ICH S1 prospective evaluation study and weight of evidence assessments: commentary from industry representatives.

Industry representatives on the ICH S1B(R1) Expert Working Group (EWG) worked closely with colleagues from the Drug Regulatory Authorities to develop an addendum to the ICH S1B guideline on carcinogenicity studies that allows for a weight-of-evidence (WoE) carcinogenicity assessment in some cases, rather than conducting a 2-year rat carcinogenicity study. A subgroup of the EWG composed of regulators have published in this issue a detailed analysis of the Prospective Evaluation Study (PES) conducted under the auspices of the ICH S1B(R1) EWG. Based on the experience gained through the Prospective Evaluation Study (PES) process, industry members of the EWG have prepared the following commentary to aid sponsors in assessing the standard WoE factors, considering how novel investigative approaches may be used to support a WoE assessment, and preparing appropriate documentation of the WoE assessment for presentation to regulatory authorities. The commentary also reviews some of the implementation challenges sponsors must consider in developing a carcinogenicity assessment strategy. Finally, case examples drawn from previously marketed products are provided as a supplement to this commentary to provide additional examples of how WoE criteria may be applied. The information and opinions expressed in this commentary are aimed at increasing the quality of WoE assessments to ensure the successful implementation of this approach.

Biocompatible metal-organic frameworks as promising platforms to eradicate HIV reservoirs ex vivo in people living with HIV.

The HIV attacks the immune system provoking an infection that is considered a global health challenge. Despite antiretroviral treatments being effective in reducing the plasma viral load in the blood to undetectable levels in people living with HIV (PLWH), the disease is not cured and has become chronic. This happens because of the existence of anatomical and cellular viral reservoirs, mainly located in the lymph nodes and gastrointestinal tract, which are composed of infected CD4+ T cells with a resting memory phenotype and inaccessible to antiretroviral therapy. Herein, a new therapeutic strategy based on nanotechnology is presented. Different combinations of antiretroviral drugs (bictegravir/tenofovir/emtricitabine and nevirapine/tenofovir/emtricitabine) and toll-like receptor agonists were encapsulated into metal-organic frameworks (MOFs) PCN-224 and ZIF-8. The encapsulation efficiencies of all the drugs, as well as their release rate from the carriers, were measured. In vitro studies about the cell viability, the hemocompatibility, and the platelet aggregation of the MOFs were carried out. Epifluorescence microscopy assays confirmed the ability of ZIF-8 to target a carboxyfluorescein probe inside HeLa cell lines and PBMCs. These results pave the way for the use of these structures to eliminate latent HIV reservoirs from anatomical compartments through the activation of innate immune cells, and a higher efficacy of the triplet combinations of antiretroviral drugs.

A mechanistic biomarker investigation of fialuridine hepatotoxicity using the chimeric TK-NOG Hu-liver mouse model and in vitro micropatterned hepatocyte cocultures.

Fialuridine (FIAU) is a nucleoside-based drug that caused liver failure and deaths in a human clinical trial that were not predicted by nonclinical safety studies. A recent report concluded that a TK-NOG humanized liver (hu-liver) mouse model detected human-specific FIAU liver toxicity, and broader use of that model could improve drug safety testing. We further evaluated this model at similar dose levels to assess FIAU sensitivity and potential mechanistic biomarkers. Although we were unable to reproduce the marked acute liver toxicity with two separate studies (including one with a "sensitized" donor), we identified molecular biomarkers reflecting the early stages of FIAU mitochondrial toxicity, which were not seen with its stereoisomer (FIRU). Dose dependent FIAU-induced changes in hu-liver mice included more pronounced reductions in mitochondrial to nuclear DNA (mtDNA/nucDNA) ratios in human hepatocytes compared to mouse hepatocytes and kidneys of the same animals. FIAU treatment also triggered a p53 transcriptional response and opposing changes in transcripts of nuclear- and mitochondrial-encoded mitochondrial proteins. The time dependent accumulation of FIAU into mtDNA is consistent with the ≥9-week latency of liver toxicity observed for FIAU in the clinic. Similar changes were observed in an in vitro micro-patterned hepatocyte coculture system. In addition, FIAU-dependent mtDNA/nucDNA ratio and transcriptional alterations, especially reductions in mitochondrially encoded transcripts, were seen in livers of non-engrafted TK-NOG and CD-1 mice dosed for a shorter period. Conclusion: These mechanistic biomarker findings can be leveraged in an in vitro model and in a more routine preclinical model (CD-1 mice) to identify nucleosides with such a FIAU-like mitochondrial toxicity mechanistic liability potential. Further optimization of the TK-NOG hu-liver mouse model is necessary before broader adoption for drug safety testing.

Early-Onset albuminuria and Associated Renal Pathology in Leucine-Rich Repeat Kinase 2 Knockout Rats.

Activating mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are associated with Parkinson disease (PD), prompting development of LRRK2 inhibitors as potential treatment for PD. However, kidney safety concerns have surfaced from LRRK2 knockout (KO) mice and rats and from repeat-dose studies in rodents administered LRRK2 inhibitors. To support drug development of this therapeutic target, we conducted a study of 26 weeks' duration in 2-month-old wild-type and LRRK2 KO Long-Evans Hooded rats to systematically examine the performance of urinary safety biomarkers and to characterize the nature of the morphological changes in the kidneys by light microscopy and by ultrastructural evaluation. Our data reveal the time course of early-onset albuminuria at 3 and 4 months in LRRK2 KO female and male rats, respectively. The increases in urine albumin were not accompanied by concurrent increases in serum creatinine, blood urea nitrogen, or renal safety biomarkers such as kidney injury molecule 1 or clusterin, although morphological alterations in both glomerular and tubular structure were identified by light and transmission electron microscopy at 8 months of age. Diet optimization with controlled food intake attenuated the progression of albuminuria and associated renal changes.

Single -and Multi-Walled Carbon Nanotubes as Nanocarriers for the Delivery of 7-Hydroxyflavone.

The research on flavonoids has exponentially grown since their first therapeutic evidence, in 1937. They are effective in vitro in a wide range of human diseases, particularly those mediated by free radicals, such as cancer, atherosclerosis, AIDS, or neuronal diseases. However, their applications have been reduced due to their low solubility, poor absorption, and rapid metabolism. Flavonoid encapsulation in nanocarriers significantly improves their oral absorption, protects the drug against degradation, decreases the first-pass hepatic effect, and makes absorption through the lymphatic system easier. In this work, carbon nanotubes were used as nanocarriers of 7-hydroxyflavone, 7-HF. The encapsulation of 7-HF into pristine single- and multi-walled carbon nanotubes, and into -COOH functionalized single-walled carbon nanotubes has been investigated. The equilibrium association constants were estimated. The structural backbone of 7-HF, two benzene rings linked through three carbon atoms that form a pyran heterocyclic ring containing a keto group, seems to play a key role in the 7-HF/CNT interactions, although other types of interactions are also at work. The in vitro release of 7-HF was studied at three pHs, 2.0, 7.4, and 9.2, mimicking the different biological barriers of the human organism.

Supramolecular Systems for Gene and Drug Delivery.

Several biomaterial-based supramolecular systems (cyclodextrins [...].

Characterization of integration frequency and insertion sites of adenovirus DNA into mouse liver genomic DNA following intravenous injection.

While generally referred to as "non-integrating" vectors, adenovirus vectors have the potential to integrate into host DNA via random, illegitimate (nonhomologous) recombination. The present study provides a quantitative assessment of the potential integration frequency of adenovirus 5 (Ad5)-based vectors following intravenous injection in mice, a common route of administration in gene therapy applications particularly for transgene expression in liver. We examined the uptake level and persistence in liver of first generation (FG) and helper-dependent (HD) Ad5 vectors containing the mouse leptin transgene. As expected, the persistence of the HD vector was markedly higher than that of the FG vector. For both vectors, the majority of the vector DNA remained extrachromosomal and predominantly in the form of episomal monomers. However, using a quantitative gel-purification-based integration assay, a portion of the detectable vector was found to be associated with high molecular weight (HMW) genomic DNA, indicating potential integration with a frequency of up to ~44 and 7000 integration events per µg cellular genomic DNA (or ~0.0003 and 0.05 integrations per cell, respectively) for the FG and HD Ad5 vectors, respectively, following intravenous injection of 1 × 1011 virus particles. To confirm integration occurred (versus residual episomal vector DNA co-purifying with genomic DNA), we characterized nine independent integration events using Repeat-Anchored Integration Capture (RAIC) PCR. Sequencing of the insertion sites suggests that both of the vectors integrate randomly, but within short segments of homology between the vector breakpoint and the insertion site. Eight of the nine integrations were in intergenic DNA and one was within an intron. These findings represent the first quantitative assessment and characterization of Ad5 vector integration following intravenous administration in vivo in wild-type mice.

Multivalent Calixarene-Based Liposomes as Platforms for Gene and Drug Delivery.

The formation of calixarene-based liposomes was investigated, and the characterization of these nanostructures was carried out using several techniques. Four amphiphilic calixarenes were used. The length of the hydrophobic chains attached to the lower rim as well as the nature of the polar group present in the upper rim of the calixarenes were varied. The lipid bilayer was formed with one calixarene and with the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE. The cytotoxicity of the liposomes for various cell lines was also studied. From the results obtained, the liposomes formed with the least cytotoxic calixarene, (TEAC12)4, were used as nanocarriers of both nucleic acids and the antineoplastic drug doxorubicin, DOX. Results showed that (TEAC12)4/DOPE/p-EGFP-C1 lipoplexes, of a given composition, can transfect the genetic material, although the transfection efficiency substantially increases in the presence of an additional amount of DOPE as coadjuvant. On the other hand, the (TEAC12)4/DOPE liposomes present a high doxorubicin encapsulation efficiency, and a slow controlled release, which could diminish the side effects of the drug.

Cationic Single-Chained Surfactants with a Functional Group at the End of the Hydrophobic Tail DNA Compacting Efficiency.

The interaction between calf-thymus DNA, ctDNA, and various single-chained surfactants with different functional groups at the end of hydrophobic tail was studied with the goal of investigating the influence of the functional group nature on surfactant DNA compacting efficiency. The surfactants investigated were dodecyltriethylammonium bromide (DTEABr), triethyl(1-phenoxydodecyl)ammonium bromide (12PhBr), triethyl(2-naphthoxydodecyl)ammonium bromide (12NBr) and 11-(isonicotinoyloxy)-N,N,N-triethyl-1-undecanaminium bromide (11PyBr). Results made evident that the surfactants' tendencies to self-aggregation is the key factor determining their efficiency to compact the nucleic acid. Subsequently, DOPE/12NBr/pEGFP-C1 lipoplexes, with different cationic surfactant molar fractions (α) and mass ratios (L/D), were prepared and characterized. DOPE is a zwitterionic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and the plasmid pEGFP-C1 carries a GFP coding sequence with the necessary regulatory elements for constitutive expression of the gene in human cells. 12NBr was chosen because it was the most efficient DNA compacting agent among the surfactants investigated. Finally, the cytotoxicity and transfection efficiency (TE) of DOPE/12NBr/pDNA lipoplexes, with different compositions, were investigated.

Potentiometric Study of Carbon Nanotube/Surfactant Interactions by Ion-Selective Electrodes. Driving Forces in the Adsorption and Dispersion Processes.

The interaction (adsorption process) of commercial ionic surfactants with non-functionalized and functionalized carbon nanotubes (CNTs) has been studied by potentiometric measurements based on the use of ion-selective electrodes. The goal of this work was to investigate the role of the CNTs' charge and structure in the CNT/surfactant interactions. Non-functionalized single- (SWCNT) and multi-walled carbon nanotubes (MWCNT), and amine functionalized SWCNT were used. The influence of the surfactant architecture on the CNT/surfactant interactions was also studied. Surfactants with different charge and hydrophobic tail length (sodium dodecyl sulfate (SDS), octyltrimethyl ammonium bromide (OTAB), dodecyltrimethyl ammonium bromide (DoTAB) and hexadecyltrimethyl ammonium bromide (CTAB)) were studied. According to the results, the adsorption process shows a cooperative character, with the hydrophobic interaction contribution playing a key role. This is made evident by the correlation between the free surfactant concentration (at a fixed [CNT]) and the critical micellar concentration, cmc, found for all the CNTs and surfactants investigated. The electrostatic interactions mainly determine the CNT dispersion, although hydrophobic interactions also contribute to this process.

Development and Application of a Transcriptomic Signature of Bioactivation in an Advanced In Vitro Liver Model to Reduce Drug-induced Liver Injury Risk Early in the Pharmaceutical Pipeline.

Early risk assessment of drug-induced liver injury (DILI) potential for drug candidates remains a major challenge for pharmaceutical development. We have previously developed a set of rat liver transcriptional biomarkers in short-term toxicity studies to inform the potential of drug candidates to generate a high burden of chemically reactive metabolites that presents higher risk for human DILI. Here, we describe translation of those NRF1-/NRF2-mediated liver tissue biomarkers to an in vitro assay using an advanced micropatterned coculture system (HEPATOPAC) with primary hepatocytes from male Wistar Han rats. A 9-day, resource-sparing and higher throughput approach designed to identify new chemical entities with lower reactive metabolite-forming potential was qualified for internal decision making using 93 DILI-positive and -negative drugs. This assay provides 81% sensitivity and 90% specificity in detecting hepatotoxicants when a positive test outcome is defined as the bioactivation signature score of a test drug exceeding the threshold value at an in vitro test concentration that falls within 3-fold of the estimated maximum drug concentration at the human liver inlet following highest recommended clinical dose administrations. Using paired examples of compounds from distinct chemical series and close structural analogs, we demonstrate that this assay can differentiate drugs with lower DILI risk. The utility of this in vitro transcriptomic approach was also examined using human HEPATOPAC from a single donor, yielding 68% sensitivity and 86% specificity when the aforementioned criteria are applied to the same 93-drug test set. Routine use of the rat model has been adopted with deployment of the human model as warranted on a case-by-case basis. This in vitro transcriptomic signature-based strategy can be used early in drug discovery to derisk DILI potential from chemically reactive metabolites by guiding structure-activity relationship hypotheses and candidate selection.

Metallo-Liposomes of Ruthenium Used as Promising Vectors of Genetic Material.

Gene therapy is a therapeutic process consisting of the transport of genetic material into cells. The design and preparation of novel carriers to transport DNA is an important research line in the medical field. Hybrid compounds such as metallo-liposomes, containing a mixture of lipids, were prepared and characterized. Cationic metal lipids derived from the [Ru(bpy)3]2+ complex, RuC11C11 or RuC19C19, both with different hydrophobic/lipophilic ratios, were mixed with the phospholipid DOPE. A relation between the size and the molar fraction α was found and a multidisciplinary study about the interaction between the metallo-liposomes and DNA was performed. The metallo-liposomes/DNA association was quantified and a relationship between Kapp and α was obtained. Techniques such as AFM, SEM, zeta potential, dynamic light scattering and agarose gel electrophoresis demonstrated the formation of lipoplexes and showed the structure of the liposomes. L/D values corresponding to the polynucleotide's condensation were estimated. In vitro assays proved the low cell toxicity of the metallo-liposomes, lower for normal cells than for cancer cell lines, and a good internalization into cells. The latter as well as the transfection measurements carried out with plasmid DNA pEGFP-C1 have demonstrated a good availability of the Ru(II)-based liposomes for being used as non-toxic nanovectors in gene therapy.

Cross-company evaluation of the human lymphocyte activation assay.

Nonclinical immunotoxicity evaluation is an important component of safety assessment for pharmaceuticals. One in vitro assay that can be applied in a weight of evidence assessment is the human lymphocyte activation (HuLA) assay, an antigen recall assay, similar in many respects to the in vivo T-cell-dependent antibody response (TDAR) in that cooperation of multiple immune cell types are needed to produce responses. This assay uses human cells and is more amenable than the TDAR to compound ranking and mechanistic studies. The HuLA assay requires less time and drug than TDAR assays, uses a relevant antigen (influenza), reflects a human immune response, and applies principles of the 3Rs to non-clinical safety assessment. Peripheral blood mononuclear cells (PBMC) from flu-immunized donors are re-stimulated with flu-vaccine in the presence of test articles, and proliferation is measured. Published data demonstrate the applicability of the HuLA assay, but it has not been evaluated for reproducibility across testing sites. To evaluate assay reproducibility, scientists from a consortium of institutions conducted the assay in parallel, using a common pool of donor PBMC, influenza vaccine, and known immunosuppressant compounds (cyclosporine A and mycophenolic acid). The HuLA assay was highly reproducible in identification of inhibition of antigen-specific responses, and there was significant agreement across testing sites in the half maximal inhibitory concentration (IC50) values. Intra-site variability was the largest contributor to the variability observed within the assay. The HuLA assay was demonstrated to be ideally suited to comparing multiple compounds (i.e. compound ranking or benchmarking) within the same assay. Overall, the data reported herein support the HuLA assay as a useful tool in mechanistic evaluations of antigen-specific immune responses.

Polyethlyene Glycol 200 Can Protect Rats Against Drug-Induced Kidney Toxicity Through Inhibition of the Renal Organic Anion Transporter 3.

MK-7680, a cyclic nucleotide prodrug, caused significant kidney tubule injury in female rats when administered orally at 1000 mg/kg/day for 2 weeks using 10% Polysorbate 80 as vehicle. However, kidney injury was absent when MK-7680 was administered at the same dose regimen using 100% Polyethylene Glycol 200 (PEG 200) as the vehicle. Subsequent investigations revealed that MK-7680 triphosphate concentrations in kidney were much lower in rats treated with MK-7680 using PEG 200 compared with 10% Polysorbate 80 vehicle, whereas plasma exposures of MK-7680 prodrug were similar. In vitro studies demonstrated that PEG 200 is an inhibitor of human renal uptake transporter organic anion transporter 3 (OAT3), of which MK-7680 is a substrate. Furthermore, PEG 200 and PEG 400 were found to interfere in vitro with human renal transporters OAT3, organic cation transporter (OCT) 2, multidrug resistance-associated protein (MRP) 2 and 4, and multidrug and toxin extrusion protein (MATE) 1 and 2K, but not OAT1. These results support a conclusion that PEG 200 may prevent MK-7680-induced kidney injury by inhibiting its active uptake into proximal tubular cells by OAT3. Caution should be exercised therefore when using PEGs as vehicles for toxicity assessment for compounds that are substrates of renal transporters.

Influence of the degree of oligomerization of surfactants on the DNA/surfactant interaction.

The interaction between calf thymus DNA, ctDNA, and a series of oligomeric surfactants derived from N-benzyl-N,N-dimethyl-N-(1-dodecyl)ammonium chloride is investigated. The influence of the surfactants' degree of oligomerization (2, 3 and 4) on the ctDNA/surfactant interaction is studied, as well as the effect of the structure of the spacer group linking the individual surfactant fragments. In particular, the effect of the distance between the positive charges and the hydrophobic chains within the oligomers on these interactions was examined, by using the three positional isomers (i.e., ortho-, meta-, and para-) with the rigid xylidene moiety as spacer. Results show that the dimeric ("gemini") surfactants are much more efficient in the inversion of the nucleic acid charge than the single-chained (monomeric) surfactant. Whereas the ortho - isomer causes a partial condensation, the meta - and para - isomers can completely condense ctDNA. The meta - and para - isomers of the trimeric surfactants can also completely condense the polynucleotide. In contrast, the tetrameric surfactant investigated does not change the morphology of the nucleic acid from an elongated coil into a compacted form, in spite of effectively inverting the nucleic acid's charge in their complex. Accordingly, the capacity for ctDNA compaction of oligomeric surfactants is not simply correlated to their degree of oligomerization, but depends on a complex balance of the number and relative distance of cationic charges and/or hydrophobic tails in the surfactants for effectively interacting with the nucleic acid to form the appropriate complex. This information will help to design more effective cationic surfactants as non-viral vectors for gene therapy.

Can Galactose Be Converted to Glucose in HepG2 Cells? Improving the in Vitro Mitochondrial Toxicity Assay for the Assessment of Drug Induced Liver Injury.

Human hepatocellular carcinoma cells, HepG2, are often used for drug mediated mitochondrial toxicity assessments. Glucose in HepG2 culture media is replaced by galactose to reveal drug-induced mitochondrial toxicity as a marked shift of drug IC50 values for the reduction of cellular ATP. It has been postulated that galactose sensitizes HepG2 mitochondria by the additional ATP consumption demand in the Leloir pathway. However, our NMR metabolomics analysis of HepG2 cells and culture media showed very limited galactose metabolism. To clarify the role of galactose in HepG2 cellular metabolism, U-13C6-galactose or U-13C6-glucose was added to HepG2 culture media to help specifically track the metabolism of those two sugars. Conversion to U-13C3-lactate was hardly detected when HepG2 cells were incubated with U-13C6-galactose, while an abundance of U-13C3-lactate was produced when HepG2 cells were incubated with U-13C6-glucose. In the absence of glucose, HepG2 cells increased glutamine consumption as a bioenergetics source. The requirement of additional glutamine almost matched the amount of glucose needed to maintain a similar level of cellular ATP in HepG2 cells. This improved understanding of galactose and glutamine metabolism in HepG2 cells helped optimize the ATP-based mitochondrial toxicity assay. The modified assay showed 96% sensitivity and 97% specificity in correctly discriminating compounds known to cause mitochondrial toxicity from those with prior evidence of not being mitochondrial toxicants. The greatest significance of the modified assay was its improved sensitivity in detecting the inhibition of mitochondrial fatty acid ß-oxidation (FAO) when glutamine was withheld. Use of this improved assay for an empirical prediction of the likely contribution of mitochondrial toxicity to human DILI (drug induced liver injury) was attempted. According to testing of 65 DILI positive compounds representing numerous mechanisms of DILI together with 55 DILI negative compounds, the overall prediction of mitochondrial mechanism-related DILI showed 25% sensitivity and 95% specificity.

Analysis of exposure margins in developmental toxicity studies for detection of human teratogens.

The draft Step 2 ICH S5(R3) guideline includes an exposure-based endpoint as an option for selecting the high-dose in reproductive and developmental toxicity studies. To help determine an appropriate exposure margin for embryofetal developmental toxicity testing, a retrospective analysis was undertaken to determine what threshold would have been sufficient to detect hazards to embryofetal development in rats and rabbits for 18 known and 4 presumed human teratogens. The analysis showed that using a high dose that provided at least a 6-fold exposure margin in the developmental toxicity studies would have been sufficient to detect the teratogenic hazard with relevance for humans for all these therapeutics. With regards to human risk assessment practices for developmental toxicity, the analysis showed that, after excluding lenalidomide and pomalidomide data in rats, all available AUC margins at the NOAEL for the induction of malformations or embryofetal lethality were <4-fold of the exposure at the MRHD for all 22 therapeutics. These data support the proposed general approach of increased level of concern for human risk when exposure margins of the NOAEL to the MRHD are <10-fold, reduced concern when the exposure margins are 10- to 25-fold, and minimal concern when the exposure margin is > 25-fold.

Use of a Bile Salt Export Pump Knockdown Rat Susceptibility Model to Interrogate Mechanism of Drug-Induced Liver Toxicity.

Inhibition of the bile salt export pump (BSEP) may be associated with clinical drug-induced liver injury, but is poorly predicted by preclinical animal models. Here we present the development of a novel rat model using siRNA knockdown (KD) of Bsep that displayed differentially enhanced hepatotoxicity to 8 Bsep inhibitors and not to 3 Bsep noninhibitors when administered at maximally tolerated doses for 7 days. Bsep KD alone resulted in 3- and 4.5-fold increases in liver and plasma levels, respectively, of the sum of the 3 most prevalent taurine conjugated bile acids (T3-BA), approximately 90% decrease in plasma and liver glycocholic acid, and a distinct bile acid regulating gene expression pattern, without resulting in hepatotoxicity. Among the Bsep inhibitors, only asunaprevir and TAK-875 resulted in serum transaminase and total bilirubin increases associated with increases in plasma T3-BA that were enhanced by Bsep KD. Benzbromarone, lopinavir, and simeprevir caused smaller increases in plasma T3-BA, but did not result in hepatotoxicity in Bsep KD rats. Bosentan, cyclosporine A, and ritonavir, however, showed no enhancement of T3-BA in plasma in Bsep KD rats, as well as Bsep noninhibitors acetaminophen, MK-0974, or clarithromycin. T3-BA findings were further strengthened through monitoring TCA-d4 converted from cholic acid-d4 overcoming interanimal variability in endogenous bile acids. Bsep KD also altered liver and/or plasma levels of asunaprevir, TAK-875, TAK-875 acyl-glucuronide, benzbromarone, and bosentan. The Bsep KD rat model has revealed differences in the effects on bile acid homeostasis among Bsep inhibitors that can best be monitored using measures of T3-BA and TCA-d4 in plasma. However, the phenotype caused by Bsep inhibition is complex due to the involvement of several compensatory mechanisms.

Preparation and Characterization of New Liposomes. Bactericidal Activity of Cefepime Encapsulated into Cationic Liposomes.

Cefepime is an antibiotic with a broad spectrum of antimicrobial activity. However, this antibiotic has several side effects and a high degradation rate. For this reason, the preparation and characterization of new liposomes that are able to encapsulate this antibiotic seem to be an important research line in the pharmaceutical industry. Anionic and cationic liposomes were prepared and characterized. All cationic structures contained the same cationic surfactant, N,N,N-triethyl-N-(12-naphthoxydodecyl)ammonium. Results showed a better encapsulation-efficiency percentage (EE%) of cefepime in liposomes with phosphatidylcholine and cholesterol than with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The presence of cholesterol and the quantity of egg-yolk phospholipid in the liposome increased the encapsulation percentage. The bactericidal activity against Escherichia coli of cefepime loaded into liposomes with phosphatidylcholine was measured. The inhibitory zone in an agar plate for free cefepime was similar to that obtained for loaded cefepime. The growth-rate constant of E. coli culture was also measured in working conditions. The liposome without any antibiotic exerted no influence in such a rate constant. All obtained results suggest that PC:CH:12NBr liposomes are biocompatible nanocarriers of cefepime that can be used in bacterial infections against Escherichia coli with high inhibitory activity.

Extended-Duration MK-8591-Eluting Implant as a Candidate for HIV Treatment and Prevention.

Regimen adherence remains a major hurdle to the success of daily oral drug regimens for the treatment and prevention of human immunodeficiency virus (HIV) infection. Long-acting drug formulations requiring less-frequent dosing offer an opportunity to improve adherence and allow for more forgiving options with regard to missed doses. The administration of long-acting formulations in a clinical setting enables health care providers to directly track adherence. MK-8591 (4'-ethynyl-2-fluoro-2'-deoxyadenosine [EFdA]) is an investigational nucleoside reverse transcriptase translocation inhibitor (NRTTI) drug candidate under investigation as part of a regimen for HIV treatment, with potential utility as a single agent for preexposure prophylaxis (PrEP). The active triphosphate of MK-8591 (MK-8591-TP) exhibits protracted intracellular persistence and, together with the potency of MK-8591, supports its consideration for extended-duration dosing. Toward this end, drug-eluting implant devices were designed to provide prolonged MK-8591 release in vitro and in vivo Implants, administered subcutaneously, were studied in rodents and nonhuman primates to establish MK-8591 pharmacokinetics and intracellular levels of MK-8591-TP. These data were evaluated against pharmacokinetic and pharmacodynamic models, as well as data generated in phase 1a (Ph1a) and Ph1b clinical studies with once-weekly oral administration of MK-8591. After a single administration in animals, MK-8591 implants achieved clinically relevant drug exposures and sustained drug release, with plasma levels maintained for greater than 6 months that correspond to efficacious MK-8591-TP levels, resulting in a 1.6-log reduction in viral load. Additional studies of MK-8591 implants for HIV treatment and prevention are warranted.