No Pharmacokinetic Interaction Between the Hepatitis C Virus Inhibitors Elbasvir/Grazoprevir and Famotidine or Pantoprazole.

Use of agents to suppress gastric acid secretion is common among patients with hepatitis C virus (HCV) infection. The aims of this open-label, three-period, fixed-sequence study were to evaluate the effect of famotidine and pantoprazole on the pharmacokinetics and safety of elbasvir/grazoprevir fixed-dose combination (FDC) in 16 healthy subjects. Elbasvir and grazoprevir each exhibited similar pharmacokinetics following single-dose administration of elbasvir/grazoprevir with or without famotidine or pantoprazole. Geometric mean ratios (GMRs) of grazoprevir AUC(0,∞), Cmax , and C24 (elbasvir/grazoprevir + famotidine or elbasvir/grazoprevir + pantoprazole vs. elbasvir/grazoprevir) ranged from 0.89-1.17. Similarly, GMRs of elbasvir AUC(0,∞), Cmax , and C24 (elbasvir/grazoprevir + famotidine or elbasvir/grazoprevir + pantoprazole vs. elbasvir/grazoprevir) ranged from 1.02-1.11. These results indicate that gastric acid-reducing agents do not modify the pharmacokinetics of elbasvir or grazoprevir in a clinically relevant manner and may be coadministered with elbasvir/grazoprevir in HCV-infected patients without restriction.

Histamine (H2) receptor-adenylate cyclase system in pig skin (epidermis).

Histamine activated adenylate cyclase in pig skin (epidermal) slices, resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of cyclic AMP-phosphodiesterase inhibitors (theophylline, papaverine). A specific H2 receptor inhibitor (metiamide) inhibited the effect of histamine completely, while other antihistamines (diphenhydramine, acetophenazine, perphenazine, fluphenazine, promethazine) inhibited the effect of histamine to various lesser degrees. It has been shown that both epinephrine and prostaglandin E stimulate epidermal adenylate cyclase. Our data using specific blocking agents indicate that histamine, epinephrine and prostaglandin E2 act independently on the epidermal adenylate cyclase system.

Specific refractoriness of adenylate cyclase in skin to epinephrine, prostaglandin E, histamine and AMP.

The cyclic AMP level in pig skin (epidermis) increases markedly after incubation with epinephrine, prostaglandin E, histamine or adenosine 5'-monophosphate. This increase is transient and "spiking" is the consistent response to these four stimulators. The "spiking" is due to a non-responsiveness or refractoriness which develops within minutes and is specific to any one stimulating hormone but not to the others. The addition of inhibitors of protein syntheses did not prevent the development of the refractoriness. Adenylate cyclase and phosphodiesterase activities measured in skin homogenates prepared from skin samples taken before, during and after the "spiking" did not change significantly. The hormone-induced refractoriness in this skin system appears to be due to a specific, localized loss of function of the adenylate cyclase system.

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase.

Adenosine, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phosphodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell surface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides had no effect on the accumulation of cyclic AMP. Among other adenine nucleotides we tested, adenosine 5'-monophosphoramidate, but not adenosine 5'-monosulfate significantly increased cyclic AMP especially with the addition of papaverine. Neither 2'- nor 3'-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.

Multiple forms of cyclic nucleotide phosphodiesterase in pig epidermis.

Pig epidermal cyclic nucleotide phosphodiesterases (EC 3.1.4.16) have been partially purified by DEAE-cellulose column chromatography. At least three different forms of the epidermal phosphodiesterases were identified. They were cyclic GMP-specific, cyclic GMP- and cyclic AMP-hydrolyzing and apparently a cyclic AMP-specific enzyme: the first two forms were soluble and the last was the particulate enzyme. The cyclic GMP-specific soluble fraction had a relatively low Km, the cyclic GMP- and cyclic AMP-hydrolyzing fraction had a high Km for the respective substrates and the third particulate enzyme had both high and low Km values for cyclic AMP. The cyclic GMP-hydrolyzing enzyme was localized almost entirely in the soluble fraction, whereas cyclic AMP-hydrolyzing enzyme was distributed to both soluble and particulate fractions. Thus, our studies show that the multiple forms of pig epidermal enzyme differ distinctly in their substrate affinity, specificity and subcellular distribution.

Inhibitors of epidermal cell DNA synthesis in surviving pig skin in vitro.

Keratome slices of domestic pig skin were used to study the DNA synthesis phase of epidermal cell DNA synthesis. Cyclic AMP and agents which elevate intracellular concentrations of cyclic AMP have no direct effect on the "S" phase of DNA synthesis. Theophylline, isobutylmethylxanthine, and adenosine inhibit DNA synthesis immediately by a mechanism which is reversible and is not dependent on cyclic AMP. This inhibition is not associated with an increase in intracellular thymidine phosphates. Hydroxyurea, however, inhibits DNA synthesis immediately and does produce an elevated pool of thymidine phosphates.

A combined alkali extraction--ethidium bromide technique for the measurement of DNA in small pieces of tissue.

Alkaline solutions (0.1--0.5 N NaOH) at elevated temperatures can be used to extract DNA from small pieces of tissue. RNA is destroyed by the treatment. In tissues which have been previously exposed to tritiated thymidine, aliquots from the extracting solution can be used directly for the determination of DNA by ethidium bromide fluorescence and radioactivity in both DNA and the nucleotide pool.

Epidermal cyclic GMP is increased in psoriasis lesions.

Cyclic GMP levels in epidermis of normal subjects and of psoriatic patients were measured with a highly sensitive radioimmunoassay method. Technical improvements for the assay are 2-fold: (1) skin samples were frozen in vivo before biopsy and local injection of any anesthetic was avoided to overcome ischemia effect which could lower cyclic GMP artificially; (2) epidermis was microdissected to avoid contamination of dermis and keratin layers. The results show that on a per mg tissue dry weight basis the cyclic GMP levels are about 200 fmol in the involved lesional epidermis and 70 fmol in the uninvolved or normal epidermis. Similarly increases in the cyclic GMP levels in the lesional epidermis are observed when the data are expressed either on a DNA or protein basis. The cyclic GMP level in normal epidermis from nonpsoriatic subjects is the same as that in the uninvolved epidermis of psoriasis patients.

Epinephrine activation of pig skin adenylate cyclase in vivo and subsequent refractoriness to activation.

Epinephrine injected intradermally activated pig skin adenylate cyclase and increased the epidermal cyclic AMP level in vivo. This biphasic response reached a maximum in 5 min and gradually decreased thereafter. The simultaneous injection of a cyclic AMP phosphodiesterase inhibitor, isobutyl methyl xanthin (IBMX) potentiated the increase. The simultaneous injection of a specific beta-adrenergic receptor inhibitor, propranolol, inhibited this accumulation of cyclic AMP. After the first activation by epinephrine in vivo, there was a marked refractoriness of the skin (epidermal) adenylate cyclase to subsequent epinephrine stimulation vivo and in vitro. This refractoriness was specific for catecholamine stimulation as responses to histamine were normal. Recovery from refractoriness started at 48 hr and was completed at 1 week after the injection of epinephrine.

Cyclic nucleotide-phosphodiesterase in the uninvolved and involved skin of psoriasis.

In the present study we have compared cyclic nucleotide-phosphodiesterase activities and affinity of phosphodiesterase for substrates (Km) in enzyme preparations obtained from the involved and uninvolved skin of psoriatic patients. With crude skin homogenates we consistently obtained two Km values (high and low) for both the involved and uninvolved, and both Km values were nearly identical between the involved and uninvolved. The same conclusion is also drawn from the Km determinations with partially purified preparations. Cyclic AMP-phosphodiesterase activities with crude homogenates showed no statistically significant differences between the involved and the uninvolved skin. However, when cyclic AMP- and cyclic GMP-phosphodiesterase activities were compared with a highly sensitive assay method in "pure" epidermal samples, which were microdissected free from stratum corneum, dermis and skin appendages, the involved skin contained 40% more activity of the low Km enzyme and 100% more of the high Km enzyme of both cyclic AMP- and cyclic GMP-phosphodiesterase. It is suggested that this may be due to a higher proportion of germinative cells in the lesional epidermis.

Cyclic AMP accumulation in psoriatic skin: differential responses to histamine, AMP, and einephrine by the uninvolved and involved epidermis.

Using the uninvolved and involved skin from psoriatic patients, we investigated the effects of histamine and AMP (or adenosine) in vitro on the intracellular cyclic AMP levels. Both agents activated adenylate cyclase of the uninvolved and involved resulting in the accumulation of cyclic AMP. Without a cyclic nucleotide phosphodiesterase (PDE) inhibitor, these responses were biphasic and the maximal accumulation was observed in 5 min. With the PDE inhibitor both responses were markedly potentiated and high levels of cyclic AMP were observed for more than 20 min. The response to histamine by the involved skin was much greater than that by the uninvolved. The degree of the response to adenosine was approximately equal. In accordance with our previous work, the response to epinephrine by the involved skin was much less than that by the uninvolved. Thus adenylate cyclases of involved skin from psoriatic patients exhibit a markedly diminished response to epinephrine while at the same time exhibiting a markedly enhanced response to histamine. This precludes the possibility that the unresponsiveness to epinephrine can be due to a generalized inability of the epidermal psoriatic plaque cell to make a functioning cell membrane.

Cyclic GMP System in epidermis: I. Effect of ischemia.

When keratome-sliced pig epidermis was floated on Hank's balanced salt solution, we observed a rapid decrease in the intracellular level of cyclic GMP. A portion of the lost cyclic GMP was detected in the incubation medium. When the epidermis was kept in air at room temperature, the cyclic GMP level also decreased rapidly but to a lesser degree. Incubating the epidermal slice at 37 degrees C in Hank's balanced salt solution with the addition of 3-isobutyl-1-methyl xanthine (IBMX) prevented the decrease. Also, after the cyclic GMP level had fallen, it could be raised to be the in vitro level by the addition of IBMX. Increased amounts of cyclic GMP were detectable in the medium in this case. These data indicate that the decrease in cyclic GMP in ischemic epidermis is due to sudden activation of epidermal cyclic GMP-phosphodiesterase and also in part due to leakage of cyclic GMP extracellularly. In contrast to the rapid decline in the cyclic GMP level, ischemia caused a rapid and transient increase in epidermal cyclic AMP. This confirms previous data by ourselves and by others (Br J Dermatol 92: 249-254, 1975; J Invest Dermatol 68:125-127, 1977). These "ischemic effects" must be avoided in order to measure the "in vivo level" of cyclic nucleotides in epidermis.

Adenylate cyclase activation by cholera toxin in pig epidermis: an obligatory role of the GTP-regulatory protein.

Cholera toxin (CT) stimulates the epidermal adenylate cyclase system in vitro. This stimulation was demonstrated in the skin (slice) floating system and the homogenate (membrane) assay system. With the floating system, the addition of CT to the incubation medium caused a marked accumulation of cAMP intracellularly, which was both dose- and time-dependent. A 1-h lag time was present before activation started. Pretreatment of the skin with CT changed the nature of the stimulatory effect caused by epinephrine and histamine, i.e., the transient accumulation of cAMP (a peak at 5 min and subsequent decrease) was no longer observed but the stimulation became persistent. With the membrane assay system in which the receptor components had been uncoupled, adenylate cyclase activities were markedly stimulated by CT (with guanosine-5'-triphosphate, GTP), guanylyl-beta,gamma-imidodiphosphate (GTP-analog, Gpp[NH]p), or sodium fluoride. The stimulation was both dose- and time-dependent without an initial time lag. Either CT or Gpp[NH]p could fully activate adenylate cyclase, and the simultaneous addition of both did not cause further additive stimulation. These data are consistent with the view that the GTP-regulatory protein plays a key role in the activation of adenylate cyclase, and that CT both activates the catalytic unit and modifies the response to receptor hormones through its action on this protein.

Epidermal cyclic AMP is not decreased in psoriasis lesions.

The cyclic AMP level in epidermis of psoriatic patients was reappraised with a highly sensitive radioimmunoassay method in conjunction with an improved skin biopsy technique to avoid any artificial rise of cyclic AMP due to ischemia. Local intradermal injection before biopsy was avoided, since even saline injection caused a clear-cut ischemia effect. The results concur with our previous study: i.e., on a tissue dry weight or protein basis, the cyclic AMP level in the involved epidermis is 20% higher than that in the uninvolved spidermis of psoriatic patients, and on a DNA basis, there was no significant difference. The cyclic AMP level in normal epidermis from non-psoriatic subjects is the same as that in the uninvolved epidermis of psoriatic patients. Such characteristics in psoriatic lesions as the increased mitosis, incomplete, differentiation and increased glycogen content cannot be simply related to a cyclic AMP deficiency.

The effects of epidermal growth factor on the cyclic nucleotide system in pig epidermis.

Incubation of pig skin slices with epidermal growth factor (EGF) caused an increase in intracellular cyclic GMP concentration. A significant increase was found after 1 hr of incubation and reached a peak by 6 hr. EGF caused no change in the cyclic AMP level nor did it affect epinephrine-induced cyclic AMP responses.

Stimulation of protein phosphorylation by epidermal growth factor in pig skin (epidermis).

Incorporation of 32P-orthophosphoric acid into pig epidermal proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The incorporation was observed in 2 major and 2 minor peaks with approximate molecular weights of 52,000 and 27,000, 17,000, and 12,000 respectively. Treatment with epidermal growth factor (EGF) consistently increased 32P incorporation into all 4 peaks. The stimulation was both time- and dose-dependent, and was not inhibited by the simultaneous addition of cycloheximide. In our previous study (submitted to J Invest Dermatol), we observed that EGF increased the cyclic GMP level in the same pig skin (epidermal) system. The possibility that the increased cyclic GMP leads to the protein phosphorylation observed in the present study was considered but ruled out because (1) the addition of cyclic GMP does not mimic the EGF treatment, (2) simultaneous addition of EGF and cyclic GMP stimulate phosphorylation to the same degree as the addition of EGF alone and (3) EGF stimulates phosphorylation in 20 min whereas the EGF induced increase in the cyclic GMP level takes 60 min.

Epidermal adenylate cyclase systems: the retention of hormone responsiveness after enzymatic separation of pure epidermis.

Although it has been shown that keratome-sliced skin contains active adenylate cyclase systems which respond to various hormones and drugs, unequivocal proof that the epidermis contains these hormone-responsive systems is still lacking. We demonstrate in this study that "pure" epidermis obtained after either collagenase or trypsin treatment does contain the hormone-sensitive adenylate cyclase systems.

Epidermal adenylate cyclase: stimulation of the histamine (H2) receptor by tolazoline.

Tolazoline (2-benzyl-2-imidazoline) activated adenylate cyclase in pig epidermal slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phosphodiesterase inhibitor, theophylline. Specific histamine (H2) receptor inhibitors (metiamide and cimetidine) completely blocked the tolazoline activation of adenylate cylase. At low concentrations (10-100 micrometer), a histamine (H1) receptor inhibitor (diphenhydramine) and a beta-adrenergic blocker (propranolol) did not inhibit this effect. The stimulation of cyclic AMP formation by the combination of tolazoline and histamine was about the same as the stimulation by histamine alone (nonadditive), whereas the stimulatory effects by tolazoline and epinephrine were additive. These data suggest that tolazoline, an alpha-adrenergic blocker, also activates adenylate cyclase at the histamine (H2) receptor site which is distinct from the beta-adrenergic receptor site. Another alpha-adrenergic blocker, phentolamine, did not have this effect.

The effect of histamine on epidermal outgrowth: its possible dual role as an inhibitor and stimulator.

We have investigated the effect of histamine on pig epidermal cell outgrowths in vitro. Histamine inhibited the epidermal cell outgrowths (and also mitosis). This inhibition was partially counteracted by a specific H2 antagonist, cimetidine. Inhibition was maximal at a histamine concentration of 10(-4) M and was less at 10(-3) M. These histamine concentrations respectively coincide with the optimal concentrations for accumulating intracellular cyclic AMP (via H2 receptors) and cyclic GMP (via H1 receptors) in the same pig epidermal slice system. 4-Methyl-histamine, a pure H2 agonist, which only increased the intracellular cyclic AMP level but not the cyclic GMP level, caused a maximal outgrowth inhibition at 10(-3) M. Attempts to counteract the histamine effects due to cyclic GMP accumulation by various H1 antagonists (so that 10(-3) M histamine would have caused maximal outgrowth inhibition) were unsuccessful, since the addition of each H1 antagonist alone strongly inhibited the outgrowth. These data strongly suggest a dual role of histamine through the cyclic nucleotide system; i.e., histamine inhibits epidermal cell growth by elevating the intracellular cyclic AMP level via an H2 receptor, while histamine at high concentrations (10(-3) M) partially counteracts the inhibition by increasing cyclic GMP via an H1 receptor.