Cationic Solid SMEDDS of Efavirenz for Improved Oral Delivery: Development by Central Composite Design, In Vitro and In Vivo Evaluation.

Efavirenz (EFV) is an anti-HIV drug with high dose and 40% oral bioavailability (BA). The aim was to improve the bioavailability by designing cationic solid SMEDDS. Solubility data, ternary phase diagrams, and central composite design were employed in design. Globule size, TEM, DSC, and SEM studies were used for characterization. Optimized L-SMEDDS contained 20 mg of EFV, 10 mg of Peceol, 43.5 mg of Tween 80, and 40 mg of Labrafac Lipophile WL-1349 and the characters included mean globule size-94 nm, PDI-0.255, and ZP-28 mV. Later, octadecylamine was added to get L-SMEDDS with + 38 mV charge. L-SMEDDS was converted into solid S-SMEDDS by adsorbing onto silica carriers. Syloid XDP was preferred based on flow and oil adsorption capacity. The % drug (EFV) release from powder, L-SMEDDS, and solid SMEDDS were 14.04, 94.47, and 85 respectively in first 30 min. TEM picture showed dispersed globules. DSC and SEM studies indicated the loss of drug crystallinity in S-SMEDDS. Pharmacokinetic (PK) studies in Wistar rats revealed 4.12 fold hike in BA for optimized cationic S-SMEDDS when compared to EFV suspension. Increased absorption could be due to the positive charge on globules. Thus, cationic S-SMEDDS emerged as a potential novel delivery system for improvement in BA and has scope for reducing the high dose for AIDS patients by future clinical studies.

Study on suitable analysis method for HIV-1 non-catalytic integrase inhibitor.

BACKGROUND: Integrase (IN) is an essential protein for HIV replication that catalyzes insertion of the reverse-transcribed viral genome into the host chromosome during the early steps of viral infection. Highly active anti-retroviral therapy is a HIV/AIDS treatment method that combines three or more antiviral drugs often formulated from compounds that inhibit the activities of viral reverse transcriptase and protease enzymes. Early IN inhibitors (INIs) mainly serve as integrase strand transfer inhibitors (INSTI) that disrupt strand transfer by binding the catalytic core domain of IN. However, mutations of IN can confer resistance to INSTI. Therefore, non-catalytic integrase inhibitors (NCINI) have been developed as next-generation INIs. METHODS: In this study, we evaluated and compared the activity of INSTI and NCINI according to the analysis method. Antiviral activity was compared using p24 ELISA with MT2 cell and TZM-bl luciferase system with TZM-bl cell. Each drug was serially diluted and treated to MT2 and TZM-b1 cells, infected with HIV-1 AD8 strain and incubated for 5 and 2 days, respectively. Additionally, to analyze properties of INSTI and NCINI, transfer inhibition assay and 3'-processing inhibition assay were performed. RESULTS: During screening of INIs using the p24 ELISA and TZM-bl luciferase systems, we found an inconsistent result with INSTI and NCINI drugs. Following infection of MT2 and TZM-bl cells with T-tropic HIV-1 strain, both INSTI and NCINI treatments induced significant p24 reduction in MT2 cells. However, NCINI showed no antiviral activity in the TZM-bl luciferase system, indicating that this widely used and convenient antiretroviral assay is not suitable for screening of NCINI compounds that target the second round of HIV-1 replication. CONCLUSION: Accordingly, we recommend application of other assay procedures, such as p24 ELISA or reverse transcription activity, in lieu of the TZM-bl luciferase system for preliminary NCINI drug screening. Utilization of appropriate analytical methods based on underlying mechanisms is necessary for accurate assessment of drug efficacy.

On the Single-Crystal Structure of Tenofovir Alafenamide Mono-Fumarate: A Metastable Phase Featuring a Mixture of Co-Crystal and Salt.

Tenofovir alafenamide (TAF) is a prodrug of tenofovir as a potent nucleotide reverse transcriptase inhibitor. It serves as the key component of Genvoya® for the first-line treatment of human immunodeficiency virus infection (HIV) and is the active component of Vemlidy® for the treatment of chronic hepatitis B. Vemlidy® is also a monotherapeutic regimen formulated as TAF hemifumarate (1; TAF:fumarate = 2:1). In this work, we report for the first time the single-crystal structure of TAF fumarate hemihydrate (2, TAF:fumarate:H2O = 2:2:1). Compound 2 is initially documented as a salt in which one proton of the fumaric acid migrates to the amine group of the adenine moiety in TAF. It was recently proposed that ca. 20-30% proton is transferred to the N atom on the aromatic adenine backbone. We herein provide definitive single-crystal X-ray diffraction results to confirm that 2, though phase pure, is formed as a mixture of co-crystal (75%) and salt (25%). It features two pairs of TAF fumarates, wherein one of the four H atoms on the fumaric acid is transferred to the N atom of the adjacent adenine moiety while the other three carboxylates remain in their intrinsic acid form. Compound 2 is a metastable phase during the preparation of 1 and can be isolated by halting the reaction during the refluxing of TAF and fumaric acid in acetonitrile (MeCN). Our report complements the previous characterizations of TAF monofumarate, and its elusive structural patterns are finally deciphered.

Enhancement of oral bioavailability of anti-HIV drug rilpivirine HCl through nanosponge formulation.

OBJECTIVE: To synthesize ß cyclodextrin nanosponges using a novel and efficient microwave mediated method for enhancing bioavailability of Rilpivirine HCl (RLP). SIGNIFICANCE: Belonging to BCS class II RLP has pH dependent solubility and poor oral bioavailability. However, a fatty meal enhances its absorption hence the therapy indicates that the dosage form be consumed with a meal. But then it becomes tedious and inconvenient to continue the therapy for years with having to face the associated gastric side effects such as nausea. METHOD: Microwave synthesizer was used to mediate the poly-condensation reaction between ß-cyclodextrin and cross-linker diphenylcarbonate. Critical parameters selected were polymer to cross-linker ratio, Watt power, reaction time and solvent volume. Characterization studies were performed using FTIR, DSC, SEM, 1H-NMR and PXRD. Molecular modeling was applied to confirm the possibility of drug entrapment. In vitro drug dissolution followed by oral bioavailability studies was performed in Sprawley rats. Samples were analyzed using HPLC. RESULTS: Microwave synthesis yields para-crystalline, porous nanosponges (∼205 nm). Drug entrapment led to enhancement of solubility and a two-fold increase in drug dissolution (P < 0.001) following Higuchi release model. Enhanced oral bioavailability was observed in fasted Sprawley rats where Cmax and AUC0-∞ increases significantly (Cmax of NS∼ 586 ± 5.91 ng/mL; plain RLP ∼310 ± 5. 74 ng/mL). CONCLUSION: The approach offers a comfortable dosing zone for AIDs patients, negating the requirement of consuming the formulation in a fed state due to enhancement in drugs' oral bioavailability.

Characteristics of adverse event reports among people living with human immunodeficiency virus (HIV) in Japan: Data mining of the Japanese Adverse Drug Event Report database.

The development of new anti-HIV drugs and advances in antiretroviral therapy (ART) regimens have enabled longer and more effective treatments in people living with HIV (PLWH). However, the aging of PLWHs is another issue that needs to be addressed. In addition to ART, many PLWHs frequently receive medications for various comorbidities. However, real-world data on the occurrence of adverse events in PLWHs and their causative drugs are rare. Therefore, this study aimed to clarify the characteristics of adverse event reports among PLWHs in Japan. PLWH cases with adverse events were comprehensively searched and analyzed using the Japanese Adverse Drug Event Report database (JADER). Despite changes in guideline-recommended ART regimens, anti-HIV drugs were the main cause of adverse events in PLWHs throughout the study period. However, considerable variations have been observed in the reporting rate of anti-HIV drug classes registered as causative drugs in JADER, especially for anchor drugs. In other words, the reporting rate of integrase strand transfer inhibitors has increased in recent years, while that of protease inhibitors and non-nucleoside reverse transcriptase inhibitors has decreased. Immune reconstitution inflammatory syndrome was the most reported adverse event and was frequently noticed by healthcare providers managing patients with HIV infections. The trends in adverse event reports for female and older patients differed from those for the overall population. This study may provide insights that can help in the establishment of optimal management strategies for PLWHs.

Understanding the Actual Use of Anti-HIV Drugs in Japan from 2016 to 2019: Demonstrating Epidemiological Relevance of NDB Open Data Japan for Understanding Japanese Medical Care.

The National Database of Health Insurance Claims and Specific Health Checkups of Japan (NDB) Open Data Japan is helpful for attaining simple and comprehensive understanding of medical care in Japan. Herein, we investigated the transition of anti-HIV-drug use in Japan over a 4-year period from fiscal year (FY) 2016 to FY 2019 using data on anti-HIV drugs that were extracted from the 3rd, 4th, 5th, and 6th NDB Open Data Japan. Then, the data were stratified by mechanism of action, single-tablet regimen (STR) or non-STR, age groups, and sex and analyzed. Throughout the study period, the prescription volume for tenofovir alafenamide fumarate as the backbone drug and integrase strand transfer inhibitors as the anchor drug increased. In FY 2019, STRs constituted approximately 44% of the total combination antiretroviral therapy regimens, 1.6 times higher than that in FY 2016 (27%). With the advent of newer drugs and regimens, the differences in anti-HIV drugs prescribed to patients of different ages and sex gradually diminished; however, differences were unremarkable in the first period, especially between sexes. The NDB Open Data Japan made it relatively easy to evaluate recent trends in anti-HIV prescription in Japan, indicating its usefulness for continuous surveys in this field.

Design, synthesis and DNA interaction studies of new fluorescent platinum complex containing anti-HIV drug didanosine.

Forming coordination complexes with nucleoside analogues may be helpful in studying anti-tumour activity of them. Therefore, to improve the clinical efficacy of nucleoside analogue and design new ones, a new fluorescent platinum (Pt) complex with anti-human immunodeficiency virus drug didanosine (ddI); K[PtCl(OCH3)2(ddI)]; was synthesized and characterized. The ultraviolet-visible (UV-vis) spectroscopy, infrared, thermogravimetric analysis, mass assignments and elemental analysis confirmed the preparation of the complex. The molecular ion peaks seen at the positive mass spectrum of Pt complex confirm coordination of the drug to metal centre. The interaction of this complex with calf thymus DNA (ct-DNA) was studied using several spectroscopic techniques such as UV absorption, fluorescence spectroscopy and dynamic viscosity measurements. Hyperchromism of the band in the UV-vis spectra and the intrinsic binding constant (0.56 ± 0.25) × 104 M-1, decreasing in Hoechst-DNA fluorescence by adding Pt complex concentration and also relatively small changes in DNA viscosity indicated that this complex could interact as a groove-binder. According to the UV spectra and the fluorescence quenching of the complex in our case seems to be primarily caused by complex formation between the Pt complex and DNA. The thermodynamic parameters showed that hydrogen bond and van der Waals interactions play main roles in the binding of Pt complex to ct-DNA. The free energy values are negative, showing the spontaneity of the Pt complex-DNA binding. The docking simulation was performed and the results confirm a preference of groove site of synthesized complex on DNA helix. The knowledge gained from this study will be helpful to further understand the DNA binding mechanism and can also provide much fruitful information for designing a new type of anti-cancer drugs.Communicated by Ramaswamy H. Sarma.

Engineered mixed oxide-based polymeric composites for enhanced antimicrobial activity and sustained release of antiretroviral drug.

Here, pH-responsive engineered polymeric composites were fabricated from sodium alginate and mixed Cu/Zn oxides. The resulting alginate-CuxZn1-xO composites were characterized by FTIR, SEM and XRD, then used as an efficient carrier for the antiretroviral drug (zidovudine, AZT) and exhibited remarkable antibacterial properties. The resulting polymeric composites had specific surface areas of 185.2-198.6 m2/g as confirmed by the Brunauer-Emmett-Teller analysis. The metal oxide distribution within the alginate matrix was confirmed from the X-ray diffraction and scanning electron microscopy analyses. The zidovudine, an antiretroviral drug was encapsulated in 30 mg of alginate-Cu0.7Zn0.3O with 68% encapsulation efficiency. The release of AZT in simulated intestinal fluid (pH 7.4) was studied, a slow and sustained release of AZT (~96.2%) was observed. The AZT release kinetics is sufficiently described by the Korsmeyer-Peppas model and follows the Fickian transport profile. Results herein demonstrated that A-Cu0.7Zn0.3O, A-Cu0.3Zn0.7O and Cu0.5Zn0.5O exhibited excellent bacterial devastation property. A dose of 8 µg/mL A-Cu0.7Zn0.3O and 13 µg/mL A-Cu0.3Zn0.7O are sufficient to completely killed E. coli DH5a and S. aureus NSUHS-151 within 24 h.

Self-assembled nanoparticles made from a new PEGylated poly(aspartic acid) graft copolymer for intravaginal delivery of poorly water-soluble drugs.

New amphiphilic PEGylated poly(aspartic acid) graft copolymer (PASP-PEG-Ph) was synthesized as a nanocarrier for intravaginal drug delivery of poorly water-soluble drugs. PASP-PEG-Ph self-assembled into negatively charged spherically shaped nanoparticles in the presence of pH 4.5 and pH 7.0 vaginal fluid simulants with a diameter of approximately 200 nm as evidenced by Zeta-potentiometer, scanning electron microscope (SEM), dynamic light scattering (DLS) analysis. A significant number of stable NPs could be maintained at pH 4.5, 37 °C for 13 days. The PASP-PEG-Ph NP showed no significant cytotoxicity toward the T-cell line SupT1 and human vaginal epithelial cell line Vk2/E6E7 up to 1 mg/mL. The highest encapsulation efficiency of the model drug coumarin 6 (C6) by PASP-PEG-Ph was 92.0 ± 5.7%. The sustained release profile of the encapsulated C6 was demonstrated by an in vitro release study. An in vitro cellular uptake study revealed strong cellular uptake of the C6 loaded NP by SupT1 cells within 2 h.

Synthesis of dual responsive carbohydrate polymer based IPN microbeads for controlled release of anti-HIV drug.

The present study aims at preparation of dual responsive interpenetrating polymer network hydrogel microbeads from sodium alginate and functionally modified guar gum. Guar gum was modified by graft copolymerization using N-vinylcaprolactam, the maximum % grafting 123.2, obtained at different optimized conditions. The graft copolymer was blended with sodium alginate to form hydrogel microbeads by emulsion crosslinking method using glutaraldehyde as crosslinker. Zidovudine, an anti-HIV drug was encapsulated with 68% encapsulation efficiency. Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance spectroscopy, scanning electron microscopy, differential scanning calorimetry and X-ray diffraction studies justified the grafting reaction, structure, morphology and polymer-drug interactions, respectively. Swelling studies ascertained that microbeads were potentially sensitive to both pH and temperature. In vitro release studies were investigated in pH 1.2 and 7.4, the release time enhanced up to 34h in pH 7.4 at 37°C.

Spectroscopic characterization and molecular structure of 3,14-dimethyl-2,6,13,17-tetraazapentacyclo[16.4.0.1(2,17).1(6,13).0(7,12)]tetracosane.

Constrained cyclam derivatives have been found to exhibit anti-HIV effects. The strength of binding to the CXCR4 receptor correlates with anti-HIV activity. The conformation of the macrocyclic compound is very important for co-receptor recognition. Therefore, knowledge of the conformation and crystal packing of macrocycles has become important in developing new highly effective anti-HIV drugs. Structural modifications of N-functionalized polyaza macrocyclic compounds have been achieved using various methods. A new synthesis affording single crystals of the title tetraazapentacyclo[16.4.0.1(2,17).1(6,13).0(7,12)]tetracosane macrocycle, C22H40N4, is reported. Formaldehyde reacts readily at room temperature with the tetraazatricyclo[16.4.0.0(2,17)]docosane precursor to yield a macropolycycle containing two five-membered rings. Characterization by elemental, spectroscopic and single-crystal X-ray diffraction analyses shows that the asymmetric unit contains half of a centrosymmetric molecule. The molecular structure shows a trans conformation for the two methylene bridges owing to molecular symmetry. The crystal structure is stabilized by intramolecular C-H...N hydrogen bonds. NMR and IR spectroscopic properties support the methylene-bridged macrocyclic structure.

Impact of HIV-1 infection pathways on susceptibility to antiviral drugs and on virus spread.

The infection routes of HIV-1 can affect several viral properties, including dissemination, pathogenesis, and immune evasion. In this study, we evaluated the inhibitory activity of a wide variety of anti-HIV drugs, focusing on the impact that different infection pathways have on their efficacy. Compared to cell-free infection, inhibitory activities were reduced in cell-to-cell productive transmission for all drugs tested. We detected weak reporter-expressing target cells after cell-to-cell transmission in the presence of integrase strand transfer inhibitors (INSTIs). Further analysis revealed that this expression was mainly due to unintegrated circular HIV (cHIV) DNAs, consisting of 1-LTR and 2-LTR circles. When in vitro-constructed cHIV DNAs were introduced into cells, the production of infectious and intercellular transmittable virions was observed, suggesting that cHIV DNA could be a source of infectious virus. These results highlight some advantages of the cell-to-cell infection mode for viral expansion, particularly in the presence of anti-retroviral drugs.

Sustained-release nanoART formulation for the treatment of neuroAIDS.

A novel approach was developed for the coencapsulation of an anti-HIV drug (tenofovir) and a latency-breaking agent (vorinostat), using magnetically guided layer-by-layer (LbL) assembled nanocarriers for the treatment of neuroAIDS. Ultrasmall iron oxide (Fe3O4) nanoparticles (10±3 nm) were synthesized and characterized. The LbL technique was used to achieve a sustained release profile, and application of 2 bilayers ([tenofovir+dextran sulphate]2+vorinostat) to magnetic nanoparticles resulted in a 2.8 times increase in drug (tenofovir) loading and also resulted in an increase in the drug release period by 30-fold, with 100% drug release in sustained manner over a period of 5 days with the simultaneous stimulation of latent HIV expression. Nanoformulation showed a good blood-brain barrier transmigration ability (37.95%±1.5%) with good in vitro antiviral efficacy (~33% reduction of p24 level) over a period of 5 days after HIV infection in primary human astrocytes, with good cell viability (>90%). Hence, LbL arrangements of drugs on magnetic nanoparticles provides sustained release and, therefore, may improve the patient's adherence to therapy and lead to better compliance.

The phenolic metabolites of the anti-HIV drug efavirenz: evidence for distinct reactivities upon oxidation with Frémy's salt.

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor administered as first line treatment against HIV-1. The major drawbacks of EFV therapy are neurotoxicity and hepatotoxicity, which may result from bioactivation to reactive metabolites capable of reacting with bionucleophiles. We investigated the in vitro oxidation of the phenolic EFV metabolites, 7-hydroxy-efavirenz (7-OH-EFV) and 8-hydroxy-efavirenz (8-OH-EFV), with Frémy's salt. A quinoline derivative, 6-chloro-2-cyclopropyl-4-(trifluoromethyl)quinolin-7-ol, presumably stemming from a radical rearrangement, was selectively obtained from 7-OH-EFV in 10% yield. In contrast, when subjected to the same oxidation conditions, 8-OH-EFV was considerably more prone to oxidative degradation and yielded multiple products. Among these, a quinone-imine derivative was tentatively identified upon LC-ESI-MS/MS analysis of the reaction mixture. These observations demonstrate a remarkable difference in the reactivities of the two phenolic EFV metabolites under oxidative conditions. Moreover, taking into consideration the toxicological significance of quinone-imine derivatives, these findings may explain earlier reports that 8-OH-EFV is a more potent toxicant than 7-OH-EFV in model test systems.

The sensitivity of transmitted/founder HIV-1 to anti-HIV drugs / 药学学报

The majority of mucosal HIV-1 infection is initially established by a few HIV-1 viral variants, followed by the development of overt systemic infection, and these viral variants are known as transmitted/founder viruses (T/F viruses). Investigation of the sensitivity of T/F virus to different anti-HIV-1 drugs will provide the best strategies of pre-exposure prophylaxis (PrEP) for high-risk groups of HIV-infected patients. Herein we constructed for the first time, a luciferase reporter system for HIV-1 T/F viruses, and then compared the drug sensitivity between T/F viruses and chronic infection virus. The result showed that the 50% inhibitory concentration (IC50) of nucleoside reverse transcriptase inhibitors (NRTIs), integrase inhibitors (INIs) and protease inhibitors (PIs) were not significantly different between the T/F viruses and chronic infection viruses of the same subtype (P>0.05), while non-nucleoside reverse transcriptase inhibitors (NNRTIs) showed a moderate resistance to T/F viruses, with a significant increase in IC50 (P<0.05). The conclusion suggests that when patients are in high-risk or in the acute infection of HIV-1, NNRTIs should be avoided in the first-line antiretroviral therapy regimens.