Comparative Bioavailability of Two Formulations of Biopharmaceutical Classification System (BCS) Class IV Drugs: A Case Study of Lopinavir/Ritonavir.

BACKGROUND: Lopinavir/ritonavir (LPV/r-A, Kaletra®), a fixed dose, co-formulated antiviral therapy for the treatment of HIV infection has been used worldwide for over two decades. Both active substances have low solubility in water and low membrane permeability. LPV/r-A tablets contain key excipients critical to ensuring acceptable bioavailability of lopinavir and ritonavir in humans. An established dog pharmacokinetic model demonstrated several generic LPV/r tablet formulations have significant oral bioavailability variability compared to LPV/r-A. METHODS: Analytical characterizations of LPV/r-B tablets were performed and a clinical study was conducted to assess the relative bioavailability of Kalidavir® (LPV/r-B) 400/100 mg tablets relative to Kaletra® (LPV/r-A) 400/100 mg tablets under fasting conditions. RESULTS: The presence of active substances were confirmed in LPV/r-B tablets in an apparent amorphous state at essentially the labeled amounts, and dissolution profiles were generally similar to LPV/r-A tablets. Excipients in the tablet formulation were found to be variable and deviate from the labeled composition. Lopinavir and ritonavir exposures (AUC) following LPV/r-B administration were approximately 90% and 20% lower compared to that of LPV/r-A. CONCLUSIONS: LPV/r-B was not shown to be bioequivalent to LPV/r-A.

Development and Palatability Assessment of Norvir® (Ritonavir) 100 mg Powder for Pediatric Population.

: Norvir® (ritonavir) is a Biopharmaceutical Classification System Class IV compound with poor solubility in water (~5 µg/mL) and limited oral bioavailability. Early stage development efforts were focused on an oral solution (OS) which provided reasonable bioavailability but exhibited taste-masking challenges and required the use of solvents with potential pediatric toxicity. Norvir® oral powder, 100 mg (NOP) was developed to replace OS. The objective of this study is to provide an overview of the development of NOP and palatability assessment strategy. Palatability of NOP was assessed using the flavor profile method: (1) As an aqueous suspension dose/response and (2) evaluation with foods. The dose/response sensory analysis indicated that NOP has strong intensity bitterness and burnt aromatics (3 on the 0⁻3 flavor profile scale) at the clinical dose (100 mg/10 mL) and the recognition threshold was determined to be 0.3 mg/10 mL. To improve palatability, 100 mg/10 mL NOP aqueous suspension was evaluated with foods. Consuming foods high in fat and/or sugar content after NOP administration successfully reduced bitterness to a 1.5 intensity. In summary, NOP provides dose flexibility, enhanced stability, eliminated solvents, and maintains consistent bioavailability, with reduced bitterness and improved palatability via administration with common food products.

Transient Receptor Potential Cation Channel Subfamily M Member 8 channels mediate the anti-inflammatory effects of eucalyptol.

BACKGROUND AND PURPOSE: Eucalyptol (1,8-cineol), the major ingredient in the essential oil of eucalyptus leaves and other medicinal plants, has long been known for its anti-inflammatory properties. Eucalyptol interacts with the TRP cation channels among other targets, but it is unclear which of these mediates its anti-inflammatory effects. EXPERIMENTAL APPROACH: Effects of eucalyptol were compared in wild-type and TRPM8 channel-deficient mice in two different models: footpad inflammation elicited by complete Freund's adjuvant (CFA) and pulmonary inflammation following administration of LPS. Oedema formation, behavioural inflammatory pain responses, leukocyte infiltration, enzyme activities and cytokine and chemokine levels were measured. KEY RESULTS: In the CFA model, eucalyptol strongly attenuated oedema and mechanical allodynia and reduced levels of inflammatory cytokines (IL-1ß, TNF-α and IL-6), effects comparable with those of ibuprofen. In the LPS model of pulmonary inflammation, eucalyptol treatment diminished leukocyte infiltration, myeloperoxidase activity and production of TNF-α, IL-1ß, IFN-γ and IL-6. Genetic deletion of TRPM8 channels abolished the anti-inflammatory effects of eucalyptol in both models. Eucalyptol was at least sixfold more potent on human, than on mouse TRPM8 channels. A metabolite of eucalyptol, 2-hydroxy-1,8-cineol, also activated human TRPM8 channels. CONCLUSION AND IMPLICATIONS: Among the pharmacological targets of eucalyptol, TRPM8 channels were essential for its anti-inflammatory effects in mice. Human TRPM8 channels are more sensitive to eucalyptol than rodent TRPM8 channels explaining the higher potency of eucalyptol in humans. Metabolites of eucalyptol could contribute to its anti-inflammatory effects. The development of more potent and selective TRPM8 agonists may yield novel anti-inflammatory agents.

Inhalation dosimetry modeling provides insights into regional respiratory tract toxicity of inhaled diacetyl.

Vapor dosimetry models provide a means of assessing the role of delivered dose in determining the regional airway response to inspired vapors. A validated hybrid computational fluid dynamics physiologically based pharmacokinetic model for inhaled diacetyl has been developed to describe inhaled diacetyl dosimetry in both the rat and human respiratory tracts. Comparison of the distribution of respiratory tract injury with dosimetry estimates provides strong evidence that regional delivered dose rather than regional airway tissue sensitivity to diacetyl-induced injury is the critical determinant of the regional respiratory tract response to this water soluble reactive vapor. In the rat, inhalation exposure to diacetyl causes much lesser injury in the distal bronchiolar airways compared to nose and large tracheobronchial airways. The degree of injury correlates very strongly to model based estimates of local airway diacetyl concentrations. According to the model, regional dosimetry patterns of diacetyl in the human differ greatly from those in the rat with much greater penetration of diacetyl to the bronchiolar airways in the lightly exercising mouth breathing human compared to the rat, providing evidence that rat inhalation toxicity studies underpredict the risk of bronchiolar injury in the human. For example, repeated exposure of the rat to 200ppm diacetyl results in bronchiolar injury; the estimated bronchiolar tissue concentration in rats exposed to 200ppm diacetyl would occur in lightly exercising mouth breathing humans exposed to 12ppm. Consideration of airway dosimetry patterns of inspired diacetyl is critical to the proper evaluation of rodent toxicity data and its relevance for predicting human risk.

Acetaminophen Attenuates House Dust Mite-Induced Allergic Airway Disease in Mice.

Epidemiologic evidence suggests that N-acetyl-para-aminophenol (APAP) may play a role in the pathogenesis of asthma, likely through pro-oxidant mechanisms. However, no studies have investigated the direct effects of APAP on the development of allergic inflammation. To determine the likelihood of a causal relationship between APAP and asthma pathogenesis, we explored the effects of APAP on inflammatory responses in a murine house dust mite (HDM) model of allergic airway disease. We hypothesized that APAP would enhance the development of HDM-induced allergic inflammation. The HDM model consisted of once daily intranasal instillations for up to 2 weeks with APAP or vehicle administration 1 hour prior to HDM during either week 1 or 2. Primary assessment of inflammation included bronchoalveolar lavage (BAL), cytokine expression in lung tissue, and histopathology. Contrary to our hypothesis, the effects of HDM treatment were substantially diminished in APAP-treated groups compared with controls. APAP-treated groups had markedly reduced airway inflammation: including decreased inflammatory cells in the BAL fluid, lower cytokine expression in lung tissue, and less perivascular and peribronchiolar immune cell infiltration. The anti-inflammatory effect of APAP was not abrogated by an inhibitor of cytochrome P450 (P450) metabolism, suggesting that the effect was due to the parent compound or a non-P450 generated metabolite. Taken together, our studies do not support the biologic plausibility of the APAP hypothesis that APAP use may contribute to the causation of asthma. Importantly, we suggest the mechanism by which APAP modulates airway inflammation may provide novel therapeutic targets for asthma.

Effects of Acetaminophen on Oxidant and Irritant Respiratory Tract Responses to Environmental Tobacco Smoke in Female Mice.

BACKGROUND: Although it is known that acetaminophen causes oxidative injury in the liver, it is not known whether it causes oxidative stress in the respiratory tract. If so, this widely used analgesic may potentiate the adverse effects of oxidant air pollutants. OBJECTIVES: The goal of this study was to determine if acetaminophen induces respiratory tract oxidative stress and/or potentiates the oxidative stress and irritant responses to an inhaled oxidant: environmental tobacco smoke (ETS). METHODS: Acetaminophen [100 mg/kg intraperitoneal (ip)] and/or sidestream tobacco smoke (as a surrogate for ETS, 5 mg/m3 for 10 min) were administered to female C57Bl/6J mice, and airway oxidative stress was assessed by loss of tissue antioxidants [estimated by nonprotein sulfhydryl (NPSH) levels] and/or induction of oxidant stress response genes. In addition, the effects of acetaminophen on airway irritation reflex responses to ETS were examined by plethysmography. RESULTS: Acetaminophen diminished NPSH in nasal, thoracic extrapulmonary, and lung tissues; it also induced the oxidant stress response genes glutamate-cysteine ligase, catalytic subunit, and NAD(P)H dehydrogenase, quinone 1, in these sites. ETS produced a similar response. The response to acetaminophen plus ETS was equal to or greater than the sum of the responses to either agent alone. Although it had no effect by itself, acetaminophen greatly increased the reflex irritant response to ETS. CONCLUSIONS: At supratherapeutic levels, acetaminophen induced oxidative stress throughout the respiratory tract and appeared to potentiate some responses to environmentally relevant ETS exposure in female C57Bl/6J mice. These results highlight the potential for this widely used drug to modulate responsiveness to oxidant air pollutants. CITATION: Smith GJ, Cichocki JA, Doughty BJ, Manautou JE, Jordt SE, Morris JB. 2016. Effects of acetaminophen on oxidant and irritant respiratory tract responses to environmental tobacco smoke in female mice. Environ Health Perspect 124:642-650; http://dx.doi.org/10.1289/ehp.1509851.

Advances in Inhalation Dosimetry Models and Methods for Occupational Risk Assessment and Exposure Limit Derivation.

The purpose of this article is to provide an overview and practical guide to occupational health professionals concerning the derivation and use of dose estimates in risk assessment for development of occupational exposure limits (OELs) for inhaled substances. Dosimetry is the study and practice of measuring or estimating the internal dose of a substance in individuals or a population. Dosimetry thus provides an essential link to understanding the relationship between an external exposure and a biological response. Use of dosimetry principles and tools can improve the accuracy of risk assessment, and reduce the uncertainty, by providing reliable estimates of the internal dose at the target tissue. This is accomplished through specific measurement data or predictive models, when available, or the use of basic dosimetry principles for broad classes of materials. Accurate dose estimation is essential not only for dose-response assessment, but also for interspecies extrapolation and for risk characterization at given exposures. Inhalation dosimetry is the focus of this paper since it is a major route of exposure in the workplace. Practical examples of dose estimation and OEL derivation are provided for inhaled gases and particulates.

Menthol attenuates respiratory irritation and elevates blood cotinine in cigarette smoke exposed mice.

Addition of menthol to cigarettes may be associated with increased initiation of smoking. The potential mechanisms underlying this association are not known. Menthol, likely due to its effects on cold-sensing peripheral sensory neurons, is known to inhibit the sensation of irritation elicited by respiratory irritants. However, it remains unclear whether menthol modulates cigarette smoke irritancy and nicotine absorption during initial exposures to cigarettes, thereby facilitating smoking initiation. Using plethysmography in a C57Bl/6J mouse model, we examined the effects of L-menthol, the menthol isomer added to cigarettes, on the respiratory sensory irritation response to primary smoke irritants (acrolein and cyclohexanone) and smoke of Kentucky reference 2R4 cigarettes. We also studied L-menthol's effect on blood levels of the nicotine metabolite, cotinine, immediately after exposure to cigarette smoke. L-menthol suppressed the irritation response to acrolein with an apparent IC50 of 4 ppm. Suppression was observed even at acrolein levels well above those necessary to produce a maximal response. Cigarette smoke, at exposure levels of 10 mg/m³ or higher, caused an immediate and marked sensory irritation response in mice. This response was significantly suppressed by L-menthol even at smoke concentrations as high as 300 mg/m³. Counterirritation by L-menthol was abolished by treatment with a selective inhibitor of Transient Receptor Potential Melastatin 8 (TRPM8), the neuronal cold/menthol receptor. Inclusion of menthol in the cigarette smoke resulted in roughly a 1.5-fold increase in plasma cotinine levels over those observed in mice exposed to smoke without added menthol. These findings document that, L-menthol, through TRPM8, is a strong suppressor of respiratory irritation responses, even during highly noxious exposures to cigarette smoke or smoke irritants, and increases blood cotinine. Therefore, L-menthol, as a cigarette additive, may promote smoking initiation and nicotine addiction.

Tissue sensitivity of the rat upper and lower extrapulmonary airways to the inhaled electrophilic air pollutants diacetyl and acrolein.

The target site for inhaled vapor-induced injury often differs in mouth-breathing humans compared with nose-breathing rats, thus complicating the use of rat inhalation toxicity data for assessment of human risk. We sought to examine sensitivity of respiratory/transitional nasal (RTM) and tracheobronchial (TBM) mucosa to two electrophilic irritant vapors: diacetyl and acrolein. Computational fluid dynamic physiologically based pharmacokinetic modeling was coupled with biomarker assessment to establish delivered dose-response relationships in RTM and TBM in male F344 rats following 6 h exposure to diacetyl or acrolein. Biomarkers included glutathione status, proinflammatory and antioxidant gene mRNA levels, and nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Modeling revealed that 0.0094-0.1653 µg acrolein/min-cm(2) and 3.9-21.6 µg diacetyl/min-cm(2) were deposited into RTM/TBM. Results indicate RTM and TBM were generally of similar sensitivity to diacetyl and acrolein. For instance, both tissues displayed induction of antioxidant and proinflammatory genes, and nuclear accumulation of Nrf2 after electrophile exposure. Hierarchical cellular response patterns were similar in RTM and TBM but differed between vapors. Specifically, diacetyl exposure induced proinflammatory and antioxidant genes concomitantly at low exposure levels, whereas acrolein induced antioxidant genes at much lower exposure levels than that required to induce proinflammatory genes. Generally, diacetyl was less potent than acrolein, as measured by maximal induction of transcripts. In conclusion, the upper and lower extrapulmonary airways are of similar sensitivity to inhaled electrophilic vapors. Dosimetrically based extrapolation of nasal responses in nose-breathing rodents may provide an approach to predict risk to the lower airways of humans during mouth-breathing.

The role of toxicological science in meeting the challenges and opportunities of hydraulic fracturing.

We briefly describe how toxicology can inform the discussion and debate of the merits of hydraulic fracturing by providing information on the potential toxicity of the chemical and physical agents associated with this process, individually and in combination. We consider upstream activities related to bringing chemical and physical agents to the site, on-site activities including drilling of wells and containment of agents injected into or produced from the well, and downstream activities including the flow/removal of hydrocarbon products and of produced water from the site. A broad variety of chemical and physical agents are involved. As the industry expands this has raised concern about the potential for toxicological effects on ecosystems, workers, and the general public. Response to these concerns requires a concerted and collaborative toxicological assessment. This assessment should take into account the different geology in areas newly subjected to hydraulic fracturing as well as evolving industrial practices that can alter the chemical and physical agents of toxicological interest. The potential for ecosystem or human exposure to mixtures of these agents presents a particular toxicological and public health challenge. These data are essential for developing a reliable assessment of the potential risks to the environment and to human health of the rapidly increasing use of hydraulic fracturing and deep underground horizontal drilling techniques for tightly bound shale gas and other fossil fuels. Input from toxicologists will be most effective when employed early in the process, before there are unwanted consequences to the environment and human health, or economic losses due to the need to abandon or rework costly initiatives.

Sex differences in the acute nasal antioxidant/antielectrophilic response of the rat to inhaled naphthalene.

Naphthalene is a nasal carcinogen, inducing respiratory adenomas in male and olfactory neuroblastomas in female rats, respectively. The reasons for the site and sex-specific tumorigenic response are unknown. Naphthalene is bioactivated to electrophilic metabolites; cytotoxicity followed by regenerative cell proliferation is likely involved in the tumorigenic response. To examine sex differences in the acute nasal response to naphthalene, male and female F344 rats were nose-only exposed to 0, 1, 3, 10, or 30 ppm naphthalene vapor for 4 or 6 h. Following exposure, respiratory/transitional mucosa (RTM) and olfactory mucosa (OM) were isolated and analyzed for markers of oxidant/electrophilic stress and/or toxicity, including reduced/oxidized glutathione levels (GSH/GSSG), mRNA levels of electrophile-responsive genes, and epithelial cytoxicity (as measured by membrane permeability to ethidium homodimer-1). Naphthalene caused significant depletion of GSH in RTM and OM with no increase in GSSG. Cytotoxicity was apparent at concentrations of 15 and 30 ppm. No consistent sex differences were observed in these responses. Sex differences were observed in the induction of antielectrophilic genes in OM: glutamyl cysteine ligase (catalytic subunit) (Gclc), NADPH quinone oxidase 1 (Nqo1), and heme oxygenase 1 (Hmox1) were all induced to a greater extent in the male OM compared with the female. No consistent sex differences were observed in the RTM. Although the mechanism of the sex difference in the RTM adenoma response remains enigmatic, sex differences in the induction of antioxidant/electrophile-responsive genes may contribute to the heightened sensitivity of the female OM to the carcinogenic effects of naphthalene.

TRPM8 is the principal mediator of menthol-induced analgesia of acute and inflammatory pain.

Menthol, the cooling natural product of peppermint, is widely used in medicinal preparations for the relief of acute and inflammatory pain in sports injuries, arthritis, and other painful conditions. Menthol induces the sensation of cooling by activating TRPM8, an ion channel in cold-sensitive peripheral sensory neurons. Recent studies identified additional targets of menthol, including the irritant receptor, TRPA1, voltage-gated ion channels and neurotransmitter receptors. It remains unclear which of these targets contribute to menthol-induced analgesia, or to the irritating side effects associated with menthol therapy. Here, we use genetic and pharmacological approaches in mice to probe the role of TRPM8 in analgesia induced by L-menthol, the predominant analgesic menthol isomer in medicinal preparations. L-menthol effectively diminished pain behavior elicited by chemical stimuli (capsaicin, acrolein, acetic acid), noxious heat, and inflammation (complete Freund's adjuvant). Genetic deletion of TRPM8 completely abolished analgesia by L-menthol in all these models, although other analgesics (acetaminophen) remained effective. Loss of L-menthol-induced analgesia was recapitulated in mice treated with a selective TRPM8 inhibitor, AMG2850. Selective activation of TRPM8 with WS-12, a menthol derivative that we characterized as a specific TRPM8 agonist in cultured sensory neurons and in vivo, also induced TRPM8-dependent analgesia of acute and inflammatory pain. L-menthol- and WS-12-induced analgesia was blocked by naloxone, suggesting activation of endogenous opioid-dependent analgesic pathways. Our data show that TRPM8 is the principal mediator of menthol-induced analgesia of acute and inflammatory pain. In contrast to menthol, selective TRPM8 agonists may produce analgesia more effectively, with diminished side effects.

Nasal dosimetry of inspired naphthalene vapor in the male and female B6C3F1 mouse.

Naphthalene vapor is a nasal cytotoxicant in the rat and mouse but is a nasal carcinogen in only the rat. Inhalation dosimetry is a critical aspect of the inhalation toxicology of inspired vapors and may contribute to the species differences in the nasal response. To define the nasal dosimetry of naphthalene in the B6C3F1 male and female mouse, uptake of naphthalene vapor was measured in the surgically isolated upper respiratory tract (URT) at inspiratory flow rates of 25 or 50 ml/min. Uptake was measured at multiple concentrations (0.5, 3, 10, 30 ppm) in controls and mice treated with the cytochrome P450 inhibitor 5-phenyl-1-pentyne. In both sexes, URT uptake efficiency was strongly concentration dependent averaging 90% at 0.5 ppm compared to 50% at 30 ppm (25 ml/min flow rate), indicating saturable processes were involved. Both uptake efficiency and the concentration dependence of uptake were significantly diminished by 5-phenyl-1-pentyne indicating inspired naphthalene vapor is extensively metabolized in the mouse nose with saturation of metabolism occurring at the higher concentrations. A hybrid computational fluid dynamic physiologically based pharmacokinetic model was developed for nasal dosimetry. This model accurately predicted the observed URT uptake efficiencies. Overall, the high URT uptake efficiency of naphthalene in the mouse nose indicates the absence of a tumorigenic response is not attributable to low delivered dose rates in this species.

In situ measurement of vapor uptake in the rodent upper respiratory tract.

The response of respiratory tract tissue target sites to inhaled vapors depends on the amount of inhaled vapor that is delivered to those sites. Direct measurement of vapor absorption within a specific airway of the living animal requires that the airway be isolated, which currently can only be performed on the upper airways. Towards this end, the upper respiratory tract (all airways anterior to the larynx) is surgically isolated in the anesthetized rat by tracheostomy and insertion of two endotracheal tubes, one leading anteriorly and the other posteriorly. The surgically manipulated animal is then placed in a nose-only inhalation chamber and test-vapor-laden air is drawn through the isolated upper airways under defined air flow conditions via the anterior endotracheal tube. The animal spontaneously respires clean air into the lungs during this procedure via the posterior endotracheal tube. The concentration of test vapor in air entering and the air exiting the upper airways is measured by gas chromatography, and the difference in concentrations provides a measure of vapor absorption in that site. A rich database is available on upper respiratory tract vapor absorption as measured by this methodology, to which newly obtained data can be compared. These data have been instrumental not only in understanding the regional airway toxicity of inspired vapors, but also for developing mathematical models to describe inhaled vapor dosimetry.

Modulation of sensory irritation responsiveness by adenosine and malodorants.

Respiratory tract reflex responses are an important defense mechanism against noxious airborne materials. This study was aimed at defining the effects of adenosine on sensory irritation responsiveness and its role in odorant-irritant interactions. These experiments were aimed at testing the hypothesis that adenosine, through the A2 receptor, enhances trigeminal nerve responses to multiple irritants and that odorants enhance responsiveness to irritants through A2 pathways in the female C57Bl/6 mouse. The adenosine precursor, AMP, immediately and markedly increased the sensory irritation response to capsaicin, cyclohexanone, and styrene, irritants that activate chemosensory nerves through differing receptor pathways. The neuromodulatory effect was blocked by the general adenosine receptor antagonist theophylline and by the A2 receptor-specific antagonist DMPX. Multiple odorants were examined, including R-carvone (spearmint), linalool (lavender), trimethylamine (rotting fish), mercaptoethanol, and ethyl sulfide (stench and rotten eggs). Of these, only mercaptoethanol and ethyl sulfide exhibited neuromodulatory effects, enhancing the sensory irritation response to styrene or cyclohexanone. This effect was blocked by theophylline and DMPX indicating the importance of adenosine A2 receptor pathways in this effect. These results highlight that trigeminal chemosensory responsiveness is not static, but can be quickly modulated by adenosine and select odors resulting in hyperresponsive states.

Biologically-based modeling insights in inhaled vapor absorption and dosimetry.

The lung is a route of entry and also a target site for inhaled vapors, therefore, knowledge of the total absorbed dose and/or the dose absorbed in each airway during inhalation exposure is essential. Vapor absorption characteristics result primarily from the fact that vapors demonstrate equilibrium/saturation behavior in fluids. Thus, during inhalation exposures blood and airway tissue vapor concentrations increase to a steady state value and increase no further no matter how long the exposure. High tissue concentrations can be obtained with highly soluble vapors, thus solubility, as measured by blood:air partition coefficient, is a fundamentally important physical/chemical characteristic of vapors. While it is classically thought that vapor absorption occurs only in the alveoli it is now understood that this is not the case. Soluble vapors can be efficiently absorbed in the airways themselves and do not necessarily penetrate to the alveolar level. Such vapors are more likely to injure the proximal than distal airways because that is the site of the greatest delivered dose. There are substantial species differences in airway vapor absorption between laboratory animals and humans making interpretation of laboratory animal inhalation toxicity data difficult. Airway absorption is dependent on vapor solubility and is enhanced by local metabolism and/or direct reaction within airway tissues. Modern simulation models that incorporate terms for solubility, metabolism, and reaction rate accurately predict vapor absorption patterns in both animals and humans and have become essential tools for understanding the pharmacology and toxicology of airborne vapors.

Menthol attenuates respiratory irritation responses to multiple cigarette smoke irritants.

Menthol, the cooling agent in peppermint, is added to almost all commercially available cigarettes. Menthol stimulates olfactory sensations, and interacts with transient receptor potential melastatin 8 (TRPM8) ion channels in cold-sensitive sensory neurons, and transient receptor potential ankyrin 1 (TRPA1), an irritant-sensing channel. It is highly controversial whether menthol in cigarette smoke exerts pharmacological actions affecting smoking behavior. Using plethysmography, we investigated the effects of menthol on the respiratory sensory irritation response in mice elicited by smoke irritants (acrolein, acetic acid, and cyclohexanone). Menthol, at a concentration (16 ppm) lower than in smoke of mentholated cigarettes, immediately abolished the irritation response to acrolein, an agonist of TRPA1, as did eucalyptol (460 ppm), another TRPM8 agonist. Menthol's effects were reversed by a TRPM8 antagonist, AMTB. Menthol's effects were not specific to acrolein, as menthol also attenuated irritation responses to acetic acid, and cyclohexanone, an agonist of the capsaicin receptor, TRPV1. Menthol was efficiently absorbed in the respiratory tract, reaching local concentrations sufficient for activation of sensory TRP channels. These experiments demonstrate that menthol and eucalyptol, through activation of TRPM8, act as potent counterirritants against a broad spectrum of smoke constituents. Through suppression of respiratory irritation, menthol may facilitate smoke inhalation and promote nicotine addiction and smoking-related morbidities.

A validated hybrid computational fluid dynamics-physiologically based pharmacokinetic model for respiratory tract vapor absorption in the human and rat and its application to inhalation dosimetry of diacetyl.

Diacetyl vapor is associated with bronchiolar injury in man but primarily large airway injury in the rat. The goal of this study was to develop a physiologically based pharmacokinetic model for inspired vapor dosimetry and to apply the model to diacetyl. The respiratory tract was modeled as a series of airways: nose, trachea, main bronchi, large bronchi, small bronchi, bronchioles, and alveoli with tissue dimensions obtained from the literature. Airborne vapor was allowed to absorb (or desorb) from tissues based on mass transfer coefficients. Transfer of vapor within tissues was based on molecular diffusivity with direct reaction with tissue substrates and/or metabolism being allowed in each tissue compartment. In vitro studies were performed to provide measures of diacetyl metabolism kinetics and direct reaction rates allowing for the development of a model with no unassigned variables. Respiratory tract uptake of halothane, acetone, ethanol and diacetyl was measured in male F344 rat to obtain data for model validation. The human model was validated against published values for inspired vapor uptake. For both the human and rat models, a close concordance of model estimates with experimental measurements was observed, validating the model. The model estimates that limited amounts of inspired diacetyl penetrate to the bronchioles of the rat (<2%), whereas in the lightly exercising human, 24% penetration to the bronchioles is estimated. Bronchiolar tissue concentrations of diacetyl in the human are estimated to exceed those in the rat by 40-fold. These inhalation dosimetric differences may contribute to the human-rat differences in diacetyl-induced airway injury.

Role of metabolic activation and the TRPA1 receptor in the sensory irritation response to styrene and naphthalene.

The current study was aimed at examining the role of cytochrome P450 (CYP450) activation and the electrophile-sensitive transient receptor potential ankyrin 1 receptor (TRPA1) in mediating the sensory irritation response to styrene and naphthalene. Toward this end, the sensory irritation to these vapors was measured in female C57Bl/6J mice during 15-min exposure via plethysmographic measurement of the duration of braking at the onset of each expiration. The sensory irritation response to 75 ppm styrene and 7 ppm naphthalene was diminished threefold or more in animals pretreated with the CYP450 inhibitor metyrapone, providing evidence of the role of metabolic activation in the response to these vapors. The sensory irritation response to styrene (75 ppm) and naphthalene (7.6 ppm) was virtually absent in TRPA1-/- knockout mice, indicating the critical role of this receptor in mediating the response. Thus, these results support the hypothesis that styrene and naphthalene vapors initiate the sensory irritation response through TRPA1 detection of their CYP450 metabolites.

Bioavailability of generic ritonavir and lopinavir/ritonavir tablet products in a dog model.

In this study, we explored the bioavailability in dogs and chemical potency of generic ritonavir and lopinavir/ritonavir tablet products manufactured by various pharmaceutical companies. Chemical potency of the products was examined by HPLC quantitation of ritonavir and lopinavir. Using a dog model, we determined point estimates for C(max) and AUC of ritonavir and lopinavir/ritonavir for eight generic products compared to Abbott's Norvir capsule and Kaletra tablet. Chemical potencies ranged from 79.0% to 104.6%. Point estimates for AUC in the generic tablet products ranged from 0.01 to 1.11, indicating that the relative bioavailability of these formulations was in the range of 1-111% compared to the branded products. This study showed significant variability in bioavailability in a dog model amongst generic tablet products containing the protease inhibitors ritonavir or lopinavir/ritonavir. The chemical potency of the generic products was not indicative of the plasma levels of ritonavir or lopinavir that were achieved. These results reinforce the need for human bioequivalence testing of generic products containing ritonavir or lopinavir/ritonavir to assure that efficacy in patients is not compromised prior to these products being made available to patients. Procurement policies of funding agencies should require such quality assurance processes.