Pharmacokinetics of diazepam administered intramuscularly by autoinjector versus rectal gel in healthy subjects: a phase I, randomized, open-label, single-dose, crossover, single-centre study.

BACKGROUND AND OBJECTIVE: Acute repetitive seizures (ARS) are a debilitating part of episodic seizure activity that can sometimes progress to status epilepticus. Currently approved treatment that can be administered by non-medical personnel to patients with ARS is a diazepam rectal gel. While effective, rectal administration can be difficult, inconvenient and objectionable. A diazepam autoinjector has been developed to deliver diazepam via an intramuscular (IM) injection. This study evaluated the dose proportionality of the diazepam autoinjector and the consequent diazepam bioavailability relative to an equivalent dose of diazepam administered rectally as a commercial gel. METHODS: This was a phase I, randomized, open-label, two-part, single-dose, crossover, single-centre pharmacokinetic study in 48 healthy young adult (aged 18-40 years) male and female subjects. Part I of the study (n = 24) evaluated the dose proportionality of three strengths of the diazepam autoinjector (5, 10 and 15 mg) administered into the mid-outer thigh via a deep IM injection. Part II (n = 24) assessed the relative bioavailability of the diazepam 10 mg autoinjector versus the diazepam 10 mg rectal gel. Parts I and II were run concurrently. Each subject completed screening up to 30 days prior to three (Part I) or two (Part II) dosing periods. Serial blood sampling for plasma diazepam and desmethyldiazepam (metabolite) concentrations, vital signs and adverse event (AE) assessments were performed at prespecified times. Treatments were separated by a 14-day washout period. RESULTS: In Part I, dose proportionality was demonstrated for the diazepam autoinjector at 5, 10 and 15 mg doses by increases in mean maximum plasma concentration (C(max)), mean area under the plasma concentration-time curve (AUC) from time zero to infinity (AUC(∞)), and mean AUC from time zero to time of last measurable concentration (AUC(last)). The median time to reach C(max) (t(max)) was consistent at 1 hour for each dose. In Part II of the study, IM administration via diazepam autoinjector (10 mg) resulted in plasma concentrations of both diazepam and desmethyldiazepam that were slightly higher and less variable than those observed following administration of diazepam rectal gel (10 mg). The geometric mean ratio (diazepam autoinjector/diazepam rectal gel) and 90% confidence intervals for diazepam C(max) and AUC(last) were 0.94 (0.84, 1.05) and 1.14 (1.08, 1.21), respectively, indicating that the overall bioavailability of the diazepam autoinjector was approximately 14% higher than that of diazepam rectal gel. Both treatments were generally well tolerated. Although the incidence of treatment-emergent AEs was higher with diazepam autoinjector compared with diazepam rectal gel (21.7% vs 13.6%), the difference can be attributed to injection site pain. Injection site pain also correlated with the diazepam autoinjector dose administered in Part I: 5 mg (4.3%), 10 mg (21.7%) and 15 mg (27.3%). However, no patients discontinued the trial due to injection site pain. No other AEs correlated with dose, and there was no evidence of respiratory depression with either administration. CONCLUSION: Results of the present study indicated that diazepam can be safely and reliably administered IM using a diazepam autoinjector.

Multiple Epstein-Barr virus strains in patients with infectious mononucleosis: comparison of ex vivo samples with in vitro isolates by use of heteroduplex tracking assays.

Recent work using a heteroduplex tracking assay (HTA) to identify resident viral sequences has suggested that patients with infectious mononucleosis (IM) who are undergoing primary Epstein-Barr virus (EBV) infection frequently harbor different EBV strains. Here, we examine samples from patients with IM by use of a new Epstein-Barr nuclear antigen 2 HTA alongside the established latent membrane protein 1 HTA. Coresident allelic sequences were detected in ex vivo blood and throat wash samples from 13 of 14 patients with IM; most patients carried 2 or more type 1 strains, 1 patient carried 2 type 2 strains, and 1 patient carried both virus types. In contrast, coresident strains were detected in only 2 of 14 patients by in vitro B cell transformation, despite screening >20 isolates/patient. We infer that coacquisition of multiple strains is common in patients with IM, although only 1 strain tends to be rescued in vitro; whether nonrescued strains are present in low abundance or are transformation defective remains to be determined.

Biology of Epstein-Barr virus during infectious mononucleosis.

Infectious mononucleosis is the clinical manifestation of primary infection with Epstein-Barr virus (EBV). We monitored primary infection during convalescence and during the establishment of persistent infection. The profiles of EBV strains in the oral cavity and in peripheral blood were determined by use of a heteroduplex tracking assay specific for the EBV gene encoding latent membrane protein 1. Multiple EBV strains were detected in most patients and persisted in and were possibly transmitted among 3 distinct compartments of infection, including the oral cavity, peripheral blood lymphocytes, and the cell-free fraction of the blood plasma. We also tracked transmission of multiple strains from an asymptomatic carrier to a patient diagnosed with primary EBV infection. These data reveal that primary EBV infection is complex, with transmission of multiple strains and clear differences in relative abundance of strains in distinct compartments.

Potential selection of LMP1 variants in nasopharyngeal carcinoma.

Seven distinct sequence variants of the Epstein-Barr virus latent membrane protein 1 (LMP1) have been identified by distinguishing amino acid changes in the carboxy-terminal domain. In this study the transmembrane domains are shown to segregate identically with the distinct carboxy-terminal amino acid sequences. Since strains of LMP1 have been shown to differ in abundance between blood and throat washes, nasopharyngeal carcinomas (NPCs) from areas of endemicity and nonendemicity with matching blood were analyzed by using a heteroduplex tracking assay to distinguish LMP1 variants. Striking differences were found between the compartments with the Ch1 strain prevalent in the NPCs from areas of endemicity and nonendemicity and the B958 strain prevalent in the blood of the endemic samples, whereas multiple strains of LMP1 were prevalent in the blood of the nonendemic samples. The possible selection against the B958 strain appearing in the tumor was highly significant (P < 0.0001). Sequence analysis of the full-length LMP1 variants revealed changes in many of the known and computer-predicted HLA-restricted epitopes with changes in key positions in multiple, potential epitopes for the specific HLA of the patients. These amino acid substitutions at key positions in the LMP1 epitopes may result in a reduced cytotoxic-T-lymphocyte response. These data indicate that strains with specific variants of LMP1 are more likely to be found in NPC. The predominance of specific LMP1 variants in NPC could reflect differences in the biologic or molecular properties of the distinct forms of LMP1 or possible immune selection.

Compartmentalization and transmission of multiple epstein-barr virus strains in asymptomatic carriers.

Infection with the Epstein-Barr virus (EBV) is often subclinical in the presence of a healthy immune response; thus, asymptomatic infection is largely uncharacterized. This study analyzed the nature of EBV infection in 20 asymptomatic immunocompetent hosts over time through the identification of EBV strain variants in the peripheral blood and oral cavity. A heteroduplex tracking assay specific for the EBV gene LMP1 precisely identified the presence of multiple EBV strains in each subject. The strains present in the peripheral blood and oral cavity were often completely discordant, indicating the existence of distinct infections, and the strains present and their relative abundance changed considerably between time points. The possible transmission of strains between the oral cavity and peripheral blood compartments could be tracked within subjects, suggesting that reactivation in the oral cavity and subsequent reinfection of B lymphocytes that reenter the periphery contribute to the maintenance of persistence. In addition, distinct virus strains persisted in the oral cavity over many time points, suggesting an important role for epithelial cells in the maintenance of persistence. Asymptomatic individuals without tonsillar tissue, which is believed to be an important source of virus for the oral cavity, also exhibited multiple strains and a cyclic pattern of transmission between compartments. This study revealed that the majority of patients with infectious mononucleosis were infected with multiple strains of EBV that were also compartmentalized, suggesting that primary infection involves the transmission of multiple strains. Both the primary and carrier states of infection with EBV are more complex than previously thought.

Identification of Epstein-Barr virus strain variants in hairy leukoplakia and peripheral blood by use of a heteroduplex tracking assay.

Epstein-Barr virus (EBV) strains can be distinguished by specific sequence variations in the LMP1 gene. In this study, a heteroduplex tracking assay (HTA) specific for LMP1 was developed to precisely identify the prototypic undeleted strain B958, other undeleted strains (Ch2, AL, NC, and Med-), and strains with the 30-bp deletion (Med+ and Ch1). This technique also provides an estimate of the relative abundance of strains in patient samples. In this study, EBV strains were identified in 25 hairy leukoplakia (HLP) biopsies and six matched peripheral blood samples and throat washes with the LMP1-HTA. To investigate the relationship of the virus found in the peripheral blood to that in the HLP lesion, the strain variants in the peripheral blood B lymphocytes and those present within the epithelial cells in the HLP lesion and in throat washes were identified. In many of the subjects, compartmental differences in the EBV strain profiles in the oral cavity and peripheral blood were readily apparent. The throat wash specimens usually had a strain profile similar to that within the corresponding HLP sample, which was distinct from the strain profile detected in the peripheral blood. These analyses reveal that the nature of EBV infection can be very dynamic, with changes in relative strain abundance over time as well as the appearance of new strains. The patterns of abundance in the blood and oral cavity provide evidence for compartmentalization and for the transmission of strains between the blood and oropharynx.