development, was identified as being part of the cortical plate. Afferents did not wait below this zone but grew into the developing cortical layers immediately after the cells that form these layers began accumulating in the loosely packed zone, starting with layer 6 on postnatal day 22 (P22). The genesis of layer 4 did not begin until P32, and these cells reached the superficial cortical plate at P54 and entered the loosely packed zone by P65. Cells of layers 5 and 6 formed the initial projection to the thalamus. Despite the protracted development of the wallaby and the large discrepancy between the time of thalamic ingrowth and genesis of layer 4, there was no extended waiting period for afferents in the subplate.

Development of the laminar distribution of thalamocortical axons and corticothalamic cell bodies in the visual cortex of the wallaby.

The distribution of afferents from the dorsal lateral geniculate nucleus (LGNd) and the lateral posterior nucleus (LP) and of cell bodies projecting to these nuclei has been studied in the visual cortex of the wallaby (Macropus eugenii) throughout development to determine how the characteristic laminar distribution of afferents and efferents of the mature cortex is achieved. Young are born after 26-28 days of gestation and do not open their eyes until around 140 days after birth. Horseradish peroxidase conjugated to wheatgerm agglutinin was injected in the visual thalamus in adults and in pouch young aged from 22 days after birth, just after thalamic axons first reach the visual cortex, to 118 days, when cortical lamination resembles the adult. From 22 to 65 days, the developing visual cortex consists of a marginal zone (MZ), cortical plate (CP), and intermediate zone (IZ) including the superficial subplate (SP), subventricular zone, and ventricular zone. There is a thin compact cell zone (CCZ) at the top of the CP and below it a less densely packed region that increases in thickness with age. Retrogradely labelled cells in two bands were first seen at 40 days, one in the CCZ and the other at the base of the CP. Two bands of cells were seen at all subsequent times if the injection covered both LGNd and LP, and by 76 days, these cells were located within cytoarchitectonically recognizable layers V and VI. Anterograde label prior to 45 days was distributed densely and evenly throughout the IZ and the CP up to the CCZ. Label in MZ was first seen at 25 days and was substantial by 54 days. Anterograde label than became gradually reduced in the IZ, whereas in the CP it remained evenly and densely distributed until 82 days. At this age, coincident with the emergence of layer IV, label within the CP first showed variations in density and by 99 days was concentrated over layer IV and, to a lesser extent, over layer VI. By 118 days label resembled the adult after injections covering both LGNd and LP, with label concentrated in layer I, IV, and VI with a less dense projection to lower layer III and upper layer V. There is a relatively earlier initial ingrowth of axons into the visual cortex in the wallaby and throughout development thalamocortical axons appear to be more widely distributed in the depth of the visual cortex than has been demonstrated for placental mammals.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of connections to and from the visual cortex in the wallaby (Macropus eugenii).

The time course of the development of connections between the visual cortex and the main subcortical visual structures, as well as intrahemispheric and interhemispheric connections, has been studied in the marsupial wallaby (Macropus eugenii) to compare its development with that of placental mammals. Pouch young are born prior to retinal innervation of the primary visual centers and spend a protracted period of development in the pouch, making them ideal for visual, developmental studies. Horseradish peroxidase conjugated to wheatgerm agglutinin was injected into either the presumptive visual cortex or the superior colliculus in young of varying ages. Thalamocortical projections from the dorsal lateral geniculate and lateral posterior nuclei reach the presumptive visual cortex between 12 and 15 days after birth. Descending cortical connections form later. Corticogeniculate axons are first detected in the geniculate and lateral posterior nucleus at 48 days after birth, while corticocollicular axons first reach the superior colliculus at 71 days and, by 81 days, have innervated the superficial layers. Intrahemispheric and interhemispheric connections form even later. By 99 days intrahemispheric axons from area 17 have accumulated in visual association areas but are yet to invade layers III and IV, their major termination zones in adult, while axons projecting back to area 17 have also reached their target area. At this time interhemispheric axons from area 17 have begun to accumulate in the opposite visual cortex, although they have not invaded the cortical layers. By 111 days cortical cells projecting to the opposite visual cortex are first labelled. These have a more widespread distribution in area 17 at 111 and 122 days compared to the adult, where they are confined to the 17/18 border. The results show that the marsupial wallaby has a timetable of similar sequence, but different relative timing, in the formation of cortical connections compared to that of placental mammals. In the first half of the period between conception and eye opening, the timing in the wallaby precedes considerably that in placental mammals. Ascending connections from the thalamus develop relatively earlier in the wallaby but descending collicular connections are delayed until the same relative time that they appear in placental mammals.