Enhancement of the transdermal delivery of zidovudine by pretreating the skin with two physical enhancers: microneedles and sonophoresis

BackgroundZidovudine (AZT) has been the most widely used drug for antiretroviral therapy. In order to improve the therapy with this drug, different alternatives have been proposed, such as the transdermal administration. The use of permeation enhancers is necessary to favor the passage of this drug through the skin, due to its physicochemical properties and to the natural permeation barrier imposed by the skin. ObjectivesTo evaluate the effect of two permeation enhancers, sonophoresis and microneedles, on the permeability of AZT through the skin.MethodsPermeation studies with an AZT solution were performed using pigskin clamped in Franz-type cells. Sonophoresis was applied under different conditions (i.e., amplitude, duty cycle and application time), selected according to an experimental design, where the response variables were the increase in temperature of the skin surface and the increase in transepidermal water loss. ATR-FTIR was also used to demonstrate the effect of enhancers on membrane components. ResultsThe permeability of AZT through intact skin was very poor, with a very long lag time. Pretreatment of the skin with sonophoresis increased AZT transport significantly, reducing the lag time. The maximum flux (27.52 µgcm⁻² h⁻¹) and the highest total amount permeated (about 624 µg/cm²) were obtained when applying sonophoresis in continuous mode, with an amplitude of 20%, and an application time of 2 min. Sonophoresis appears to have an impact on stratum corneum proteins. The use of microneedles further increased the flux (30.41 µgcm⁻² h⁻¹) and the total amount permeated (about 916 µg/cm²), relative to sonophoresis. ConclusionThe results are encouraging in terms of promoting AZT transport through the skin using sonophoresis or microneedles as permeation enhancers.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s40199-021-00402-y.     

Molecular characterization of virulence and antimicrobial resistance profile of Shigella species isolated from children with moderate to severe diarrhea in northeastern Brazil

Molecular characterization of virulence and antimicrobial resistance profiles were determined for Shigella species isolated from children with diarrhea in Fortaleza, Brazil. Fecal specimens were collected along with socioeconomic and clinical data from children with moderate to severe diarrhea requiring emergency care. Shigella spp. were isolated by standard microbiological techniques, and we developed 4 multiplex polymerase chain reaction assays to detect 16 virulence-related genes (VRGs). Antimicrobial susceptibility tests were performed using disk diffusion assays. S. flexneri and S. sonnei were the predominant serogroups. S. flexneri was associated with low monthly incomes; more severe disease; higher number of VRGs; and presence of pic, set, and sepA genes. The SepA gene was associated with more intense abdominal pain. S. flexneri was correlated with resistance to ampicillin and chloramphenicol, whereas S. sonnei was associated with resistance to azithromycin. Strains harboring higher numbers of VRGs were associated with resistance to more antimicrobials. We highlight the correlation between presence of S. flexneri and sepA, and increased virulence and suggest a link to socioeconomic change in northeastern Brazil. Additionally, antimicrobial resistance was associated with serogroup specificity in Shigella spp. and increased bacterial VRGs.     

Report of the symposium on the use of intravenous gammaglobulin in adults infected with the human immunodeficiency virus.

On July 27, 1989, the International Conference on Molecular Aspects of Immune Response and Infectious Diseases devoted a symposium to the subject of the use of intravenous gamma globulin (IVIG) in acquired immunodeficiency syndrome (AIDS). The information presented confirmed that IVIG benefits human immunodeficiency virus (HIV)-infected children with recurrent infections and that much remains to be learned about the influence of IVIG in adult AIDS. The symposium participants recognized the urgent need to develop randomized clinical trials using a control group to assess the efficacy of a treatment with IVIG in PGL (persistent generalized lymphadenopathy), ARC (AIDS-related complex), and AIDS. To prepare this report, a committee was established, including individuals with expertise in immunology, immunopharmacology, microbiology, virology, infectious diseases, general medicine, and pediatrics and representing research experience in academia and hospitals. After an introduction to the report with a summary of immunotherapeutic agents under evaluation to treat HIV infection, section 1 lays out the present understanding of the disease pathogenesis. Section 2 then outlines the treatment of HIV-seropositive individuals, discussing the uncertainties that any treatment entails. Section 3 discusses the rationale for treating HIV-infected individuals with IVIG, and Section 4 examines the major differences between IVIG and hyperimmunoglobulins for the treatment of HIV infection. Section 5 looks at IVIG as a mean to delay the emergence of opportunistic infections and restore immunocompetence in AIDS and related illnesses, and Sections 6 and 7 suggest a pilot protocol on the use of IVIG in association with low-dose or standard-dose zidovudine (AZT).(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydroxyurea enhances 3′-azido-3′-deoxythymidine (AZT) cytotoxicity in human chronic myeloid leukemia models*

Abstract: In this report we have evaluated the cytotoxic activity of 3′-azido-3′-deoxythymidine (AZT) used in combination with hydroxyurea (HU), an agent which disrupts de novo thymidylate synthesis. In 2 chronic myeloid leukemia (CML) cell lines, K.562 and RWLeu4, the IC50 of AZT was 8 (μmol/l and 28 μmol/l respectively, after a 5-day exposure, and the IC50 of HU was 80 μmol/l and 70 μmol/l respectively. In the presence of various concentrations of HU (1 μmol/l-100 μmol/l) the IC50 of AZT in both cell lines was significantly reduced and subsequent isobologram analysis revealed synergistic activity. Similarly, analysis of [3H]AZT incorporation into the DNA fraction of these cells indicated that exposure to AZT + HU resulted in an increased incorporation of AZT into DNA when compared to incubation in AZT alone. Biochemically, this effect appeared to be related to a decrease in dTTP pools caused by HU. The combination AZT + HU has also been demonstrated to exert a synergistic effect in inhibiting colony growth of bone marrow granulocyte-macrophage progenitors (CFU-GM) from patients affected by Ph1⁺ CML in chronic phase. These results are promising in view of a possible in vivo utilization of this drug combination.     

In Vitro and in Vivo Transport of Zidovudine (AZT) Across the Blood–Brain Barrier and the Effect of Transport Inhibitors

The transport of the antiviral nucleoside analogue zidovudine (3-azido-3-deoxythymidine; AZT) into the central nervous system (CNS) was characterized in vitro and in vivo. The in vitro model consisted of primary cultures of isolated bovine capillary endothelial cells. The transport rate of AZT across the monolayer, expressed as endothelial permeability P, was determined following luminal and abluminal administration. P did not differ between the two administration sites (luminal, 1.65 ± 0.44 cm/min/10³; abluminal, 1.63 ± 0.28 cm/min/10³). The transport of AZT across the endothelial cell monolayer was found to be concentration independent in the range between 0.4 and 50 µg/mL. AZT transport was not affected by pre-treatment of the cells with either metabolic inhibitors (DODG and DODG/NaN₃) or probenecid. This suggests that AZT passes the monolayer mainly by passive diffusion. The in vivo transport of AZT across the blood–brain barrier and the blood–CSF barrier was studied in male Wistar rats after coadministration of potential inhibitors of active transport of AZT: probenecid (organic anion transport) and thymidine (nucleoside transport). Intracerebroventricular and intravenous coadministration of probenecid caused a significant (P < 0.001) increase in the CSF/plasma concentration ratio compared to the control phase, indicating that the organic anion carrier is involved in AZT transport from CSF to blood. Since there was no effect of probenecid on the transport of AZT in vitro, it is suggested that this carrier is located at the choroid plexus. Coadministration of thymidine did not affect the CSF/plasma concentration ratio, suggesting that a nucleoside carrier system is not involved in AZT transport into or out of the CNS.     

AZT-induced mitochondrial myopathy

Histochemical, electron microscopy and biochemical studies were performed on muscle biopsy specimens from 11 AIDS patients treated with zidovudine. A peculiar association of structural abnormalities and mitochondrial dysfunction was found. Focal cytochrome c oxidase (COX) deficiency was evident in muscle sections from 9 patients, 8 of whom had received long-term treatment while one had been treated for 1 month only. Electron microscopy showed changes in number, size and structure of mitochondria. Biochemical studies proved partial COX and succinate cytochrome c reductase (SCR) deficiency in 4 patients; one patient had only reduced SCR activity. Our data confirm that AZT therapy can cause toxic myopathy with mitochondrial dysfunction.Paper presented at the National Congress at Sorrento in 1991 and selected by the Editorial Board of the Journal     

Analysis of Zidovudine Distribution to Specific Regions in Rabbit Brain Using Microdialysis

The distribution of zidovudine (3-azido-3-deoxythymidine; AZT) into two regions of rabbit brain was investigated in crossover using microdialysis. Six rabbits had guide cannulas surgically implanted in the lateral ventricle and thalamus by stereotaxic placement. After recovery, microdialysis probes were positioned and i.v. bolus doses of 5, 10, 20, and 30 mg/kg were administered to each animal over a period of 2 weeks. Blood was drawn via a marginal ear vein catheter for 8 hr. Brain dialysate was collected at 3 µl/min from ventricle and thalamus dialysis probes every 10 min. Simulated cerebrospinal fluid (CSF), to which 3-azido-2,3-dideoxyuridine (AZdU) was added, was used as perfusate. AZdU loss, which was measured during simultaneous retrodialysis, served as a marker for in vivo recovery of AZT. AZT concentrations in plasma, as well as in ventricle and thalamus dialysate, were determined using a sensitive HPLC assay, and AZdU was simultaneously analyzed in the dialysates. Calculation of in vivo recovery of AZT was based on loss of AZdU from the perfusate during retrodialysis and was used to estimate the concentration of drug at both sites in the brain. In vitro loss of AZdU and recovery of AZT showed good agreement, demonstrating a bivariate regression slope of 0.99. The half-lives and AUCs (normalized to dose) achieved in the plasma, ventricle, or thalamus were not significantly different for the four doses. The AUC ratios, which represent the ratio of clearances into and from the CNS, were not significantly different among the doses studied (AUC_v/AUC_p range, 0.16–0.19; AUC_t/AUC_p range, 0.05–0.09), providing further evidence that the kinetics of distribution into the thalamus and CSF are linear. The results also demonstrate that the time-averaged concentrations of AZT in thalamus ECF are about half of those in the CSF.     

AZT对白血病细胞株KG-1a细胞增殖和端粒酶活性的影响

本研究探索端粒酶抑制剂3’-叠氮-2’,3’-脱氧胸腺核苷(AZT)对人急性髓系白血病细胞株KG-1a细胞增殖和端粒酶活性的影响。用MTT法检测不同浓度AZT分别作用24、48、72 h时KG-1a细胞增殖水平;流式细胞术检测AZT对KG-1a细胞周期及细胞凋亡的影响;TRAP-PCR-ELISA法检测细胞端粒酶活性;RT-PCR法检测细胞端粒逆转录酶(hTERT)mRNA的表达。结果表明,AZT能抑制KG-1a细胞增殖,抑制作用具有时间和浓度依赖性;随AZT浓度的增加,处于S期的细胞数目减少,G2/M期细胞数目增加,且出现凋亡峰;AZT作用后实验组细胞的端粒酶活性降低,hTERT-mRNA表达下降,下调程度与AZT浓度呈正相关。结论:AZT在体外能明显抑制KG-1a细胞增殖,并诱导其凋亡,其机制与降低端粒酶活性、下调hTERT mRNA表达有关。