1 H NMR study of the phase behavior of aqueous eutectic solutions using the inverse Laplace transform analysis of T2 relaxation.

High-field 1 H NMR T2 relaxation studies were used to characterize the changes in the physical phases of water, NaCl, and dextrose solutions over a temperature range of -65 to 15 °C. The data were analyzed with the inverse Laplace transform and with a linear fit to the logarithm of the time domain signal. Two liquid phases were detected for the NaCl and dextrose solutions at lower temperatures and assigned to low and high concentrated solution domains. The high concentrated solution domain was found to be present between -30 and -5 °C in the NaCl solution and between -55 and -5 °C in the dextrose solution. Copyright © 2017 John Wiley & Sons, Ltd.

Paclitaxel nanosuspensions for targeted chemotherapy - nanosuspension preparation, characterization, and use.

OBJECTIVE: The purpose of this work was to prepare a stable paclitaxel nanosuspension and test it for potential use as a targeted chemotherapeutic. Different particle coatings were employed to assess their impact on cellular uptake in vitro. In vivo work was then performed to demonstrate efficacy in tumor-bearing mouse models. MATERIALS AND METHOD: Paclitaxel nanosuspensions were prepared using a homogenization process and coated with excipients. Surface charge was measured by zeta potential, potency by high-performance liquid chromatography, and solubility using an in-line UV probe. Cellular uptake studies were performed via flow cytometry. In vivo experiments were performed to determine residence time, maximum tolerated dose, and the efficacy of paclitaxel nanosuspensions (Paclitaxel-NS). RESULTS: A stable paclitaxel nanosuspension was prepared and coated with various excipients. Studies in mice showed that the nanosuspension was well-tolerated and at least as effective as the IV Taxol control in prolonging mouse survival in a head and neck cancer model as well as an ovarian cancer model with a lower overall drug dose than the traditional IV administration route. CONCLUSIONS: The paclitaxel nanosuspension is suitable for cellular uptake. The nanosuspension was effective in prolonging life in two separate xenograft orthotopic murine cancer models through two separate routes of administration.

Macrophage delivery of nanoformulated antiretroviral drug to the brain in a murine model of neuroAIDS.

Antiretroviral therapy (ART) shows variable blood-brain barrier penetration. This may affect the development of neurological complications of HIV infection. In attempts to attenuate viral growth for the nervous system, cell-based nanoformulations were developed with the focus on improving drug pharmacokinetics. We reasoned that ART carriage could be facilitated within blood-borne macrophages traveling across the blood-brain barrier. To test this idea, an HIV-1 encephalitis (HIVE) rodent model was used where HIV-1-infected human monocyte-derived macrophages were stereotactically injected into the subcortex of severe combined immunodeficient mice. ART was prepared using indinavir (IDV) nanoparticles (NP, nanoART) loaded into murine bone marrow macrophages (BMM, IDV-NP-BMM) after ex vivo cultivation. IDV-NP-BMM was administered i.v. to mice resulting in continuous IDV release for 14 days. Rhodamine-labeled IDV-NP was readily observed in areas of HIVE and specifically in brain subregions with active astrogliosis, microgliosis, and neuronal loss. IDV-NP-BMM treatment led to robust IDV levels and reduced HIV-1 replication in HIVE brain regions. We conclude that nanoART targeting to diseased brain through macrophage carriage is possible and can be considered in developmental therapeutics for HIV-associated neurological disease.

Aerosol based detectors for the investigation of phospholipid hydrolysis in a pharmaceutical suspension formulation.

A high performance liquid chromatography (HPLC) method was developed to quantify free fatty acids (FFA) in a pharmaceutical suspension formulated with phospholipids as stabilizing agents. Specifically, a suspension of crystalline itraconazole microparticles stabilized with Lipoid E80 was used as a model system to study the physicochemical stability of an aqueous, phospholipid-based suspension for injection. The hydrolysis of the phospholipids during storage at elevated temperatures (40 degrees C) necessitated the development of a suitable HPLC method for the determination of free fatty acid content in the suspension samples. HPLC methods using two types of aerosol detectors were investigated for the above purpose. Reversed-phase separation coupled with either an evaporative light scattering detector (ELSD) or a Corona(Plus) charged aerosol detector (CAD) was used. A comparison of the methods indicated that the CAD method provided better sensitivity, precision, recovery, and linearity for the parameters evaluated. As a result, this method was chosen for the stability study of itraconazole suspension and has been incorporated in subsequent formulation studies.

Physicochemical stability of phospholipid-dispersed suspensions of crystalline itraconazole.

The physicochemical stability of an aqueous, phospholipid-based dispersion of itraconazole microcrystals was studied as a model water-insoluble drug suspension. The particle size, phospholipid concentrations, free fatty acid (FFA) content, pH, and zeta potential of two test suspensions were followed over 63 days at 5 and 40 degrees C storage conditions. Hydrolysis of a control suspension containing Lipoid E80 led to rapid FFA formation, pH drop, and subsequent particle aggregation. In the second suspension, sodium oleate used in conjunction with Lipoid E80 significantly enhanced the suspension physicochemical stability. Oleate anions effectively (1) increased the anionic charge of the phospholipid surface layer, (2) buffered the suspension near pH 7, and (3) reduced the specific production of oleic acid as a phosphatidylcholine (PC) degradant. The observed hydrolysis rate constants k(obs) approximately 2 x 10(-7) (Lipoid only) and k(obs) approximately 5 x 10(-8) (Lipoid and oleate) were consistent with the pH dependent behavior reported for saturated soybean PC solutions. Mechanistically, FFA formed initially in the control suspension partitioned to the aqueous phase with limited influence on the phospholipid microenvironment at the itraconazole particle surface. Phospholipid stabilization of water-insoluble drugs was demonstrated with clear benefits from fatty acid anions as co-additives to influence the surface microenvironment, reduce hydrolysis kinetics, and enhance suspension physicochemical stability.

Itraconazole suspension for intravenous injection: determination of the real component of complete refractive index for particle sizing by static light scattering.

The purpose of this study is to assess the impact of real refractive indices, using different itraconazole suspensions, on the associated particle size distributions. Instrumental particle size measurement remains the practical option for determining the particle size distribution of a suspension. In this study, the suspension particle size distribution was measured by static light scattering, which requires knowledge of both the real and imaginary components of the complete refractive index. The real refractive indices of micronized itraconazole raw material, as well as vacuum-dried itraconazole suspension samples obtained from different formulations, polymorphs, manufacturing methods and particle size distributions, were determined using the Becke line method. Identical samples were analyzed by two contract laboratories in order to assess consistency. For the static light scattering equipment used in this study, the complete relative refractive index (RRI = n(particte) / n(dispersant) - ik) input required for software calculation is denoted by a refractive index kernel (RRI input) comprising a relative real component and an imaginary component. The reported real refractive indices for the itraconazole raw material as well as vacuum dried itraconazole suspension samples were different, ranging from 1.608 to 1.65 (selected kernel range of 120A010I to 124A010I). The imaginary component of itraconazole suspension was determined in a previous study to be 010I. The average real refractive index was calculated to be 1.62 (122A010I). The particle size distributions obtained using 120A010I and 124A010I were in good agreement with one generated using 122A010I. Therefore, itraconazole suspensions that were produced using different manufacturing methods/formulations or exhibited different particle size distributions/polymorphic forms may use 122A010I in determining particle size distribution. The particle size distributions determined using RRI input outside the range of 120A010I to 124A010I may not be reliable. However, it is recommended that similar investigations be conducted for other drug suspensions on a case-by-case basis.

Intra-articular (IA) ropivacaine microparticle suspensions reduce pain, inflammation, cytokine, and substance p levels significantly more than oral or IA celecoxib in a rat model of arthritis.

Current therapeutic treatment options for osteoarthritis entail significant safety concerns. A novel ropivacaine crystalline microsuspension for bolus intra-articular (IA) delivery was thus developed and studied in a peptidoglycan polysaccharide (PGPS)-induced ankle swelling rat model. Compared with celecoxib controls, both oral and IA, ropivacaine IA treatment resulted in a significant reduction of pain upon successive PGPS reactivation, as demonstrated in two different pain models, gait analysis and incapacitance testing. The reduction in pain was attended by a significant reduction in histological inflammation, which in turn was accompanied by significant reductions in the cytokines IL-18 and IL-1ß. This may have been due to inhibition of substance P, which was also significantly reduced. Pharmacokinetic analysis indicated that the analgesic effects outlasted measurable ropivacaine levels in either blood or tissue. The results are discussed in the context of pharmacologic mechanisms both of local anesthetics as well as inflammatory arthritis.

Anion effects on electrostatic charging of sterically stabilized, water insoluble drug particles.

Water-insoluble suspensions of itraconazole and budesonide were sterically stabilized using nonionic polymers (poloxamer 188 or polysorbate 80) and probed for polymer-anion interactions by measuring changes in particle zeta potential. Anions comprising a range of functionalities and aqueous solubilities were examined. Results showed that the more hydrophobic anions partitioned to the particle interface, and a simple model for predicting anion adsorption was developed from their calculated properties. Anions with a calculated Klopman water solubility less than 10 microM or a calculated log P>3.5 were adsorbed to the particle-polymer interface effectively increasing the overall particle charge. Anions of similar hydrophobicities with sulfonate or sulfate functionalities showed a much higher degree of particle charging compared with their carboxylate and phosphonate analogs at pH 9.5. In addition, the electrostatic charging of particles occurred at lower solution concentrations of sulfonate derivatives. These results suggest that the relative basicity of the oxoanion functionality may influence protonation or ion-pairing phenomena of the anions when adsorbed at the particle-polymer interface. Cetyltetramethylammonium bromide (CTAB) produced a positively charged particle consistent with the model developed for anion adsorption. Particle charging of sterically stabilized drug suspensions appeared largely independent of drug and polymer type. Anion hydrophobicity (solubility) and headgroup functionality dictated the observed charging behavior.

NanoART synthesis, characterization, uptake, release and toxicology for human monocyte-macrophage drug delivery.

BACKGROUND: Factors limiting the efficacy of conventional antiretroviral therapy for HIV-1 infection include treatment adherence, pharmacokinetics and penetration into viral sanctuaries. These affect the rate of viral mutation and drug resistance. In attempts to bypass such limitations, nanoparticles containing ritonavir, indinavir and efavirenz (described as nanoART) were manufactured to assess macrophage-based drug delivery. METHODS: NanoART were made by high-pressure homogenization of crystalline drug with various surfactants. Size, charge and shape of the nanoparticles were assessed. Monocyte-derived macrophage nanoART uptake, drug release, migration and cytotoxicity were determined. Drug levels were measured by reverse-phase high-performance liquid chromatography. RESULTS: Efficient monocyte-derived macrophage cytoplasmic vesicle uptake in less than 30 min based on size, charge and coating was observed. Antiretroviral drugs were released over 14 days and showed dose-dependent reduction in progeny virion production and HIV-1 p24 antigen. Cytotoxicities resulting from nanoART carriage were limited. CONCLUSION: These results support the continued development of macrophage-mediated nanoART carriage for HIV-1 disease.

Laboratory investigations for the morphologic, pharmacokinetic, and anti-retroviral properties of indinavir nanoparticles in human monocyte-derived macrophages.

The effectiveness of anti-retroviral therapies (ART) depends on its ultimate ability to clear reservoirs of continuous human immunodeficiency virus (HIV) infection. We reasoned that a principal vehicle for viral dissemination, the mononuclear phagocytes could also serve as an ART transporter and as such improve therapeutic indices. A nanoparticle-indinavir (NP-IDV) formulation was made and taken up into and released from vacuoles of human monocyte-derived macrophages (MDM). Following a single NP-IDV dose, drug levels within and outside MDM remained constant for 6 days without cytotoxicity. Administration of NP-IDV when compared to equal drug levels of free soluble IDV significantly blocked induction of multinucleated giant cells, production of reverse transcriptase activity in culture fluids and cell-associated HIV-1p24 antigens after HIV-1 infection. These data provide "proof of concept" for the use of macrophage-based NP delivery systems for human HIV-1 infections.

Development of a macrophage-based nanoparticle platform for antiretroviral drug delivery.

Complex dosing regimens, costs, side effects, biodistribution limitations, and variable drug pharmacokinetic patterns have affected the long-term efficacy of antiretroviral medicines. To address these problems, a nanoparticle indinavir (NP-IDV) formulation packaged into carrier bone marrow-derived macrophages (BMMs) was developed. Drug distribution and disease outcomes were assessed in immune-competent and human immunodeficiency virus type 1 (HIV-1)-infected humanized immune-deficient mice, respectively. In the former, NP-IDV formulation contained within BMMs was adoptively transferred. After a single administration, single-photon emission computed tomography, histology, and reverse-phase-high-performance liquid chromatography (RP-HPLC) demonstrated robust lung, liver, and spleen BMMs and drug distribution. Tissue and sera IDV levels were greater than or equal to 50 microM for 2 weeks. NP-IDV-BMMs administered to HIV-1-challenged humanized mice revealed reduced numbers of virus-infected cells in plasma, lymph nodes, spleen, liver, and lung, as well as, CD4(+) T-cell protection. We conclude that a single dose of NP-IDV, using BMMs as a carrier, is effective and warrants consideration for human testing.