Proper Accounting for Surface Area to Solution Volume Ratios in Exaggerated Extractions.

When drug products contact plastic manufacturing components, packaging systems, and/or delivery devices, leachables from the plastics can accumulate in the drug product, potentially affecting its key quality attributes. Given practical issues associated with screening drug products for leachables, potential leachables are frequently surfaced as extractables revealed in extraction studies. To facilitate extractables discovery and identification and to shorten extraction times, extraction studies can be exaggerated and/or accelerated. One means of exaggerating an extraction is to increase the test article's extracted surface area to extraction solution volume ratio (SA/V), as it is generally accepted that an extractable's concentration in an extract is proportional to SA/V in a 1 to 1 manner. However, as the relationship between an extractable's concentration and SA/V depends on the extractable's plastic/solvent partition coefficient (kp/l), the effect of SA/V on the extractable's concentrations can be either under- or over-estimated if a 1 to 1 proportion is used. This article presents the theoretical relationship between SA/V, concentration, and kp/l; illustrates theory with a case study; and suggests proper exaggeration strategies.LAY ABSTRACT: When drug products are manufactured, stored, or delivered in systems that contain plastics, substances can be leached from the plastics and remain in the drug product, where they might affect the product's key quality attributes. To discover and identify these leached substances, the plastics are extracted under laboratory conditions and the extracts are appropriately tested. To facilitate this process, extracts may be generated under laboratory conditions that exaggerate or accelerate the drug product's clinical conditions of manufacturing or use. The proper use of the ratio of the extracted item's surface area to the volume of the extracting solution as an exaggeration parameter is discussed in this paper.

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.

An enantiomerically pure formulation of esmolol attenuates hypotension and preserves heart rate control in dogs.

BACKGROUND: Esmolol is marketed as a racemate (RS-esmolol) with hypotension being the most frequently reported adverse event. Previously, it has been shown that the S-enantiomer (S-esmolol) possesses all of the heart rate (HR) control. The authors studied whether S-esmolol alone mitigates hypotension at similar degrees of HR control compared with RS-esmolol. METHODS: The effects of RS- and S-esmolol on blood pressure (BP) were compared at multiple infusion rates producing similar HR control in dogs (N=21). Differences in BP were further interrogated by monitoring global cardiovascular function and included the R-enantiomer (R-esmolol) (N=3). RESULTS: S-esmolol at half the rate (µg kg min) of RS-esmolol provided the same degree of HR control over all infusion rates. RS-esmolol lowered BP by 3, 6, 11, 20, and 38 mmHg at 90, 300, 600, 1,000, and 2,000 µg kg min, compared with 2, 4, 5, 10, and 16 mmHg at 45, 150, 300, 500, and 1,000 µg kg min for S-esmolol. Decreased BP with RS-esmolol was attributed to decreases in left ventricular developed pressure (LVDP) (-34 mmHg), LVdP/dt+max (-702 mmHg/s), and cardiac output (-1 l/min). R-esmolol also decreased BP (-10 mmHg), LVDP (-10 mmHg), LVdP/dt+max (-241 mmHg/s), and cardiac output (to -0.2 l/min). S-esmolol reversed these trends toward pre-esmolol values by increasing BP (+13 mmHg), LVDP (+12 mmHg), LVdP/dt+max (+76 mmHg/s), and cardiac output (+0.4 l/min). CONCLUSIONS: R-enantiomer provided no HR control, but contributed to the hypotension with RS-esmolol, which appears to be due to negative inotropy. Thus, an S-enantiomer formulation of esmolol may provide similar HR control with less hypotension.

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.

NMR of heparin API: investigation of unidentified signals in the USP-specified range of 2.12-3.00 ppm.

This article addresses the identification and quantification of the chemical species resulting in resonances at 2.17 and 2.25 ppm in the (1)H nuclear magnetic resonance (NMR) spectrum of pharmaceutical-grade heparin sodium. The NMR signals in question were first confirmed to arise from chemical moieties covalently attached to the heparin molecule through NMR diffusion experiments as well as chemical treatment of heparin active pharmaceutical ingredient (API) containing the resonances. The material responsible for the extra NMR signals was then demonstrated by NMR spiking studies to be something other than oversulfated chondroitin sulfate and was finally identified as an O-acetylation product of heparin through (13)C labeling experiments with subsequent NMR analysis. The extent of O-acetylation was quantified using three orthogonal techniques: (1)H NMR, ion chromatography, and headspace gas chromatography/mass spectrometry. The results of this work showed good agreement between the three quantitative methods developed to analyze the signals in the United States Pharmacopeia-specified region of 2.12-3.00 ppm for heparin API.

Pharmacotoxicology of monocyte-macrophage nanoformulated antiretroviral drug uptake and carriage.

Limitations inherent to antiretroviral therapy (ART) in its pharmacokinetic properties remain despite over 15 years of broad use. Our laboratory has pioneered a means to improve ART delivery through monocyte-macrophage carriage of nanoformulated drug-encapsulated particles (nanoART). To this end, our prior works sought to optimize nanoART size, charge, and physical properties for cell uptake and antiretroviral activities. To test the functional consequences of indinavir, ritonavir, and efavirenz formulations we investigated relationships between human monocyte and macrophage cytotoxicities and nanoART dose, size, surfactant, and preparation. Wet-milled particles were more cytotoxic to monocytes-macrophages than those prepared by homogenization; with concurrent induction of tumor necrosis factor-alpha. Interestingly, pure suspensions of indinavir and ritonavir at 0.5 mM, and efavirenz at 0.1 mM and 0.5 mM also proved cytotoxic. Individual surfactants and formulated fluconazole neither affected cell function or viability. Although nanoART did not alter brain tight junction proteins ZO-2 and occludin, 0. 5mM ritonavir formulations did alter brain transendothelial electric resistance. These results underscore the potential importance of evaluating the physicochemical and functional properties of nanoART before human evaluations.

Nanoformulated antiretroviral drug combinations extend drug release and antiretroviral responses in HIV-1-infected macrophages: implications for neuroAIDS therapeutics.

We posit that improvements in pharmacokinetics and biodistributions of antiretroviral therapies (ART) for human immunodeficiency virus type one-infected people can be achieved through nanoformulationed drug delivery systems. To this end, we manufactured nanoparticles of atazanavir, efavirenz, and ritonavir (termed nanoART) and treated human monocyte-derived macrophages (MDM) in combination therapies to assess antiretroviral responses. This resulted in improved drug uptake, release, and antiretroviral efficacy over monotherapy. MDM rapidly, within minutes, ingested nanoART combinations, at equal or similar rates, as individual formulations. Combination nanoART ingested by MDM facilitated individual drug release from 15 to >20 days. These findings are noteworthy as a nanoART cell-mediated drug delivery provides a means to deliver therapeutics to viral sanctuaries, such as the central nervous system during progressive human immunodeficiency virus type one infection. The work brings us yet another step closer to realizing the utility of nanoART for virus-infected people.

Nanosuspension formulation of itraconazole eliminates the negative inotropic effect of SPORANOX in dogs.

Previously, it was observed that a nanosuspension formulation of itraconazole was more efficacious and yet less acutely toxic in rats as compared with the conventional solution formulation, SPORANOX (itraconazole) Injection. The present study compares the two formulations with respect to specifically myocardial contractility in conscious dogs. Motivation for doing so is highlighted by the black-box warning in the package insert for SPORANOX (itraconazole) Injection, which warns of negative inotropic effects. Conscious dogs, instrumented with a high-fidelity pressure transducer in the left ventricle, were placed in a sling for dosing and cardiac monitoring. Test and control articles were administered intravenously via a peripheral vein, and left ventricular parameters were measured continuously through 60 min from the start of dosing. As expected, SPORANOX (itraconazole) Injection caused a significant reduction in myocardial contractility as determined by the contractility index. In contrast, the itraconazole nanosuspension administered at twice the dose and at twice the rate of infusion did not result in significant changes in myocardial contractility. A novel formulation technology applied to itraconazole completely prevented the negative inotropic effect observed in conscious dogs as compared with SPORANOX (itraconazole) Injection.

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.

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.

Facilitated monocyte-macrophage uptake and tissue distribution of superparmagnetic iron-oxide nanoparticles.

BACKGROUND: We posit that the same mononuclear phagocytes (MP) that serve as target cells and vehicles for a host of microbial infections can be used to improve diagnostics and drug delivery. We also theorize that physical and biological processes such as particle shape, size, coating and opsonization that affect MP clearance of debris and microbes can be harnessed to facilitate uptake of nanoparticles (NP) and tissue delivery. METHODS: Monocytes and monocyte-derived macrophages (MDM) were used as vehicles of superparamagnetic iron oxide (SPIO) NP and immunoglobulin (IgG) or albumin coated SPIO for studies of uptake and distribution. IgG coated SPIO was synthesized by covalent linkage and uptake into monocytes and MDM investigated related to size, time, temperature, concentration, and coatings. SPIO and IgG SPIO were infused intravenously into naïve mice. T(2) measures using magnetic resonance imaging (MRI) were used to monitor tissue distribution in animals. RESULTS: Oxidation of dextran on the SPIO surface generated reactive aldehyde groups and permitted covalent linkage to amino groups of murine and human IgG and F(ab')(2) fragments and for Alexa Fluor(R) 488 hydroxylamine to form a Schiff base. This labile intermediate was immediately reduced with sodium cyanoborohydride in order to stabilize the NP conjugate. Optical density measurements of the oxidized IgG, F(ab')(2), and/or Alexa Fluor(R) 488 SPIO demonstrated approximately 50% coupling yield. IgG-SPIO was found stable at 4 degrees C for a period of 1 month during which size and polydispersity index varied little from 175 nm and 200 nm, respectively. In vitro, NP accumulated readily within monocyte and MDM cytoplasm after IgG-SPIO exposure; whereas, the uptake of native SPIO in monocytes and MDM was 10-fold less. No changes in cell viability were noted for the SPIO-containing monocytes and MDM. Cell morphology was not changed as observed by transmission electron microscopy. Compared to unconjugated SPIO, intravenous injection of IgG-SPIO afforded enhanced and sustained lymphoid tissue distribution over 24 hours as demonstrated by MRI. CONCLUSIONS: Facilitated uptake of coated SPIO in monocytes and MDM was achieved. Uptake was linked to particle size and was time and concentration dependent. The ability of SPIO to be rapidly taken up and distributed into lymphoid tissues also demonstrates feasibility of macrophage-targeted nanoformulations for diagnostic and drug therapy.

Ingress of blood-borne macrophages across the blood-brain barrier in murine HIV-1 encephalitis.

Blood-borne macrophage ingress into brain in HIV-1 associated neurocognitive disorders governs the tempo of disease. We used superparamagnetic iron-oxide particles loaded into murine bone marrow-derived macrophages (BMM) injected intravenously into HIV-1 encephalitis mice to quantitatively assess BMM entry into diseased brain regions. Magnetic resonance imaging tests were validated by histological coregistration and enhanced image processing. The demonstration of robust BMM migration into areas of focal encephalitis provide 'proof of concept' for the use of MRI to monitor macrophage ingress into brain.

Suspensions for intravenous (IV) injection: a review of development, preclinical and clinical aspects.

There has been growing interest in nanoparticles as an approach to formulate poorly soluble drugs. Besides enhanced dissolution rates, and thereby, improved bioavailability, nanoparticles can also provide targeting capabilities when injected intravenously. The latter property has led to increased research and development activities for intravenous suspensions. The first intravenously administered nanoparticulate product, Abraxane (a reformulation of paclitaxel), was approved by the FDA in 2006. Additional clinical trials have been conducted or are ongoing for multiple other indications such as oncology, infective diseases, and restenosis. This article reviews various challenges associated with developing intravenous nanosuspension dosage forms. In addition, various formulation considerations specific to intravenous nanosuspensions as well as reported findings from various clinical studies have been discussed.

Itraconazole IV nanosuspension enhances efficacy through altered pharmacokinetics in the rat.

The goal of this research was to evaluate an intravenous itraconazole nanosuspension dosage form, relative to a solution formulation, in the rat. Itraconazole was formulated as a nanosuspension by a tandem process of microcrystallization followed by homogenization. Acute toxicity, pharmacokinetics, and distribution were studied in the rat, and compared with a solution formulation of itraconazole. Efficacy was studied in an immunocompromised rat model, challenged with a lethal dose of either itraconazole-sensitive or itraconazole-resistant C. albicans. Itraconazole nanosuspension was tolerated at significantly higher doses compared with a solution formulation. Pharmacokinetics of the nanosuspension were altered relative to the solution formulation. C(max) was reduced and t(1/2) was much prolonged. This occurred due to distribution of the nanosuspension to organs of the monocyte phagocytic system (MPS), followed by sustained release from this IV depot. The higher dosing of the drug, enabled in the case of the nanosuspension, led to higher kidney drug levels and reduced colony counts. Survival was also shown to be superior relative to the solution formulation. Thus, formulation of itraconazole as a nanosuspension enhances efficacy of this antifungal agent relative to a solution formulation, because of altered pharmacokinetics, leading to increased tolerability, permitting higher dosing and resultant tissue drug levels.

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.

Nanotechnology: a focus on nanoparticles as a drug delivery system.

This review will provide an in-depth discussion on the previous development of nanoparticle-based drug delivery systems (DDS) and discuss original research data that includes the therapeutic enhancement of antiretroviral therapy. The use of nanoparticle DDS will allow practitioners to use drugs to target specific areas of the body. In the treatment of malignancies, the use of nanoparticles as a DDS is making measurable treatment impact. Medical imaging will also utilize DDS to illuminate tumors, the brain, or other cellular functions in the body. The utility of nanoparticle DDS to improve human health is potentially enormous.

Pharmacokinetics of drugs administered in nanosuspension.

Extract: Nanosuspensions are submicron-sized crystalline drug particles that are stabilized by coatings of surfactant (a surface-active agent which reduces surface tension) to produce stable pharmaceutical formulations. Their development arose in response to the evolving needs of the medicinal chemist over the last twenty years. During this period, the implementation of high throughput screening tests has enabled the identification of molecular drug candidates with greater affinity for protein receptor targets. In general, such lead compounds have proved to be larger and more hydrophobic (water-hating) than previous candidates, thus permitting the exclusion of water from the receptor surface and increasing the hydrophobic interaction with the target. While effective, as demonstrated in in-vitro binding assays, such compounds often lack sufficient water solubility, a parameter required for successful, subsequent development.

Nanosuspensions in drug delivery.

A surprisingly large proportion of new drug candidates emerging from drug discovery programmes are water insoluble, and therefore poorly bioavailable, leading to abandoned development efforts. These so-called 'brickdust' candidates can now be rescued by formulating them into crystalline nanosuspensions. In the process of overcoming issues involving solubility, additional pharmacokinetic benefits of the drugs so formulated have come to be appreciated. As such, insolubility issues of the past have provoked a paradigm change, which now offers novel solutions for innovative drugs of the future.