Molecular pharmaceutics [journal]
- Transporters for Antiretroviral Drugs in Colorectal CD4+ T Cells and Circulating α4β7 Integrin CD4+ T cells: Implications for HIV Microbicides. [JOURNAL ARTICLE]
- Mol Pharm 2016 Jul 28.
CD4+ T lymphocytes in the colorectal mucosa are key in HIV-1 transmission and dissemination. As such they are also the primary target for anti-retroviral (ARV)-based rectal microbicides for pre-exposure prophylaxis. Drug transporters expressed in mucosal CD4+ T cells determine ARV distribution across the cell membrane and, most likely, efficacy of microbicides. We describe transporters for antiretroviral drugs in colorectal mucosal CD4+ T lymphocytes and compare gene expression with circulating α4β7+CD4+ T cells which traffic to the intestine and have been shown to be preferentially infected by HIV-1. Purified total CD4+ T cells were obtained from colorectal tissue and blood samples by magnetic separation. CD4+ T cells expressing α4β7 integrin were isolated by fluorescence-activated cell sorting from peripheral blood mononuclear cells of healthy volunteers. Expressions of 15 efflux and uptake drug transporter genes were quantified using Taqman qPCR assays. Expression of efflux transporters MRP3, MRP5, BCRP and uptake transporter CNT2 was significantly higher in colorectal CD4+ T cells compared to circulating CD4+ T cells (p=0.01-0.03). Conversely, circulating α4β7+CD4+ T cells demonstrated significantly higher expression of OATPD compared to colorectal CD4+ T cells (p=0.001). To the best of our knowledge this is the first report of drug transporter gene expression in colorectal CD4+ and peripheral α4β7+CD4+ T cells. The qualitative and quantitative differences in drug transporter gene expression profiles between α4β7+CD4+ T cells and total mucosal CD4+ T cells may have significant implications for the efficacy of rectally delivered ARV-microbicides. Most notably, we have identified efflux drug transporters that could be targeted by selective inhibitors or beneficial drug-drug interactions to enhance intracellular accumulation of antiretroviral drugs.
- Identification and Quantitative Assessment of Uremic Solutes as Inhibitors of Renal Organic Anion Transporters, OAT1 and OAT3. [JOURNAL ARTICLE]
- Mol Pharm 2016 Jul 28.
One of the characteristics of chronic kidney disease (CKD) is the accumulation of uremic solutes in the plasma. Less is known about the effects of uremic solutes on transporters that may play critical roles in pharmacokinetics. We evaluated the effect of 72 uremic solutes on organic anion transporter 1 and 3 (OAT1 and OAT3) using a fluorescent probe substrate, 6-carboxyfluorescein. A total of 12 and 13 solutes were identified as inhibitors of OAT1 and OAT3, respectively. Several of them inhibited OAT1 or OAT3 at clinically relevant concentrations and reduced the transport of other OAT1/3 substrates in vitro. Review of clinical studies showed that the active secretion of most drugs that are known substrates of OAT1/3 deteriorated faster than the renal filtration in CKD. Collectively, these data suggest that through inhibition of OAT1 and OAT3, uremic solutes contribute to the decline in renal drug clearance in patients with CKD.
- Inhalable particles for 'pincer therapeutics' targeting nitazoxanide as bactericidal and host-directed agent to macrophages in a mouse model of tuberculosis. [JOURNAL ARTICLE]
- Mol Pharm 2016 Jul 27.
Nitazoxanide (NTX) has moderate mycobactericidal activity and is also an inducer of autophagy in mammalian cells. High-payload (40-50% w/w) inhalable particles containing NTX alone or in combination with anti-tuberculosis (TB) agents isoniazid (INH) and rifabutin (RFB) were prepared with high incorporation efficiency of 92%. In vitro drug release was corrected for drug degradation during the course of study and revealed first-order controlled release. Particles were efficiently taken up in vitro by macrophages and maintained intracellular drug concentrations at one order of magnitude higher than NTX in solution for six hours. Dose dependent killing of Mtb and restoration of lung and spleen architecture were observed in experimentally-infected mice treated with inhalations containing NTX. Adjunct NTX with INH and RFB cleared culturable bacteria from the lung and spleen and markedly healed tissue architecture. NTX can be used in combination with INH-RFB to kill the pathogen and heal the host.
- Control over particle size distribution by autoclaving poloxamer-stabilized trimyristin nanodispersions. [JOURNAL ARTICLE]
- Mol Pharm 2016 Jul 27.
Lipid nanoparticles are under investigation as delivery systems for poorly water-soluble drugs. The particle size in these dispersions strongly influences important pharmaceutical properties like biodistribution and drug loading capacity; it should be below 500 nm for direct injection into the bloodstream. Consequently, small particles with a narrow particle size distribution are desired. Hitherto, there are, however, only limited possibilities for the preparation of monodisperse, pharmaceutically relevant dispersions. In this work, the effect of autoclaving at 121 °C on the particle size distribution of lipid nanoemulsions and -suspensions consisting of the pharmaceutically relevant components trimyristin and poloxamer 188 was studied. Additionally, the amount of emulsifier needed to stabilize both untreated and autoclaved particles was assessed. In our study, four dispersions of mean particle sizes from 45 to 150 nm were prepared by high-pressure melt homogenization. The particle size distribution before and after autoclaving was characterized using static and dynamic light scattering, differential scanning calorimetry and transmission electron microscopy. Asymmetrical flow field-flow-fractionation was used for particle size distribution analyses and for the determination of free poloxamer 188. Upon autoclaving, the mean particle size increased to up to 200 nm, but not proportionally to the initial size. At the same time, the particle size distribution width decreased remarkably. Heat treatment thus seems to be a promising approach to achieve the desired narrow particle size distribution of such dispersions. Related to the lipid content, suspension particles needed more emulsifier for stabilization than emulsion droplets, and smaller particles more than larger ones.
- Incorporation of a nuclear localization signal in pH responsive LAH4-L1 peptide enhances transfection and nuclear uptake of plasmid DNA. [JOURNAL ARTICLE]
- Mol Pharm 2016 Jul 26.
The major intracellular barriers associated with DNA delivery using non-viral vectors are inefficient endosomal/lysosomal escape and poor nuclear uptake. LAH4-L1, a pH responsive cationic amphipathic peptide, is an efficient DNA delivery vector that promotes the release of nucleic acid into cytoplasm through endosomal escape. Here we further enhance the DNA transfection efficiency of LAH4-L1 by incorporating nuclear localizing signal (NLS) to promote nuclear importation. Four NLSs were investigated: Simian virus 40 (SV40) large T-antigen derived NLS, nucleoplasmin targeting signal, M9 sequence and the reverse SV40 derived NLS. All peptides tested were able to form positively charged nano-sized complexes with DNA. Significant improvement in DNA transfection was observed in slow-dividing epithelial cancer cells (Calu-3), macrophages (RAW264.7), dendritic cells (JAWSII), as well as thymidine-induced growth-arrested cells, but not in rapidly dividing cells (A549). Among the four NLS-modified peptides, PK1 (modified with SV40 derived NLS) and PK2 (modified with reverse SV40 derived NLS) were the most consistent in improving DNA transfection; up to a 10-fold increase in gene expression was observed for PK1 and PK2 over the unmodified LAH4-L1. Additionally PK1 and PK2 were shown to enhance cellular uptake as well as nuclear entry of DNA. Overall, we show that the incorporation of SV40 derived NLS, in particular, to LAH4-L1 is a promising strategy to improve DNA delivery efficiency in slow-dividing cells and dendritic cells, with development potential for in vivo applications and as a DNA vaccine carrier.
- Potential of translationally controlled tumor protein-derived protein transduction domains as antigen carriers for nasal vaccine delivery. [JOURNAL ARTICLE]
- Mol Pharm 2016 Jul 25.
Nasal vaccination offers a promising alternative to intramuscular (i.m) vaccination, because it can induce mucosal and systemic immunity. However, its major drawback is poor absorption of large antigens in the nasal epithelium. Protein transduction domains (PTDs), also called cell-penetrating peptides, have been proposed as vehicles for nasal delivery of therapeutic peptides and proteins. Here, we evaluated the potential of a mutant PTD derived from translationally controlled tumor protein (designated TCTP-PTD 13) as an antigen carrier for nasal vaccines. We first compared the L- and D-forms of TCTP-PTD 13 isomers (L- or D-TCTP-PTD 13) as antigen carriers. Studies in mice demonstrated that nasally administered mixtures of the model antigen ovalbumin (OVA) and D-TCTP-PTD 13 induced higher plasma IgG titers and secretory IgA levels in nasal washes than nasally administered OVA alone, OVA/L-TCTP-PTD 13, or i.m-injected OVA. Plasma IgG subclass responses (IgG1 and IgG2a) of mice nasally administered OVA/D-TCTP-PTD 13 showed that the predominant IgG subclass was IgG1, indicating a Th2-biased immune response. We also used synthetic CpG oligonucleotides (CpG) as a Th1 immune response-inducing adjuvant. Nasally administered CpG plus OVA/D-TCTP-PTD 13 was superior in eliciting systemic and mucosal immune responses compared to those induced by nasally administered OVA/D-TCTP-PTD 13. Furthermore, the OVA/CpG/D-TCTP-PTD 13 combination skewed IgG1 and IgG2a profiles of humoral immune responses towards a Th1 profile. These findings suggest that TCTP-derived PTD is a suitable vehicle to efficiently carry antigens and to induce more powerful antigen-specific immune responses and a more balanced Th1/Th2 response when combined with a DNA adjuvant.
- Differences in intestinal hydrolytic activities between cynomolgus monkeys and humans: evaluation of substrate specificities using recombinant carboxylesterase 2 isozymes. [JOURNAL ARTICLE]
- Mol Pharm 2016 Jul 25.
Cynomolgus monkeys, used as an animal model to predict human pharmacokinetics, occasionally show different oral absorption patterns to humans due to differences in their intestinal metabolism. In this study, we investigated the differences between intestinal hydrolytic activities in cynomolgus monkeys and humans, in particular the catalyzing activities of their carboxylesterase 2 (CES2) isozymes. For this purpose we used both human and monkey microsomes and recombinant enzymes derived from a cell culture system. Monkey intestinal microsomes showed lower hydrolytic activity than human microsomes for several substrates. Interestingly, in contrast to human intestinal hydrolysis which is not enantioselective, monkey intestine showed preferential R-form hydrolysis of propranolol derivatives. Recombinant CES2 isozymes from both species, mfCES2v3 from monkeys and human hCE2, showed similar metabolic properties to their intestinal microsomes when expressed in HEK293 cells. Recombinant hCE2 and mfCES2v3 showed similar Km values for both enantiomers of all propranolol derivatives tested. However, recombinant mfCES2v3 showed extreme R-enantioselective hydrolysis, and both hCE2 and mfCES2v3 showed lower activity for O-3-methyl-n-butyryl propranolol than for O-valeryl and O-2-methyl-n-butyryl propranolol. This lower hydrolytic activity was characterized by lower Vmax values. Docking simulations of the protein-ligand complex demonstrated that the enantioselectivity of mfCES2v3 for propranolol derivatives was possibly caused by the orientation of its active site being deformed by an amino acid change of Leu107 to Gln107 and the insertion of Met309, compared with hCE2. In addition, molecular dynamics simulation indicated the possibility that the inter-atomic distance between the catalytic triad and the substrate was elongated by a 3-positioned methyl in the propranolol derivatives. Overall, these findings will help us to understand the differences in intestinal hydrolytic activities between cynomolgus monkeys and humans.
- Zinc-stabilized chitosan-chondroitin sulfate nanocomplexes for HIV-1 infection inhibition application. [JOURNAL ARTICLE]
- Mol Pharm 2016 Jul 25.
Polyelectrolyte complexes (PECs) constituted of chitosan and chondroitin sulfate (ChonS) were formed by the one-shot addition of default amounts of polyanion to an excess of polycation. Key variables of the formulation process (e.g. degree of depolymerization, charge mixing ratio, the concentration and pH of polyelectrolyte solutions) were optimized based on the PECs sizes and polydispersities. The PECs maintained their colloidal stability at physiological salt concentration and pH thanks to the complexation of polyelectrolytes with zinc(II)ion during the nanoPECs formation process. The PECs were capable of encapsulating an antiretroviral drug tenofovir(TF) with a minimal alteration on the colloidal stability of the dispersion. Moreover, the particles interfaces could efficiently be functionalized with anti-OVA or anti-α4β7 antibodies with conservation of the antibody bio-recognition properties over one week of storage in PBS at 4 °C. In-vitro cytotoxicity studies showed that zinc(II) stabilized chitosan-ChonS nanoPECs were non-cytotoxic to human peripheral blood mononuclear cells (PBMCs) and in-vitro antiviral activity test demonstrated that nanoparticles formulations led to a dose-dependent reduction of HIV-1 infection. Using nanoparticles as a drug carrier system decrease the IC50 (50% inhibitory concentration) from an aqueous TF of 4.35 μmol•L-1 to 1.95 μmol•L-1. Significantly, zinc ions in this system also exhibited a synergistic effect in the antiviral potency. These data suggest that chitosan-ChonS nanoPECs can be promising drug delivery system to improve the antiviral potency of drugs to the viral reservoirs for the treatment of HIV infection.
- Multidrug Delivery Systems Based on Human Serum Albumin for Combination Therapy With 3 Anticancer Agents. [JOURNAL ARTICLE]
- Mol Pharm 2016 Jul 24.
When administering several anticancer drugs within a single carrier, it is important to regulate their spatial distribution so as to avoid possible mutual interference and to thus enhance the drugs' selectivity and efficiency. To achieve this, we proposed to develop human serum albumin (HSA)-based multidrug delivery systems for combination anticancer therapy. We used 3 anticancer agents (an organic drug [5-fluorouracil, or 5FU], a metallic agent [2-benzoylpyridine thiosemicarbazide copper II, or BpT], and a gene agent [AS1411]) to treat liver cancer and confirm our hypothesis. The structure of the HSA-palmitic acid (PA)-5FU-BpT complex revealed that 5FU and BpT respectively bind to the IB and IIA subdomains of HSA. Our MALDI-TOF-MS spectral data show that one AS1411 molecule is conjugated to Cys-34 of the HSA-5FU-BpT complex via a linker. Compared with unregulated 3-drug combination therapy, the HSA-5FU-BpT-AS1411 complex enhances cytotoxicity in Bel-7402 cells approximately 7-fold in vitro; however, in normal cells it does not raise cytotoxicity levels. Importantly, our in vivo results demonstrate that the HSA-5FU-BpT-AS1411 complex is superior to the unregulated 3-drug combination in enhancing targeting ability, inhibiting liver tumor growth, and causing fewer side effects.
- Physiologically Based Absorption Modeling for Amorphous Solid Dispersion Formulations. [JOURNAL ARTICLE]
- Mol Pharm 2016 Jul 21.
Amorphous solid dispersion (ASD) formulations are routinely used to enable the delivery of poorly soluble compounds. This type of formulations can enhance bioavailability due to higher kinetic solubility of the drug substance and increased dissolution rate of the formulation, by the virtue of the fact that the drug molecule exists in the formulation in a high energy amorphous state. In this paper we report the application of physiologically based absorption models to mechanistically understand the clinical pharmacokinetics of solid dispersion formulations. Three case studies are shown here to cover a wide range of ASD bioperformance in human and modeling to retrospectively understand their in-vivo behavior. Case study 1 is an example of fairly linear PK observed with dose escalation and the use of amorphous solubility to predict bioperformance. Case study 2 demonstrates the development of a model that was able to accurately predict the decrease in fraction absorbed (%Fa) with dose escalation thus demonstrating that such model can be used to predict the clinical bioperformance in the scenario where saturation of absorption is observed. Finally, case study 3 shows the development of an absorption model with the intent to describe the observed incomplete and low absorption in clinic with dose escalation. These case studies highlight the utility of physiologically based absorption modeling in gaining a thorough understanding of ASD performance and the critical factors impacting performance to drive design of a robust drug product that would deliver the optimal benefit to the patients.