Pulmonary Delivery of Liposomal Drugs

Abstract

This overview will discuss our studies of liposomes aerosols to treat diseases of the lung and will entail (i) formulation and characterization of liposome aerosols, including dry liposome powder aerosols, (ii) modulation of the pharmacokinetic profile of liposomal drugs delivered by aerosol or intratracheal instillation, (iii) liposome-alveolar macrophage interactions in vitro and in vivo, and (iv) safety of liposome aerosols in vivo in mice, sheep and healthy human volunteers. Water-soluble agents can be retained in liposomes during aerosolization with air-pressure nebulizers within certain limitations of liposome composition, size, and operating conditions. Dry powder liposome aerosols have been formulated and deliver water-soluble encapsulated substances efficiently. Pharmacokinetic profiles of liposomal drugs delivered via intratracheal instillation exhibit typical slow release plasma profiles indicating that the carrier is the rate-limiting barrier for release. Accordingly, pulmonary mean residence times are significantly prolonged and systemic concentrations remain low. Liposomes do not inhibit the phagocytic activity of alveolar macrophages in vitro and in vivo, have no apparent histopathologic effects on lung architecture even after chronic administration, and do not alter dynamic compliance, lung resistance, p_aO₂ and p_aCO₂ in awake, unanesthetized sheep and in healthy human volunteers. In conclusion, liposomes are a promising innocuous aerosol delivery system for drugs to achieve prolonged localized drug concentrations in the lung or intracellular drug targeting to alveolar macrophages.     

Multiple mechanisms of reflex bronchospasm in guinea pigs.

The mechanisms of histamine- and bradykinin-induced reflex bronchospasm were determined in anesthetized guinea pigs. With intravenous administration, both autacoids evoked dose-dependent increases in tracheal cholinergic tone. Vagotomy or atropine prevented these tracheal reflexes. When delivered as an aerosol, bradykinin readily increased tracheal cholinergic tone, whereas histamine aerosols were much less effective at inducing tracheal reflexes. Also, unlike histamine, bradykinin could evoke profound increases in cholinergic tone without directly or indirectly (e.g., prostanoid dependent) inducing measurable airway smooth muscle contraction resulting in bronchospasm. Neither autacoid required de novo synthesis of prostanoids or nitric oxide to induce reflex tracheal contractions. Combined cyclooxygenase inhibition and tachykinin-receptor antagonism did, however, abolish all effects of bradykinin in the airways, whereas responses to histamine were unaffected by these pretreatments. The data indicate that histamine and bradykinin initiate reflex bronchospasm by differential activation of vagal afferent nerve subtypes. We speculate that selective activation of either airway C fibers or airway rapid adapting receptors can initiate reflex bronchospasm.     

Calcium chelators increase airway responsiveness

To test the effect of calcium chelation on airway responsiveness to methacholine, purebred Basenji dogs were pretreated with a calcium-chelating aerosol (edetate disodium, Na2EDTA) or a placebo aerosol (saline or CaNa2-EDTA) and then challenged with methacholine bromide aerosols. The lowest dose of methacholine (0.15 mg/ml) produced no change in pulmonary resistance (RL) following pretreatment with the placebo aerosols, but RL increased (P less than 0.05) by 5.1 +/- 1.2 (SE) cmH2O X l-1 X s following pretreatment with Na2EDTA. The highest dose of methacholine (1.5 mg/ml) increased RL in all animals, but the increase was greater (P less than 0.01) following pretreatment with Na2EDTA (9.5 +/- 1.9 cm H2O X l-1 X s) than following pretreatment with a placebo aerosol (6.4 +/- 1.5 cmH2O X l-1 X s). These studies show that calcium-chelating aerosols significantly increase airway responsiveness and suggest that a localized calcium deficit may contribute to hyperresponsive airway disease.     

Experimental study of a gentamicin aerosol]

The study of gentamicin aerosol showed its relative innocuousness: it did not inhibit the growth and development of young animals, did not induce pathological changes in the upper respiratory tract, kidneys, liver, heart and spleen on its prolonged use. Pathohistological examination revealed slight irritating effect of the gentamicin aerosol in the lungs after its use in a dose of 8 or 25 mg/kg for 6 weeks. A procedure for investigating the effect of the aerosol on the activity of the trachea ciliated epithelium of warm blooded animals was developed. The gentamicin aerosols prepared from solutions of different concentrations (1 to 50 mg/ml) induced ingibition of the ciliated epithelium function at average from 15 to 35 per cent which was associated with the solution acidity (pH 4.54 to 4.82). Such a decrease in the function of the ciliated epithelium due to the antibiotic aerosol use was a factor prolonging the antibiotic retention time in the respiratory organs. It was found that aqueous solutions of drugs used for inhalation, such as ephedrin, euphelin, dimedrol, N-acetyl-L-cystein and others had no effect on the activity of gentamicin and may be used with it in a form of aerosols.     

Chemotherapy of Campylobacter infections]

A total of 600 patients with suspected alimentary food poisoning were hospitalized. The ++clinico-laboratory findings showed that 27 (4.5 per cent) of them had Campylobacter infection. The cultures of Campylobacter jejuni and Campylobacter coli were isolated from 24 and 3 patients, respectively. The patients underwent complex pathogenetic treatment with oral rehydration saline solutions, symptomatic agents, enzymatic preparations and diet (the basic therapy) supplemented with biological bacterial preparations in less severe cases (8 patients). Antibacterial drugs such as furazolidone (roxytromycin), gentamicin and levomycetin, as well as fluoroquinolones such as ofloxacin and ciprofloxacin were additionally used in the treatment of both the patients with the generalized infection and those with more severe processes of the disease, pronounced diarrhea with blood traces and persisting isolation of Campylobacter (12 patients). The most favourable results in the treatment of more severe patients with Campylobacter infection were obtained with the fluoroquinolones used after inadequately efficient therapy with furazolidone or antibiotics especially in the cases with repeated isolation of campylobacteria.     

Pulmonary drug delivery and retention: A computational study to identify plausible parameters based on a coupled airway-mucus flow model

Pulmonary drug delivery systems rely on inhalation of drug-laden aerosols produced from aerosol generators such as inhalers, nebulizers etc. On deposition, the drug molecules diffuse in the mucus layer and are also subjected to mucociliary advection which transports the drugs away from the initial deposition site. The availability of the drug at a particular region of the lung is, thus, determined by a balance between these two phenomena. A mathematical analysis of drug deposition and retention in the lungs is developed through a coupled mathematical model of aerosol transport in air as well as drug molecule transport in the mucus layer. The mathematical model is solved computationally to identify suitable conditions for the transport of drug-laden aerosols to the deep lungs. This study identifies the conditions conducive for delivering drugs to the deep lungs which is crucial for achieving systemic drug delivery. The effect of different parameters on drug retention is also characterized for various regions of the lungs, which is important in determining the availability of the inhaled drugs at a target location. Our analysis confirms that drug delivery efficacy remains highest for aerosols in the size range of 1-5 μm. Moreover, it is observed that amount of drugs deposited in the deep lung increases by a factor of 2 when the breathing time period is doubled, with respect to normal breathing, suggesting breath control as a means to increase the efficacy of drug delivery to the deep lung. A higher efficacy also reduces the drug load required to be inhaled to produce the same health effects and hence, can help in minimizing the side effects of a drug.     

Effect of formulation on the stability and aerosol performance of a nebulized antibody

Most monoclonal antibodies (mAbs) are administered to patients intravenously to ensure high bioavailability as rapidly as possible. The airways, however, are an attractive delivery route for mAbs for the treatment of lung diseases, making it possible to increase their concentration in the target organ while limiting their systemic passage. Several challenges must be overcome for translation into clinical practice. For example, the drug and device must be paired for the efficient and reliable deposition of a pharmacologically active and safe mAb in the lung region of interest. Mesh nebulizers appear to be the most effective aerosol-producing devices for delivering large amounts of biopharmaceutical while limiting protein instability during nebulization. We used metrological and analytic methods to analyze the effect of both antibody concentration and surfactant addition on aerosol performance and antibody integrity. These two factors had a limited effect on aerosol performance, but affected antibody aggregation. The addition of surfactants to antibody formulations at concentrations appropriate for lung administration markedly reduced the formation of medium or large aggregates, as shown by dynamic light scattering and fluorescence microscopy. Aggregation was also dependent on the type of mesh nebulizer, highlighting the need to optimize drug and device together.     

Effect of formulation on the stability and aerosol performance of a nebulized antibody

Most monoclonal antibodies (mAbs) are administered to patients intravenously to ensure high bioavailability as rapidly as possible. The airways, however, are an attractive delivery route for mAbs for the treatment of lung diseases, making it possible to increase their concentration in the target organ while limiting their systemic passage. Several challenges must be overcome for translation into clinical practice. For example, the drug and device must be paired for the efficient and reliable deposition of a pharmacologically active and safe mAb in the lung region of interest. Mesh nebulizers appear to be the most effective aerosol-producing devices for delivering large amounts of biopharmaceutical while limiting protein instability during nebulization. We used metrological and analytic methods to analyze the effect of both antibody concentration and surfactant addition on aerosol performance and antibody integrity. These two factors had a limited effect on aerosol performance, but affected antibody aggregation. The addition of surfactants to antibody formulations at concentrations appropriate for lung administration markedly reduced the formation of medium or large aggregates, as shown by dynamic light scattering and fluorescence microscopy. Aggregation was also dependent on the type of mesh nebulizer, highlighting the need to optimize drug and device together.     

Skin as a potential source of infectious foot and mouth disease aerosols

This review examines whether exfoliated, virus-infected animal skin cells could be an important source of infectious foot and mouth disease virus (FMDV) aerosols. Infectious material rafting on skin cell aerosols is an established means of transmitting other diseases. The evidence for a similar mechanism for FMDV is: (i) FMDV is trophic for animal skin and FMDV epidermis titres are high, even in macroscopically normal skin; (ii) estimates for FMDV skin cell aerosol emissions appear consistent with measured aerosol emission rates and are orders of magnitude larger than the minimum infectious dose; (iii) the timing of infectious FMDV aerosol emissions is consistent with the timing of high FMDV skin concentrations; (iv) measured FMDV aerosol sizes are consistent with skin cell aerosols; and (v) FMDV stability in natural aerosols is consistent with that expected for skin cell aerosols. While these findings support the hypothesis, this review is insufficient, in and of itself, to prove the hypothesis and specific follow-on experiments are proposed. If this hypothesis is validated, (i) new FMDV detection, management and decontamination approaches could be developed and (ii) the relevance of skin cells to the spread of viral disease may need to be reassessed as skin cells may protect viruses against otherwise adverse environmental conditions.     

Electrical cardioversion-effectiveness of quinidine prophylaxis following countershock

Direct current countershock was given to 46 patients with chronic atrial fibrillation of diverse causes, with successful conversion in 87 per cent of them. Patients were observed while receiving quinidine prophylaxis, with serial determination of serum quinidine levels, for up to 22 months. Two quinidine preparations were employed, one a long-acting form, in various dosage regimens and both preparations were found to be effective prophylactic agents for maintaining reliable serum quinidine levels. Fifty per cent of patients remained in normal sinus rhythm at three months, 28 per cent at six months and 13 per cent at twelve months.     

Electrical Cardioversion—Effectiveness of Quinidine Prophylaxis Following Countershock

Direct current countershock was given to 46 patients with chronic atrial fibrillation of diverse causes, with successful conversion in 87 per cent of them. Patients were observed while receiving quinidine prophylaxis, with serial determination of serum quinidine levels, for up to 22 months. Two quinidine preparations were employed, one a long-acting form, in various dosage regimens and both preparations were found to be effective prophylactic agents for maintaining reliable serum quinidine levels. Fifty per cent of patients remained in normal sinus rhythm at three months, 28 per cent at six months and 13 per cent at twelve months.     

Isoelectric fractionation of sulfated glycoproteins on polytetrafluoroethylene-coated columns

The isolation of the acidic glycoproteins in mucous secretions is impeded by their low solubility and high viscosity in aqueous media. Mucin preparations have therefore been treated with alkali (1), detergents or reducing agents (2), boiling (3) or proteolytic enzymes (4,5) prior to conventional fractionation procedures. However, such agents may irreversibly alter the protein structure (1–5). In the present paper a fractionation method based on isoelectric focusing, and without sample pretreatment, is described.     

大气气溶胶增加对作物的影响研究进展

大气气溶胶是指悬浮在大气中的固态和液态颗粒物总称。近年来空气污染带来了一些区域大气气溶胶浓度上升明显,一定程度上对作物生长环境带来影响。国内外关于大气气溶胶增加可能对作物产生的影响表现在:(1)大气气溶胶增加会导致直接辐射减少,而散射辐射可能会有一定程度增加;散射辐射增加有利于一些作物整个冠层光合能力的提高。(2)大气气溶胶带来辐射的改变也会影响近地面小气候环境,尤其是大气昼夜温度变化,从而影响作物干物质积累;而辐射与温度的改变同时也会影响农田蒸散和最终水分利用效率。(3)大气气溶胶形成的干沉降会停留在叶片表面,减少光合有效辐射到达叶片的量,同时对作物叶片结构和功能产生直接影响。在总结国内外研究进展基础上,提出未来关于大气气溶胶增加对作物影响需要进一步明确大气气溶胶带来的作物生长环境改变对作物碳同化、积累和消耗的影响以及直接和散射辐射比例改变如何影响作物光合和蒸腾的相互关系,通过全面系统的研究大气气溶胶对作物产量形成的影响机制,提出有针对性的田间应对技术。     

Repeated antigen challenge induced airway hyperresponsiveness to neurokinin A and vagal non-adrenergic, non-cholinergic (NANC) stimulation in guinea pigs.

The effect of repeated weekly antigen challenges by aerosol on bronchopulmonary responses to ACh, histamine, neurokinin A or atropine-resistant (NANC) component of vagal stimulation, has been studied in guinea pigs. Bronchospastic responses were measured in anaesthetized animals, 7 days after the last challenge with antigen (or vehicle). No difference was observed between control and antigen challenged guinea pigs in their responsiveness to acetylcholine (1-300 mumol kg-1 i.v.) or histamine (1-300 mumol kg-1 i.v.). On the other hand, amplitude of bronchospasm induced by neurokinin A (1-3 mumol kg-1 i.v.) or NANC vagal stimulation (20 Hz, 1 msec, 10 V, trains of 5-20 sec) was significantly increased in guinea pigs previously challenged with antigen, as compared to controls. These results suggest that repetitive antigen exposure in sensitized guinea pigs generates an increase in the responsiveness to exogenously administered or endogenously released tachykinins, at a time when no generalized hyperresponsiveness to other spasmogens could be observed.     

Whole-Body Nanoparticle Aerosol Inhalation Exposures

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle inhalation toxicology studies, the aerosols in a chamber housing the experimental animals must have: 1) a steady concentration maintained at a desired level for the entire exposure period; 2) a homogenous composition free of contaminants; and 3) a stable size distribution with a geometric mean diameter < 200 nm and a geometric standard deviation σ_g < 2.5 ⁵. The generation of aerosols containing nanoparticles is quite challenging because nanoparticles easily agglomerate. This is largely due to very strong inter-particle forces and the formation of large fractal structures in tens or hundreds of microns in size ⁶, which are difficult to be broken up. Several common aerosol generators, including nebulizers, fluidized beds, Venturi aspirators and the Wright dust feed, were tested; however, none were able to produce nanoparticle aerosols which satisfy all criteria ⁵.A whole-body nanoparticle aerosol inhalation exposure system was fabricated, validated and utilized for nano-TiO₂ inhalation toxicology studies. Critical components: 1) novel nano-TiO₂ aerosol generator; 2) 0.5 m³ whole-body inhalation exposure chamber; and 3) monitor and control system. Nano-TiO₂ aerosols generated from bulk dry nano-TiO₂ powders (primary diameter of 21 nm, bulk density of 3.8 g/cm³) were delivered into the exposure chamber at a flow rate of 90 LPM (10.8 air changes/hr). Particle size distribution and mass concentration profiles were measured continuously with a scanning mobility particle sizer (SMPS), and an electric low pressure impactor (ELPI). The aerosol mass concentration (C) was verified gravimetrically (mg/m³). The mass (M) of the collected particles was determined as M = (M_post-M_pre), where M_preand M_post are masses of the filter before and after sampling (mg). The mass concentration was calculated as C = M/(Q*t), where Q is sampling flowrate (m³/min), and t is the sampling time (minute). The chamber pressure, temperature, relative humidity (RH), O₂ and CO₂ concentrations were monitored and controlled continuously. Nano-TiO₂ aerosols collected on Nuclepore filters were analyzed with a scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis.In summary, we report that the nano-particle aerosols generated and delivered to our exposure chamber have: 1) steady mass concentration; 2) homogenous composition free of contaminants; 3) stable particle size distributions with a count-median aerodynamic diameter of 157 nm during aerosol generation. This system reliably and repeatedly creates test atmospheres that simulate occupational, environmental or domestic ENM aerosol exposures.     

EPINEPHRINE HAND NEBULIZER IN ASTHMA—Technique of Use,Clinical Aspects

It behooves the physician seeking relief for asthmatic patients not to be casual about the epinephrine hand nebulizer and the manner in which it is used. Patients who claim to get no relief from the nebulizer should be asked to demonstrate their technique. If the nebulizer produces a hardly visible mist, it should be discarded. For many patients, the goal in spraying by hand must be the production of more or less continuous and voluminous aerosol, regardless of the phase of respiration, in order to effect relief. If the respiratory pattern has not deviated too far from normal in rate and depth, inhalations may be carried out in courses or cycles of about a half-minute duration and spaced a few minutes apart. If the respiratory pattern is abnormal during the act of spraying, it must be corrected.Inhaled epinephrine aerosols as constituted today appear to be somewhat irritating to the mouth, throat, and upper portion of the pulmonary tract of some persons, but it has not been convincingly demonstrated that serious and permanent damage to the lower respiratory tract of humans can occur from long-continued use of inhaled epinephrine.     

Epinephrine hand nebulizer in asthma; technique of use,clinical aspects

It behooves the physician seeking relief for asthmatic patients not to be casual about the epinephrine hand nebulizer and the manner in which it is used. Patients who claim to get no relief from the nebulizer should be asked to demonstrate their technique. If the nebulizer produces a hardly visible mist, it should be discarded. For many patients, the goal in spraying by hand must be the production of more or less continuous and voluminous aerosol, regardless of the phase of respiration, in order to effect relief. If the respiratory pattern has not deviated too far from normal in rate and depth, inhalations may be carried out in courses or cycles of about a half-minute duration and spaced a few minutes apart. If the respiratory pattern is abnormal during the act of spraying, it must be corrected. Inhaled epinephrine aerosols as constituted today appear to be somewhat irritating to the mouth, throat, and upper portion of the pulmonary tract of some persons, but it has not been convincingly demonstrated that serious and permanent damage to the lower respiratory tract of humans can occur from long-continued use of inhaled epinephrine.     

MILK LET-DOWN—The Use of Intranasal Oxytocin for Nursing Mothers

A study was carried out to determine whether intranasal spraying with a solution of oxytocin was an effective way to increase flow of milk in mothers who wished to breast-feed their babies.A hundred such women were given the drug intramuscularly for two days before they were to begin nursing. Then administration by that means was discontinued and 50 of the hundred were given oxytocin nasal spray kits for use at home. In general the patients receiving the spray kits were those who were apprehensive about sufficient lactation, those who had had previous difficulty and those who had flat, inverted or tender nipples.Results were not much different between the 50 women who used the spray and the 50 controls, but since the former group included the “difficult” cases, some benefit may be attributed to the aerosol therapy. Ninety per cent of those who used it said they would be willing to use it again.     

Subtotal gastric resection; an appraisal of a means of treatment of benign peptic ulceration of the stomach and duodenum

In a series of 400 cases of subtotal gastric resection for the treatment of benign ulceration of the stomach and duodenum, the mortality, morbidity and recurrence rate was acceptably low. Fifty-six per cent of the patients had a perfect result, 38 per cent satisfactory, and 6 per cent unsatisfactory. However, the postoperative nutritional status was sufficiently interfered with in a number of patients whose preoperative weight was subnormal that the routine adoption of 75 per cent gastric resection must be questioned. Vagotomy with either pyloroplasty or partial resection may prove to be the most valuable procedure for patients of this type. In properly selected patients, however, gastric resection is a rewarding procedure for both patient and surgeon.     

SUBTOTAL GASTRIC RESECTION—An Appraisal of a Means of Treatment of Benign Peptic Ulceration of the Stomach and Duodenum

In a series of 400 cases of subtotal gastric resection for the treatment of benign ulceration of the stomach and duodenum, the mortality, morbidity and recurrence rate was acceptably low. Fifty-six per cent of the patients had a perfect result, 38 per cent satisfactory, and 6 per cent unsatisfactory. However, the postoperative nutritional status was sufficiently interfered with in a number of patients whose preoperative weight was subnormal that the routine adoption of 75 per cent gastric resection must be questioned. Vagotomy with either pyloroplasty or partial resection may prove to be the most valuable procedure for patients of this type. In properly selected patients, however, gastric resection is a rewarding procedure for both patient and surgeon.