Distribution of lamivudine into lymph node HIV reservoir.

Efficient delivery of antiretroviral agents to lymph nodes is important to decrease the size of the HIV reservoir within the lymphatic system. Lamivudine (3TC) is used in first-line regimens for the treatment of HIV. As a highly hydrophilic small molecule, 3TC is not predicted to associate with chylomicrons and therefore should have negligible uptake into intestinal lymphatics following oral administration. Similarly, negligible amounts of 3TC are predicted to be transported into peripheral lymphatics following subcutaneous (SC) injection due to the faster flow rate of blood in comparison to lymph. In this work, we performed pharmacokinetic and biodistribution studies of 3TC in rats following oral lipid-based, oral lipid-free, SC, and intravenous (IV) administrations. In the oral administration studies, mesenteric lymph nodes (MLNs) had significantly higher 3TC concentrations compared to other lymph nodes, with mean tissue:serum ratios ranging from 1.4 to 2.9. However, cells and chylomicrons found in mesenteric lymph showed low-to-undetectable concentrations. In SC studies, administration-side (right) draining inguinal and popliteal lymph nodes had significantly higher concentrations (tissue:serum ratios as high as 3.2) than corresponding left-side nodes. In IV studies, lymph nodes had lower mean tissue:serum ratios ranging from 0.9 to 1.4. We hypothesize that following oral or SC administration, slower permeation of this hydrophilic molecule into blood capillaries may result in considerable passive 3TC penetration into lymphatic vessels. Further studies will be needed to clarify the mechanism of delivery of 3TC and similar antiretroviral drugs into the lymph nodes.

Development of lipophilic ester prodrugs of dolutegravir for intestinal lymphatic transport.

The establishment of latent cellular and anatomical viral reservoirs is a major obstacle to achieving a cure for people infected by HIV. Mesenteric lymph nodes (MLNs) are one of the most important anatomical reservoirs of HIV. Suboptimal levels of antiretroviral (ARVs) drugs in these difficult-to-penetrate viral reservoirs is one of the limitations of current antiretroviral therapy (ART) regimens. This study aimed to design and assess highly lipophilic ester prodrugs of dolutegravir (DTG) formulated with long-chain triglyceride (LCT) for delivery of DTG to the viral reservoir in mesenteric lymph and MLNs. A number of alkyl ester prodrugs of DTG were designed based on the predicted affinity to chylomicrons (CM), and the six most promising prodrugs were selected and synthesised. The synthesised prodrugs were further assessed for their intestinal lymphatic transport potential and biotransformation in biorelevant media in vitro and ex vivo. DTG and the most promising prodrug (prodrug 5) were then assessed in pharmacokinetic and biodistribution studies in rats. Although oral administration of 5 mg/kg of unmodified DTG (an allometrically scaled dose from humans) with or without lipids achieved concentrations above protein binding-adjusted IC90 (PA-IC90) (64 ng/mL) in most tissues, the drug was not selectively targeted to MLNs. The combination of lipophilic ester prodrug and LCT-based formulation approach improved the targeting selectivity of DTG to MLNs 4.8-fold compared to unmodified DTG. However, systemic exposure to DTG was limited, most likely due to poor intestinal absorption of the prodrug following oral administration. In vitro lipolysis showed a good correlation between micellar solubilisation of the prodrug and systemic exposure to DTG in rats in vivo. Thus, it is prudent to include in vitro lipolysis in the early assessment of orally administered drugs and prodrugs in lipidic formulations, even when intestinal lymphatic transport is involved in the absorption pathway. Further studies are needed to clarify the underlying mechanisms of low systemic bioavailability of DTG following oral administration of the prodrug and potential ways to overcome this limitation.

Patient-led rapid titration of basal insulin in gestational diabetes is associated with improved glycaemic control and lower birthweight.

AIMS: Elevated fasting blood glucose in gestational diabetes (GDM) is a key predictor of high birthweight babies and adverse pregnancy outcomes but is hard to treat. We implemented a simple, patient-led, insulin dose titration algorithm aiming to improve fasting glycaemic control in GDM. METHODS: In women with GDM, initiating basal insulin, we recommended a daily four-unit dose increase after every fasting glucose value ≥5.0 mmol/mol (90 mg/dl). This approach augmented our pre-existing intensive (weekly) specialist nursing input. Using a before-and-after retrospective observational study design, we examined insulin doses and glucose values at 36 weeks gestation and maternal and neonatal outcomes in 105 women completing pregnancy before and 93 women after the intervention. RESULTS: The baseline characteristics of women in the before and after groups were the same. Women initiated on insulin after implementation (n = 30 before, n = 43 after) achieved substantially higher doses at 36 weeks (53 vs. 36 units/day; 0.56 vs. 0.37 units/kg/day; p = 0.027). 36-week mean fasting glucose was lower in those on insulin after implementation (4.6 vs. 5.1 mmol/L [83 vs. 92 mg/dl]; p = 0.031). Birthweight was significantly reduced (birthweight Z-scores 0.34 vs. 0.92; p = 0.005). There was no significant difference in macrosomia (after; 2% vs. before; 17% p = 0.078) or caesarean sections (after; 33% vs. before; 47%; p = 0.116). No women experienced severe hypoglycaemia. There were no outcome differences before versus after intervention in women not treated with insulin. CONCLUSIONS: Patient-led daily insulin titration in gestational diabetes leads to higher insulin dose use lower fasting glucose and is associated with lower birthweight without causing significant hypoglycaemia.

Multicenter Survey on Staff Understanding of Preoperative Fasting Guidelines.

PURPOSE: To assess the knowledge of nursing staff regarding pediatric preoperative fasting in a tertiary pediatric center and a general hospital. DESIGN: Anonymous electronic survey with nine questions modified to each institution. METHODS: This was a prospective quantitative study. Nursing staff at a tertiary pediatric center and pediatric nursing staff at a general hospital with pediatric services were eligible for participation. An anonymous electronic survey with nine questions via Survey Monkey was used over a 2-month period. FINDINGS: There were 295 participants from the tertiary pediatric center and 24 from the general hospital which represented 10% of overall nursing staff at the tertiary pediatric center and approximately 50% of pediatric nursing staff at the general hospital. At both the tertiary pediatric center and the general hospital, 50 to 80% of participants correctly answered most questions. More participants were correct for the fasting times for infants less than 6 month of age than for those over 6 months old. For clear fluids, 61 (20.7%) and 13 (4.4%) considered jelly and breast milk as clear fluids respectively at the tertiary pediatric center. CONCLUSIONS: Preoperative fasting continues to be a core area of pediatric care that is not completely understood. Our survey showed that although the majority of staff claim to be able to access the hospital guidelines, knowledge of these guidelines can be improved. In light of ongoing changing evidence, it is clear that education is a key factor in reducing morbidity and improving patient experience related to preoperative fasting.

Pevonedistat targets malignant cells in myeloproliferative neoplasms in vitro and in vivo via NFκB pathway inhibition.

Targeted inhibitors of JAK2 (eg ruxolitinib) often provide symptomatic relief for myeloproliferative neoplasm (MPN) patients, but the malignant clone persists and remains susceptible to disease transformation. These observations suggest that targeting alternative dysregulated signaling pathways may provide therapeutic benefit. Previous studies identified NFκB pathway hyperactivation in myelofibrosis (MF) and secondary acute myeloid leukemia (sAML) that was insensitive to JAK2 inhibition. Here, we provide evidence that NFκB pathway inhibition via pevonedistat targets malignant cells in MPN patient samples as well as in MPN and patient-derived xenograft mouse models that are nonredundant with ruxolitinib. Colony forming assays revealed preferential inhibition of MF colony growth compared with normal colony formation. In mass cytometry studies, pevonedistat blunted canonical TNFα responses in MF and sAML patient CD34+ cells. Pevonedistat also inhibited hyperproduction of inflammatory cytokines more effectively than ruxolitinib. Upon pevonedistat treatment alone or in combination with ruxolitinib, MPN mouse models exhibited reduced disease burden and improved survival. These studies demonstrating efficacy of pevonedistat in MPN cells in vitro as well as in vivo provide a rationale for therapeutic inhibition of NFκB signaling for MF treatment. Based on these findings, a Phase 1 clinical trial combining pevonedistat with ruxolitinib has been initiated.

Adenoviral Infections in Singapore: Should New Antiviral Therapies and Vaccines Be Adopted?

BACKGROUND: A number of serious human adenovirus (HAdV) outbreaks have been recently reported: HAdV-B7 (Israel, Singapore, and USA), HAdV-B7d (USA and China), HAdV-D8, -D54, and -C2 (Japan), HAdV-B14p1 (USA, Europe, and China), and HAdV-B55 (China, Singapore, and France). METHODS: To understand the epidemiology of HAdV infections in Singapore, we studied 533 HAdV-positive clinical samples collected from 396 pediatric and 137 adult patients in Singapore from 2012 to 2018. Genome sequencing and phylogenetic analyses were performed to identify HAdV genotypes, clonal clusters, and recombinant or novel HAdVs. RESULTS: The most prevalent genotypes identified were HAdV-B3 (35.6%), HAdV-B7 (15.4%), and HAdV-E4 (15.2%). We detected 4 new HAdV-C strains and detected incursions with HAdV-B7 (odds ratio [OR], 14.6; 95% confidence interval [CI], 4.1-52.0) and HAdV-E4 (OR, 13.6; 95% CI, 3.9-46.7) among pediatric patients over time. In addition, immunocompromised patients (adjusted OR [aOR], 11.4; 95% CI, 3.8-34.8) and patients infected with HAdV-C2 (aOR, 8.5; 95% CI, 1.5-48.0), HAdV-B7 (aOR, 3.7; 95% CI, 1.2-10.9), or HAdV-E4 (aOR, 3.2; 95% CI, 1.1-8.9) were at increased risk for severe disease. CONCLUSIONS: Singapore would benefit from more frequent studies of clinical HAdV genotypes to identify patients at risk for severe disease and help guide the use of new antiviral therapies, such as brincidofovir, and potential administration of HAdV 4 and 7 vaccine.

Genomic signature of early T-cell response is associated with lower antibody titer threshold for sterilizing immunity.

Vaccination is an effective approach to reduce disease burden. High vaccination coverage blocks pathogen transmission to ensure herd immunity. However, the concept of herd immunity assumes that vaccinated individuals cannot be infected and mediate silent pathogen transmission. While the correlates of vaccine-mediated protection against disease have been examined, the correlates of sterilizing immunity that prevents infection have not been systematically defined. Here, we used full genome expression profiling to explore the molecular correlates of serological response and non-response to measles, mumps and rubella (MMR) vaccination as surrogates of infection and sterilizing immunity, respectively. We observed that the antibody titers needed to sterilize infection with the vaccine strains were higher than current WHO disease protection thresholds. In subjects with baseline antibodies below such sterilizing immunity thresholds, serological non-response to MMR vaccination was associated with gene expression profile indicative of early T-cell activation and signalling. Specifically, genes that regulate T-cell function and response were induced at day 1 post-vaccination in non-responders but not in responders. These findings suggest that rapid T-cell response prevented MMR vaccine infection to limit antigenic presentation and hence serological response. Collectively, our findings suggest an important role for T-cells in engendering sterilizing immunity.

A phase II randomized study to determine the safety and immunogenicity of the novel PIKA rabies vaccine containing the PIKA adjuvant using an accelerated regimen.

BACKGROUND: Human Rabies infection continues to be potentially fatal despite the availability of post-exposure prophylaxis with rabies vaccine. The PIKA Rabies vaccine adjuvant is a TLR3 agonist and has been shown to be safe and immunogenic in clinical phase I studies. METHODS: We conducted a phase II, open label, randomized study in healthy adults to assess the safety and immunogenicity of the PIKA rabies vaccine under an accelerated regimen. 126 subjects were randomized into two groups: control vaccine classic regimen ("control-classic") and PIKA vaccine accelerated regimen ("PIKA-accelerated"). Subjects were followed up for safety and rabies virus neutralizing antibodies (RVNA). RESULTS: Both the control and PIKA vaccines were generally well tolerated. 57.6% of subjects in the PIKA vaccine group, compared with 43.8% of subjects in the control-classic group, achieved the target RVNA titer of ≥0.5 IU/mL by Day 7. All subjects achieved the target RVNA titer by Day 14. The RVNA geometric mean titer at Day 7 was 0.60 IU/ml in the PIKA vaccine group and 0.39 IU/ml in the control-classic group. At Day 14, the RVNA geometric mean titer was 18.25 IU/ml in the PIKA-accelerated group and 19.24 IU/ml in the control-classic group. The median time taken to reach the target RVNA titer level of ≥0.5 IU/mL was 7.0 days (95% CI: 7.0-42.0 days) in the PIKA-accelerated group and 14.0 days (95% CI: 7.0-42.0 days) in the control-classic group. CONCLUSION: The accelerated regimen using the investigational PIKA Rabies vaccine was well-tolerated and demonstrated non-inferior immunogenicity compared to the classic regimen using the commercially available vaccine in healthy adults. Clinical trial registry: The study was registered with clinicaltrials.gov (NCT02956421).

An accelerated rabies vaccine schedule based on toll-like receptor 3 (TLR3) agonist PIKA adjuvant augments rabies virus specific antibody and T cell response in healthy adult volunteers.

BACKGROUND: Rabies is a fatal disease where post-exposure prophylaxis (PEP) is crucial in preventing infection. However, deaths even after appropriate PEP, have been reported. The PIKA Rabies vaccine adjuvant is a TLR3 agonist that activates B and T cells leading to a robust immune response. METHODS: We conducted a phase I, open label, randomized study in healthy adults to assess the safety and immunogenicity of the PIKA Rabies vaccine and an accelerated vaccine regimen. Thirty-seven subjects were randomized into 3 groups: control vaccine classic regimen, PIKA vaccine classic regimen and PIKA vaccine accelerated regimen. Subjects were followed up for safety, rabies virus neutralizing antibodies (RVNA) and T cell responses. RESULTS: Both the control and PIKA Rabies vaccine were well tolerated. All adverse events (AEs) were mild and self-limiting. Seventy-five percent of subjects in the PIKA accelerated regimen achieved a RVNA titer ⩾0.5IU/mL on day 7, compared to 53.9% in the PIKA classic regimen (p=0.411) and 16.7% in control vaccine classic regimen (p=0.012). The PIKA rabies vaccine elicited multi-specific rabies CD4 mediated T cell response already detectable ex vivo at day 7 after vaccination and that was maintained at day 42. CONCLUSION: The investigational PIKA rabies vaccine was well tolerated and more immunogenic than the commercially available vaccine in healthy adults. Clinical trial registry: The study was registered with clinicaltrials.gov NCT02657161.