Plasma Protein Binding Determination for Unstable Ester Prodrugs: Remdesivir and Tenofovir Alafenamide.

Remdesivir (RDV) and tenofovir alafenamide (TAF) are prodrugs designed to be converted to their respective active metabolites. Plasma protein binding (PPB) determination of these prodrugs is important for patients with possible alteration of free fraction of the drugs due to plasma protein changes in renal impairment, hepatic impairment, or pregnancy. However, the prodrugs' instability in human plasma presents a challenge for accurate PPB determination. In this research work, two approaches were used in the method development and qualification for PPB assessment of RDV and TAF. For RDV, dichlorvos was used to inhibit esterase activity to stabilize the prodrug in plasma during equilibrium dialysis (ED). The impact of dichlorvos on protein binding was evaluated and determined to be insignificant by comparing the unbound fraction (fu) determined by the ED method with dichlorvos present and the fu determined by an ultrafiltration method without dichlorvos. In contrast to RDV, TAF degradation in plasma is ∼3-fold slower, and TAF stability cannot be improved by dichlorvos. Fit-for-purpose acceptance criteria for the TAF PPB method were chosen, and an ED method was developed based on these criteria. These two methods were then qualified and applied for PPB determinations in clinical studies.

The Impacts of Slc19a3 Deletion and Intestinal SLC19A3 Insertion on Thiamine Distribution and Brain Metabolism in the Mouse.

The Thiamine Transporter 2 (THTR2) encoded by SLC19A3 plays an ill-defined role in the maintenance of tissue thiamine, thiamine monophosphate, and thiamine diphosphate (TDP) levels. To evaluate the impact of THTR2 on tissue thiamine status and metabolism, we expressed the human SLC19A3 transgene in the intestine of total body Slc19a3 knockout (KO) mice. Male and female wildtype (WT) and transgenic (TG) mice were fed either 17 mg/kg (1×) or 85 mg/kg (5×) thiamine hydrochloride diet, while KOs were only fed the 5× diet. Thiamine vitamers in plasma, red blood cells, duodenum, brain, liver, kidney, heart, and adipose tissue were measured. Untargeted metabolomics were performed on the brain tissues of groups with equivalent plasma thiamine. KO mice had ~two- and ~three-fold lower plasma and brain thiamine levels than WT on the 5× diet. Circulating vitamers were sensitive to diet and equivalent in TG and WT mice. However, TG had 60% lower thiamine but normal brain TDP levels regardless of diet, with subtle differences in the heart and liver. The loss of THTR2 reduced levels of nucleic acid and amino acid derivatives in the brain. Therefore, mutation or inhibition of THTR2 may alter the brain metabolome and reduce the thiamine reservoir for TDP biosynthesis.

The Effect of Trimethoprim on Thiamine Absorption: A Transporter-Mediated Drug-Nutrient Interaction.

Trimethoprim is predicted to inhibit several thiamine transporters, including the primary thiamine intestinal absorptive transporter, ThTR-2, and the hepatic and renal organic cation transporters, OCT1, OCT2, and MATEs. To investigate the effect of trimethoprim on thiamine absorption, studies were conducted in cells, mice, and healthy volunteers and supported by use of real-world data. In a randomized, crossover clinical study, seven healthy volunteers were given a single oral dose of thiamine or thiamine plus trimethoprim, followed by blood sampling. The thiamine area under the curve (AUC) increased with trimethoprim co-administration (P value = 0.031). Similar results were seen in mice. Trimethoprim appeared to act on thiamine absorption through inhibition of hepatic OCT1 as evidenced from its ability to modulate levels of isobutyrylcarnitine and propionylcarnitine, OCT1 biomarkers identified from metabolomic analyses. Real-world data further supported this finding, showing an association between trimethoprim use and higher levels of triglycerides, LDL cholesterol, and total cholesterol, consistent with OCT1 inhibition (P values: 2.2 × 10-16 , 5.75 × 10-7 , and 5.82 × 10-7 , respectively). These findings suggest that trimethoprim increases plasma levels of thiamine by inhibiting hepatic OCT1. Trimethoprim reduced urinary excretion and clearance of biomarkers for OCT2 and MATEs, consistent with inhibition of renal organic cation transporters. This inhibition did not appear to play a role in the observed increases in thiamine levels. This study highlights the potential for drug-nutrient interactions involving transporters, in addition to transporters' established role in drug-drug interactions.

Reversal of cancer gene expression identifies repurposed drugs for diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive incurable brainstem tumor that targets young children. Complete resection is not possible, and chemotherapy and radiotherapy are currently only palliative. This study aimed to identify potential therapeutic agents using a computational pipeline to perform an in silico screen for novel drugs. We then tested the identified drugs against a panel of patient-derived DIPG cell lines. Using a systematic computational approach with publicly available databases of gene signature in DIPG patients and cancer cell lines treated with a library of clinically available drugs, we identified drug hits with the ability to reverse a DIPG gene signature to one that matches normal tissue background. The biological and molecular effects of drug treatment was analyzed by cell viability assay and RNA sequence. In vivo DIPG mouse model survival studies were also conducted. As a result, two of three identified drugs showed potency against the DIPG cell lines Triptolide and mycophenolate mofetil (MMF) demonstrated significant inhibition of cell viability in DIPG cell lines. Guanosine rescued reduced cell viability induced by MMF. In vivo, MMF treatment significantly inhibited tumor growth in subcutaneous xenograft mice models. In conclusion, we identified clinically available drugs with the ability to reverse DIPG gene signatures and anti-DIPG activity in vitro and in vivo. This novel approach can repurpose drugs and significantly decrease the cost and time normally required in drug discovery.

The Impact of the COVID-19 Pandemic on Internet Use and the Use of Digital Health Tools: Secondary Analysis of the 2020 Health Information National Trends Survey.

BACKGROUND: The COVID-19 pandemic increased the use of digital tools in health care (eg, patient portal, telemedicine, and web-based scheduling). Studies have shown that older individuals, racial/ethnic minority groups, or populations with lower educational attainment or income have lower rates of using digital health tools. Digitalization of health care may exacerbate already existing access barriers in these populations. OBJECTIVE: This study evaluated how use of digital tools to asynchronously communicate with clinicians, schedule appointments, and view medical records changed near the beginning of the pandemic. METHODS: Using 2020 Health Information National Trends Survey (HINTS) data, we examined internet use and 7 digital health technology use outcomes (electronic communication with a provider, electronic appointment scheduling, electronic test result viewing, patient portal access, portal use to download health records, portal use for patient-provider communication, and portal use to view test results). The HINTS surveyors designated surveys received after March 11, 2020, as postpandemic responses. Using weighted logistic regression, we investigated the impact of the pandemic after adjusting for sociodemographic traits (age, race/ethnicity, income, education, and gender), digital access (having ever used the internet and smartphone/tablet ownership), and health-related factors (insurance coverage, caregiver status, having a regular provider, and chronic diseases). To explore differences in changes in outcomes among key sociodemographic groups, we tested for significant interaction terms between the pandemic variable and race/ethnicity, age, income, and educational attainment. RESULTS: There were 3865 respondents (1437 prepandemic and 2428 postpandemic). Of the 8 outcomes investigated, the pandemic was only significantly associated with higher odds (adjusted odds ratio 1.99, 95% CI 1.18-3.35) of using electronic communication with a provider. There were significant interactions between the pandemic variable and 2 key sociodemographic traits. Relative to the lowest income group (

Resistant starch wheat increases PYY and decreases GIP but has no effect on self-reported perceptions of satiety.

Dietary fiber has numerous health benefits, such as increasing satiety, and is regularly included in healthy dietary recommendations. However, different types and sources of fiber vary in their chemical properties and biological effects. This double-blind, randomized, placebo-controlled, crossover study investigated the effects of resistant starch type 2 (RS2) from wheat on self-reported perceptions of satiety and associated gut hormones in 30 healthy adults ages 40-65 years of age. Participants consumed rolls made using either RS2-enriched wheat flour or a wild-type flour for one week before a test day during which they ate a mixed meal containing the same roll type. Both self-reported perceptions of satiety and plasma concentrations of gut hormones were measured following the meal to assess whether the RS2-enriched wheat enhanced satiety and suppressed hunger for a longer period than the control wheat. Exploratory analysis indicated that fasting and peak concentration of peptide YY3-36 (PYY3-36; qfast = 0.02, qpeak = 0.02) increased, while peak concentration and iAUC of glucose-dependent insulinotropic peptide (GIP; qpeak < 0.001, qiAUC < 0.001) decreased after ingesting RS2-enriched wheat. However, self-reported perceptions of hunger or fullness using visual analog scales (VAS) did not differ following the test meal.

Diagnostic accuracy of a liquid chromatography-tandem mass spectrometry assay in small hair samples for rifampin-resistant tuberculosis drug concentrations in a routine care setting.

BACKGROUND: Treatment monitoring of drug-resistant tuberculosis (DR-TB) in resource-limited settings is challenging. We developed a multi-analyte assay for eleven anti-TB drugs in small hair samples as an objective metric of drug exposure. METHODS: Small hair samples were collected from participants at various timepoints during directly observed RR-TB treatment at an inpatient tertiary referral facility in South Africa (DR-TB cohort). We assessed qualitative determination (i.e., detection above limit of detection) of bedaquiline, linezolid, clofazimine, pretomanid, levofloxacin, moxifloxacin, pyrazinamide, isoniazid, ethambutol, ethionamide, and prothionamide in an LC-MS/MS index panel assay against a reference standard of inpatient treatment records. Because treatment regimens prior to hospitalization were not available, we also analyzed specificity (for all drugs except isoniazid) using an external cohort of HIV-positive patients treated for latent TB infection with daily isoniazid (HIV/LTBI cohort) in Uganda. RESULTS: Among the 57 DR-TB patients (58% with pre-XDR/XDR-TB; 70% HIV-positive) contributing analyzable hair samples, the sensitivity of the investigational assay was 94% or higher for all drugs except ethionamide (58.5, 95% confidence interval [CI], 40.7-99.9). Assay specificity was low across all tested analytes within the DR-TB cohort; conversely, assay specificity was 100% for all drugs in the HIV/LTBI cohort. CONCLUSIONS: Hair drug concentrations reflect long-term exposure, and multiple successive regimens commonly employed in DR-TB treatment may result in apparent false-positive qualitative and falsely elevated quantitative hair drug levels when prior treatment histories within the hair growth window are not known.

OCTAD: an open workspace for virtually screening therapeutics targeting precise cancer patient groups using gene expression features.

As the field of precision medicine progresses, treatments for patients with cancer are starting to be tailored to their molecular as well as their clinical features. The emerging cancer subtypes defined by these molecular features require that dedicated resources be used to assist the discovery of drug candidates for preclinical evaluation. Voluminous gene expression profiles of patients with cancer have been accumulated in public databases, enabling the creation of cancer-specific expression signatures. Meanwhile, large-scale gene expression profiles of cellular responses to chemical compounds have also recently became available. By matching the cancer-specific expression signature to compound-induced gene expression profiles from large drug libraries, researchers can prioritize small molecules that present high potency to reverse expression of signature genes for further experimental testing of their efficacy. This approach has proven to be an efficient and cost-effective way to identify efficacious drug candidates. However, the success of this approach requires multiscale procedures, imposing considerable challenges to many labs. To address this, we developed Open Cancer TherApeutic Discovery (OCTAD; http://octad.org ): an open workspace for virtually screening compounds targeting precise groups of patients with cancer using gene expression features. Its database includes 19,127 patient tissue samples covering more than 50 cancer types and expression profiles for 12,442 distinct compounds. The program is used to perform deep-learning-based reference tissue selection, disease gene expression signature creation, drug reversal potency scoring and in silico validation. OCTAD is available as a web portal and a standalone R package to allow experimental and computational scientists to easily navigate the tool.

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples.

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide's metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Case Report: Analytically Confirmed Severe Albenzadole Overdose Presenting with Alopecia and Pancytopenia.

Internet-facilitated self-diagnosis and treatment is becoming more prevalent, putting individuals at risk of toxicity when drugs are acquired without medical oversight. We report a patient with delusional parasitosis who consumed veterinary albendazole purchased on the Internet, leading to pancytopenia, transaminase elevation, and alopecia. A 53-year-old man was sent to the emergency department (ED) by his gastroenterologist because of abnormal laboratory results. The patient had chronic abdominal pain and believed he was infected with parasites. He purchased two bottles of veterinary-grade albendazole on the Internet, and over the 3 weeks before his ED visit, he consumed 113.6 g of albendazole (a normal maximal daily dose is 800 mg). Five days before admission, he noticed hair loss and a rash on his face. His examination was notable for significant scalp hair loss and hyperpigmentation along the jaw line. Laboratory studies were remarkable for pancytopenia (most notably a white blood cell count (WBC) of 0.4 × 103 cells/mm3, with an absolute neutrophil count (ANC) of 0 × 103 cells/mm3) and transaminase elevation (aspartate aminotransferase [AST] 268 IU/L, alanine aminotransferase [ALT] 89 IU/L). He developed a fever and was treated with antibiotics and colony-stimulating factors for presumed neutropenic bacteremia. Over the course of 1 week, his hepatic function normalized and his ANC increased to 3,000 × 103 cells/mm3. Serial albendazole and albendazole sulfoxide concentrations were measured in serum and urine by liquid chromatography-quadruple time-of-flight mass spectrometry. On day 2, his serum concentrations were 20.7 ng/mL and 4,257.7 ng/mL for albendazole and albendazole sulfoxide, respectively. A typical peak therapeutic concentration for albendazole sulfoxide occuring at 2-5 hours post-ingestion is 220-1,580 ng/mL. Known adverse effects of albendazole include alopecia, transaminase elevation, and neutropenia. Pancytopenia leading to death from septic shock is reported. In our patient, prolonged use of high-dose albendazole resulted in a significant body burden of albendazole and albendazole sulfoxide, leading to pancytopenia, transaminase elevation, and alopecia. He recovered with supportive therapy.

Isoniazid concentrations in hair and plasma area-under-the-curve exposure among children with tuberculosis.

We measured hair and plasma concentrations of isoniazid among sixteen children with tuberculosis who underwent personal or video-assisted directly observed therapy and thus had 100% adherence. This study therefore defined typical isoniazid exposure parameters after two months of treatment among fully-adherent patients in both hair and plasma (plasma area under the concentration-time curve, AUC, estimated using pharmacokinetic data collected 0, 2, 4, and 6 hours after drug administration). We found that INH levels in hair among highly-adherent individuals did not correlate well with plasma AUC or trough concentrations, suggesting that each measure may provide incremental and complementary information regarding drug exposure in the context of TB treatment.

Synthetic Cannabinoid and Mitragynine Exposure of Law Enforcement Agents During the Raid of an Illegal Laboratory - Nevada, 2014.

Synthetic cannabinoids (SCs), commonly known by the street name "Spice," are designer drugs of abuse that mimic the psychoactive effects of marijuana. Intentional SC use has resulted in multiple toxicities (1,2), but little is known about occupational SC exposure. After a federal agency's law enforcement personnel in Nevada reported irritability and feeling "high" after raiding illegal SC laboratories and processing seized SCs, a request for a health hazard evaluation was made by the agency to CDC's National Institute for Occupational Safety and Health (NIOSH) in 2014 to evaluate agents' occupational SC exposures. After making the request for a health hazard evaluation, federal agents conducted a raid of an illegal SC laboratory, with assistance from local law enforcement and Drug Enforcement Administration (DEA) personnel and with NIOSH investigators observing from a distance. After the raid, agents collected and processed material evidence. NIOSH investigators tested agents' urine for SC levels before and after the raid and measured SCs in the air and on surfaces after the raid. DEA determined that AB-PINACA (an SC compound) and mitragynine (a plant material with opium-like effects, also known as "kratom") were present in the illegal laboratory. AB-PINACA, its metabolites, and mitragynine were not detected in agents' urine before the raid; however, one or more of these substances was found in the urine of six of nine agents after the raid and processing of the SC evidence. AB-PINACA was detected in one surface wipe sample from the SC laboratory; none was detected in the air in the laboratory or in the offices of the law enforcement agency where the materials were processed after the raid. No policies were in place regarding work practices and use of personal protective equipment (PPE) during raids and evidence processing. To protect agents from SC exposures, NIOSH recommended that the agency require agents to wear a minimum level of PPE (e.g., protective gloves and disposable clothing) and undergo training in PPE and in handling and storing of contaminated evidence from SC laboratory raids. Showers and locker rooms also need to be provided so that agents can reduce contamination and prevent take-home exposure.

Quantifying Isoniazid Levels in Small Hair Samples: A Novel Method for Assessing Adherence during the Treatment of Latent and Active Tuberculosis.

BACKGROUND: Tuberculosis (TB) is the leading cause of death from an infectious pathogen worldwide and the most prevalent opportunistic infection in people living with HIV. Isoniazid preventive therapy (IPT) reduces the incidence of active TB and reduces morbidity and mortality in HIV-infected patients independently of antiretroviral therapy. However, treatment of latent or active TB is lengthy and inter-patient variability in pharmacokinetics and adherence common. Current methods of assessing adherence to TB treatment using drug levels in plasma or urine assess short-term exposure and pose logistical challenges. Drug concentrations in hair assess long-term exposure and have demonstrated pharmacodynamic relevance in HIV. METHODS: A large hair sample from a patient with active TB was obtained for assay development. Methods to pulverize hair and extract isoniazid were optimized and then the drug detected by liquid chromatography/ tandem mass spectrometry (LC/MS-MS). The method was validated for specificity, accuracy, precision, recovery, linearity and stability to establish the assay's suitability for therapeutic drug monitoring (TDM). Hair samples from patients on directly-observe isoniazid-based latent or active TB therapy from the San Francisco Department of Public Health TB clinic were then tested. RESULTS: Our LC/MS-MS-based assay detected isoniazid in quantities as low as 0.02ng/mg using 10-25 strands hair. Concentrations in spiked samples demonstrated linearity from 0.05-50ng/mg. Assay precision and accuracy for spiked quality-control samples were high, with an overall recovery rate of 79.5%. In 18 patients with latent or active TB on treatment, isoniazid was detected across a wide linear dynamic range. CONCLUSIONS: An LC-MS/MS-based assay to quantify isoniazid levels in hair with performance characteristics suitable for TDM was developed and validated. Hair concentrations of isoniazid assess long-term exposure and may be useful for monitoring adherence to latent or active TB treatment in the setting of HIV.