Delivery of Clodronate Liposomes to the Lung

We are in the process of reviewing the publications related to pulmonary delivery of clodronate liposomes. This discussion will be updated with any pertinent information as we continue the review.

Summary A clodronate liposome suspension can be delivered directly to the lungs in order to deplete alveolar macrophages and other pulmonary phagocytic cells (i.e. interstitial macrophages) without any reported effects on phagocytic cells outside the lungs. Unlike  systemic administration, intratracheal administration of free clodronate at higher doses has also been shown to also deplete alveolar macrophages. However, endothelial cells were morphologically changed after free clodronate treatment introducing the possibility of inflammatory responses from these cells which could confound data from these studies. Therefore, liposomal clodronate remains as the preferred method for selectively depleting alveolar macrophages.

Another complicating factor with the free clodronate response is the fact that at least 1 method of pulmonary delivery (aerosolization by nebulizer) is known to disrupt liposomes similar to those used to encapsulate clodronate. Therefore, appropriate control experiments should be included when using aerosols produced with nebulizers.

Secondly, several studies have shown that various formulations of clodronate liposomes leak especially when introduced into a biological medium such as serum. Thus, all macrophage depletion studies should take into account the potential for free clodronate being released from liposomes immediately after dosing with liposomal clodronate.

Intravenous dosing has also been shown to deplete over 60% of alveolar macrophages in mice 48 hours post-treatment, while a combination of intracheal injection and i.v. dosing resulted in over 90% depletion.

There are 4 general methods for direct administration of clodronate liposomes to the lung. Aerosol administration, intratracheal admininstration, nasal instillation, and pharyngeal aspiration and their variations are discussed in detail. All of these methods have pros and cons, but ≥ 90% depletion of alveolar macrophages is attainable in several models either with pulmonary administration alone or in combination with systemic administration.

Introduction. The lining of the lungs is a mucosal surface directly accessible by environmental toxins and pathogens. Therefore, phagocytic cells patrol the pulmonary space awaiting attack by particulate materials, virus, bacteria or chemical agents. Alveolar macrophages (AM) are the most common targets for depletion, although pulmonary dendritic cells (DC) and interstitial macrophages (IM) have been reported to be depleted by clodronate liposomes [1]. Although known to phagocytose liposomes, neutrophils (PMN) are not killed by clodronate liposomes [2], but explanations as to why PMN are not killed remain hypothetical. Reports vary as to whether or not other types of phagocytic cells are killed by clodronate liposomes. This may have to do with the fact that other cell types only become phagocytic under certain conditions such as complement activation [3] or that clodronate liposomes do not have access to some phagocytic cells in vivo [4].

Free clodronate depletes as well, but with a caveat. A major difference in the delivery of clodronate, free or liposomal, to the lung is that free clodronate, at sufficient concentrations, has been shown to be just as effective as encapsulated clodronate in depleting alveolar macrophages. However, the free clodronate can also induces morphological changes in pulmonary endothelial cells and may even increase the influx of PMN compared to clodronate liposomes, perhaps due to the release of inflammatory mediators by the damaged endothelial cells [5]. Free clodronate depletion was confirmed in vitro for cultured rat alveolar macrophages by these authors and free clodronate also has demonstrated in vitro growth inhibition to the RAW 264 cell line [6]. While the in vitro results are not unexpected, the in vivo effects of free clodronate in the lung may require some changes in the dosing paradigm. Incidental release of clodronate from liposomes or dying macrophages have been postulated as potential sources of free clodronate with systemic adminstration, but free clodronate’s preclinical and clinical profile from oral or i.v. dosing confirms that it has very limited bioavailability and no substantial effects on tissue other than bone [8]. Berg, et. al. speculate that free clodronate does not affect other cells when dosed systemically due to its extremely short half-life in serum, but free clodronate does not readily cross the relatively tight and selective blood-gas barrier in the lung. Therefore, free clodronate may establish a sufficient concentration to either kill or adversely effect susceptible cells in the lung. If this speculation were true, there are at least two other factors which may contribute to the accumulation of free clodronate in the lung.

Beware of the effects of nebulization on clodronate liposomes. First, none the authors employing nebulization confirmed that their clodronate liposome formulations were stable to the process; more details are presented below. Based on nebulization studies referenced in that discussion, it is possible, if not probable, that the liposome preparations from each of these studies will release some of the encapsulated clodronate during nebulization. Although the total clodronate concentration delivered should not change, less than 95-100% of the clodronate will be encapsulated depending on what proportion of the liposomes are disrupted by nebulization. It is impossible to predict the resulting ratio of free-to-encapsulated clodronate because not only are the liposomes being disrupted, they are also reforming at different sizes and number of lamellae. Thus, the amount of clodronate encapsulated, which is dependent on the morphology of the liposomes (primarily size distribution and lamellarity), as well as the concentration of clodronate in which the liposomes form, will almost certainly be different from the initial preparation (pre-nebulization). In summary, both free and encapsulated clodronate is dosed in the aerosol. If the concentration of free clodronate is in the range shown to not only kill macrophages, but also affect other (i.e. endothelial, other?) pulmonary cells, any results could be confounded by the potential contribution of inflammatory responses from those cells. Hashimoto, et. al. [7] state that they see no histological anomalies with either free or encapsulated clodronate, but their estimation of the total amount of clodronate delivered to the lung in their study is much less than the amount of free clodronate Berg, et. al. dosed by intratracheal instillation.

Clodronate liposomes are inherantly “leaky”…and should be. Second, early studies on liposomal and free clodronate biodistribution in mice confirm that free clodronate is not retained in liver, spleen, lungs or blood [9]. Furthermore, in vitro leakage assessment of clodronate from liposomal formulations confirm that 20% or more of the clodronate is released from liposomes either in storage (presumably at 4°C) or immediately upon dilution (1:20) in saline or human serum. While serum did initially have a substantial influence on clodronate leakage (+ 15%), this effect diminished with time so that the % clodronate released from the liposomes at 3 hours either in buffer or serum were similar. Therefore, the van Rooijen clodronate liposome formulation used in the majority of published studies on macrophage depletion released at least 35% of the entrapped clodronate immediately after injection or instillation and clodronate continued to leak from the liposomes so that ~60% was released at 30 minutes. The leakage profiles presented in this paper strongly suggest that clodronate would continue to leak rapidly in an “infinite dilution” environment such as after intravenous injection. While leaky liposomes have been given the so-called “bad name”, this is another case (the number of cases continues to grow) where there is demonstrated efficacy despite the rather rapid release of the drug in the bloodstream. In fact, clodronate liposomes belongs to a more elite group of liposome formulations in which leakiness corresponds to efficacy. Mönkkönen, et. al. [10, 11] reported a systematic study on DSPC/DSPG/Chol liposomes encapsulating clodronate investigating the effects of size and charge (modulated by increasing amounts of DSPG) on encapsulation and efficacy. A very important result from this study was that DSPC/Chol clodronate liposomes (shown to leak very little in serum) did not inhibit the growth of the RAW 264 cells up to 100 µM clodronate (29 µg/ml) while the IC 50 (clodronate concentration at which 50% cell growth is inhibited compared to control) of the DSPC/DSPG/Chol formulations were all less than 10 µM.

More details on clodronate leak… Of the several interesting results from Mönkönnen, et. al., these are most pertinent to this discussion

  1. DSPC/Chol (66:33) clodronate liposomes are not effective at inhibiting the growth of RAW 264 macrophages 100 µM clodronate (29 µg/ml).
  2. DSPC/DSPG/Chol clodronate liposomes, in which (DSPC+DSPG) = 66 mole% / Chol = 33 mole% AND DSPG ≥ 25 mole% in the (DSPC+DSPG) mixture, completely inhibit the growth of RAW 264 macrophages at 100 µM clodronate.
  3. DSPC/Chol (66:33) liposomes loaded with calcein/buffer as the same osmotic strength as encapsulated clodronate (~600 mOsm) release <20% (all within the first few seconds) of their contents when incubated in culture medium, human serum or synovial fluid and monitored for 200 s. (Calcein is a fluorescent dye which may or may not leak at the same rate as clodronate, however this assay is often used to estimate the stability of a liposomal formulation.)
  4. The analogous formulations containing DSPG (as described in point 2.) under the conditions described in point 3. release 40-80% of their contents over this time period.
  5. The leakage in the DSPG-containing formulations were completely ameliorated by the addition of 4 mM EDTA to the incubation media suggesting that Ca 2+ /Mg 2+ was responsible for the leakage. (Multvalent cations bind to negatively charged lipids, such as DSPG, causing both inter- and intra-liposomal membrane disruption due to the bridging or clustering of the negatively charged lipid molecules.)
  6. When the liposomes described in points 3 and 4 were prepared with isoosmotic calcein, no leakage occurred in any formulation except 100 mole% DSPG further suggesting that the hyperosmotic nature of the encapsulated clodronate contributes to the “leakiness” of various formulations. (However, the earlier Buiting, et. al. leakage study claims that osmotic stress did not contribute to the leakage of those liposomes.)
  7. When the DSPG clodronate liposomes were extruded (avg diameter reduced to 128 nm), they were able to inhibit the growth of L929 fibroblasts in vitro in the 5-100 µM range. (This suggests that small liposomes may be endocytosed by cells other than phagocytes reducing the selective nature of the depletion.)

When the van Rooijen EPC/Chol clodronate liposome formulation was included in this RAW 264 growth inhibition assay, no effect was observed in the 0-100 µM range. It is difficult to directly compare van Rooijen’s RAW 264 growth inhibition assay results to these because van Rooijen, et.al. (1988) do not state the actual clodronate concentration used in these assays, nor did they report an IC 50 . If we assume that the clodronate concentration in the preps based on the typical 20 mg clodronate/4 ml liposomes, their clodronate concentration range is 7-277 µM and the IC 50 is just under 10 µM.  While this value is in the same range that Mönkkönen, et. al. reported for their DSPC/DSPG/Chol preparations (and no activity for the van Rooijen formulation), we are assuming the clodronate concentration and the IC 50 is in the steep region of the growth curve (IC 40 = 4 µM, IC 60 = 24 µM).

Methods for Depletion of Pulmonary Phagocytes

Studies have shown that intravenous administration of clodronate liposomes for systemic macrophage depletion also depletes a significant portion of alveolar macrophages. For example, Koay, et. al. reported that intravenous injection of clodronate liposomes (200 µl X 5 mg/ml) resulted in a significant depletion (66%) of AM in bronchial alveolar lavage fluid (BALF) 48 hours post-treatment, while intratracheal injection (100 µl X 4-5 mg/ml depleted 77% of the AM [12]. When the C57BL/6 mice were dosed by both routes (100 µl i.t. + 200 µl i.v.), 90% of the AM were depleted. Therefore, systemic administration of clodronate liposomes can supplement intrapulmonary administration if sufficient depletion is not achieved by pulmonary dosing alone as has been discussed for other methods of local depletion.

There are 4 categories of published methods for delivering clodronate liposomes to the lung as listed in the tabs below. The number of papers listed for each method is taken from the database which was last updated on 09/23/11. A review comparing some of these methods provides useful descriptions and limitations of each method [13], but the conclusions are somewhat different than the data in these database papers provide. Since the author did not include a discussion on the delivery of  liposomes or other nanoparticles, the conclusions are expected to be skewed from those including pulmonary delivery of particles. One method which has not been reported as utilized to deliver clodronate liposomes, direct visual instillation (DVI), appears to warrant further investigation as it is much more efficient in delivering 125 I-albumin to the lung when compared to nasal instillation and intratracheal injection (92%, 77% and 48% respectively) [14]. This method also demonstrates an almost complete access to all parts of the lung by Evans blue dye when the appropriate parameters are applied to this endoscopic technique.

[tabs] [tab title=” Aerosol Administration “] Aerosol administration is performed either by nasal inhalation of nebulized (Aerotech II, 2 papers) clodronate liposomes or intratracheal aerosolization with the Penn-Century Microsprayer device (3 papers). A variation that essentially combines these two methods is referred to as intratracheal inhalation (ITIH) in which the animal’s trachea is cannulated followed by aerosol delivery of the liposomes via an external nebulizer (1 paper). Whole-body inhalation, which involves the delivery of the nebulized aerosol to a gas-tight chamber large enough for the animals to move about freely, is also demonstrated in this paper.

  • The main advantage of nebulization is that the aerosolized droplet size delivered is 10X smaller than that produced by the Microsprayer. Nebulized droplets are characterized with devices (cascade impactors) which allow the determination of the mean mass aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the droplets. The MMAD determined by the nebulization methods used for clodronate liposomes was 1.6 µm while the Microsprayer manufacturer [21] states that the MMAD of the particles delivered with the device used in those studies is 16-25 µm (do not to comfuse MMAD with liposome size). It has been shown that the smaller droplet will penetrate more effectively into the distal alveolar spaces [15]. However, unless the lab already has the necessary equipment and expertise to administer nebulized liposomes to animals, a significant financial investment in equipment as well as a substantial learning curve is required to effectively accomplish nebulized liposome dosing. Detailed reviews of the parameters which must be considered when nebulizing liposomes have been published [16-18] and nebulized liposome formulations are in late Phase III clinical trials [19]. An obvious issue in aerosolizing clodronate liposomes prepared by the standard protocol [2] is that the diameter of many, if not the majority, of the liposomes is larger than the MMAD of the nebulized droplet. Thus, either the liposomes will not be nebulized (remain in the chamber) or more commonly, the liposomes will be disrupted by the nebulization process. Some groups actually use nebulization as a method of reducing the size of liposomes before the unencapsulated drug is removed [20]. The rationale is that the liposomes will be disrupted and reform at progressively smaller sizes until they can be included within the aerosol droplet. Since the liposomes are reforming in a solution of drug, drug will be encapsulated in the reformed liposomes. In effect, nebulization is replacing the extrusion process and will, by default, generate an optimally sized liposome for aerosol delivery. The presence of unencapsulated drug was not an issue for their investigations; the encapsulated drug was more effective and the presence of unencapsulated drug was not toxic to the lung or the animal. This is not true for clodronate liposomes, therefore the investigator should determine the effect of nebulization on the clodronate liposome formulation prior to dosing the aerosol. Given these issues, delivery of nebulized clodronate liposomes by nasal inhalation of the aerosol was reported to deplete >95% of alveolar macrophages in 2 publications [7, 22]. Hashimoto, et. al. further report that the mice receive an estimated 0.1-0.5% of the aerosolized dose for a total of 0.5-0.25 µmoles of clodronate (initially encapsulated in liposomes). They compare this delivered dose to that of Berg, et. al. who reports as much as 2 µmoles clodronate delivered by intratracheal instillation, yet only 70% of the AM were depleted by counts in BALF; immunohistochemical staining gave an 83% depletion. This supports the general experience with aerosolization being a much more effective method of pulmonary delivery for drugs despite the inefficiency in the method. Most importantly, aerosol delivery appears to avoid the neutrophilia commonly reported with intratracheal instillation.
  • While the Penn-Century Microspray device [21] does generate a larger droplet size, the aerosolization (atomization) occurs within the trachea, so that the entire volume dosed is directly delivered to the lung. The Microsprayer consists of a gas-tight stainless steel syringe fitted with either a blunted, rigid, stainless steel tube or a flexible, blunted, stainless steel tube for direct insertion into the trachea through the animal’s mouth. This is sometimes accomplished with a small animal otolaryngoscope ( Penn-Century, USA) which can help in ensuring that the trachea rather than the esophagus is being accessed. The device is relatively inexpensive and requires little training. No other special animal restraints or other equipment is needed although assistive instruments are available from Penn-Century. Several studies conclude that the Microspray device is preferable to intratracheal instillation with regard better distribution of the dose throughout the lung and less trauma due to delivery of bulk liquid into the lung. The only clodronate liposome study which reported PMN counts after using the Microspray device shows a detectable, but statistically insignificant, increase. Therefore, it is tempting to conclude that neutrophila post-treatment with clodronate liposomes is related to the droplet size of the instilled suspension. However, further direct comparisons of these methods to intratracheal instillation is necessary to test this hypothesis. Limited studies on liposomes (not clodronate-based)  delivered with the Microsprayer did not identify changes in preparations as a result of the Microsprayer atomization (personal communication), however data on microsprayed clodronate liposomes has not been collected. One report of 78% and another of >95% depletion of AM indicates that additional data is needed to fully evaluate this method.
  • Elder, et. al. present a hybrid technique for efficiently delivering aerosolized clodronate liposomes to the lung which they call intratracheal inhalation (ITIH) [22]. Intratracheal cannulae are inserted into anesthetized animals followed by delivery of nebulized liposomal clodronate through the cannulae. The immediately obvious advantages include the simultaneous dosing of several animals (assuming that the manifold delivers a uniform dose to each animal by a parallel configuration) and virtually 100% of the dose is deposited in the lungs as with the Microspray device. The flow rate of this device is much slower than that used for nose-only or whole-body aerosol delivery, so it is possible that this method is less disruptive to liposome formulations. [custom_button text=”Publications: Aerosol Administration” title=”Publications: Aerosol Administration” url=”https://www.clodrosome.com/routes-of-administration/intrapulmonary/publications-reporting-aerosol-administration/” size=”large” bg_color=”#002dcf” text_color=”#FFFFFF” align=”right” target=”_blank”]Again, a significant investment in equipment and training is required to accomplish this delivery method. The study compares the results of ITIH with whole-body inhalation and intratracheal instillation (described below). Despite a significantly larger dose of clodronate (186 µg/rat), the depletion achieved in this study (86%) is underwhelming. As might be expected, whole-body inhalation of nebulized clodronate liposomes was very inefficient at delivering clodronate liposomes (3.8 µg clodronate/rat) and likewise inefficient at AM depletion (37%). Although this study appears to be very thorough and systematic, the intratracheal instillation portion of the study demonstrated an increase in AM by 6% and PMN by 70% which is atypical of this method.[/tab]
[tab title=” Intranasal Administration “] Intranasal administration can be an effective method of delivery to the lungs provided that the administration method is optimized for delivery of the instilled suspension to the lower respiratory tract. At least 48 publications report using intranasal instillation for depleting alveolar macrophages. Southam, et. al. [24] found that larger volumes (optimal in this study = 35 µl) instilled (by Pipetman) into lightly anesthetized mice resulted in 50% or more of the total recovered dose appearing in the lungs. This study was performed by tracking a radioactive sulfur colloid; the authors did not report the efficiency of the delivery (amount recovered/amount delivered) therefore, do not confuse the 50% recovered dose with 50% of the delivered dose. These results generally support some of the findings in earlier reports on intranasal administration of Evans blue dye [25] and tetanus toxin [26]. Of particular importance in all studies, the administration of small volumes (5 µl) did not deliver any instillate to the lungs. The proportion of colloid appearing in the lung increased as a function of volume dosed almost linearly until an apparent saturation between 25 and 50 µl dosed. Details of the method of intranasal administration are often not disclosed in papers, therefore when planning to deplete alveolar macrophages using intranasal instillation, careful consideration of the volume instilled and other details is necessary. As Southam, et.al. point out in their paper, particles other than the sulfur colloid may behave differently when instilled intranasally and [custom_button text=”Publications: Intranasal Administration” title=”Publications: Intranasal Administration” url=”https://www.clodrosome.com/routes-of-administration/intrapulmonary/publications-reporting-intranasal-administration-intrapulmonary/” size=”large” bg_color=”#002dcf” text_color=”#FFFFFF” align=”right” target=”_blank”]Visweswaraiah, et. al. confirm the interspecies variability in their study. Ideally, pilot experiments evaluating the efficiency of delivery to the lung using fluorescent clodronate (or clodronate control) liposomes should be performed to optimize and/or validate the chosen method especially when translating the method to another species. [/tab] [tab title=” Intratracheal Administration “] Intratracheal administration is used in a few more papers (51) than intranasal instillation. This term encompasses variations of two methods which would seem to have very different potential physiological responses. A review of the pros and cons of intratracheal instillation compares this method to inhalation delivery to the lung for toxicological evaluations [27]. This review does suggest that this first method is prohibitively invasive. However, if frequent repeated dosing is required, any of these methods may result in inflammation at the site of administration. In some descriptions of both methods, the administration of the liquid bolus is followed by the instillation of air (about 2x the volume of the liquid bolus) to ensure that the liquid is transferred from the trachea to the lungs. Chest auscultation (listening) to confirm intake of liquid into the lungs is commonly reported as well.

  • The first method is essentially a tracheotomy and may also be described as an intratracheal injection or invasive intratracheal instillation. Using sterile technique, a shallow incision is made in the skin covering the anesthestized animal’s trachea exposing the trachea. A bolus of the  liposome suspension is injected directly into the trachea by piercing the translucent tissue between the cartilaginous rings. Usually, the injection is made with a 1 cc syringe fitted with a small gauge needle which is inserted into the trachea and aimed downward into the lungs. The incision is closed with a surgical clip and the animal is allowed to awaken. The animal should breathe and behave normally-no gurgling or wheezing sounds. If the animal exhibits any breathing anomalies, it should probably be sacrificed or otherwise excluded from the experimental groups. If several animals have breathing difficulties, the surgical protocol should be reassessed, along with the volume injected and the liposome suspension itself. It’s possible that the liposome suspension is contaminated causing an anaphylactic reaction in the animals. However, with experienced technicians, this method is usually quite robust with few unsuccessful injections. Some papers report utilizing a cannula instead of a needle for injection.
  • The non-surgical method of intratracheal instillation involves delivering the bolus of liposome suspension by accessing the trachea through the mouth of the animal (transorally).  These tracheal access techniques are identical to those described above for administering aerosols within the trachea. A cannula is sometimes inserted into the trachea through which the bolus is instilled, but often a syringe with a blunted [custom_button text=”Publications: Intratracheal Administration” title=”Publications: Intratracheal Administration” url=”https://www.clodrosome.com/routes-of-administration/intrapulmonary/publications-reporting-intratracheal-administration/” size=”large” bg_color=”#002dcf” text_color=”#FFFFFF” align=”right” target=”_blank”]needle (or small gavage needle) attached is used to instill the liposome suspension. Several papers describe detailed methods for intratracheal instillation [28, 29].
  • Another method of intratracheal administration, ITIH, is described above under Aerosol Administration.
[/tab] [tab title=” Pharyngeal Administration “] Pharyngeal administration, or more appropriately, pharyngeal aspiration, of clodronate liposomes has been reported in one paper. This method is based upon the (usually) accidental aspiration of material from the pharyngeal space into the trachea. It is a relatively simple method in which the liquid or suspension is deposited into the pharyngeal space (in the throat) of an anesthetized animal restrained in a “nearly upright” position. Prior to delivering the bolus, the animal’s tongue is extended using padded forceps and held in extension “until the suspension was aspirated into the lungs” [30]. Both the tongue extension and anesthesia suppress the natural swallowing response presumably increasing the portion of the dose that enters the trachea. Pharyngeal aspiration models have been used to examine the effects of accidental aspiration of solids and liquids entering through the mouth and nose or regurgitated stomach contents, as well as incidental aspiration due to various pathologies such as esophageal reflux conditions, partial paralysis by stroke and mechanical ventilation. It is also widely used as a method of introducing various particulates found in the environment into the lung for toxicological studies. Surprisingly, aspiration of sterile buffer does not appear to cause distress in the mice post-anesthesia, nor do the control mice develop aspiration pneumonia as evidenced by lack of clinical symptoms as well as negative cytological and histopathological results for inflammation [31]. The authors who used aspiration to deliver clodronate liposomes to the lung report >80% [custom_button text=”Publications: Pharyngeal Administration” title=”Publications: Pharyngeal Administration” url=”https://www.clodrosome.com/2010-lo-6367/” size=”large” bg_color=”#002dcf” text_color=”#FFFFFF” align=”right” target=”_blank”]depletion of alveolar macrophages on day 3 post-treatment. Although liposomal PBS controls were included, the only data presented was related to the effect of IL-17A abolition. Therefore further investigation is required to evaluate and compare pharyngeal aspiration to other methods for delivering clodronate liposomes to the lung.[/tab] [/tabs]

A note on nomenclature. In many papers, the authors have used the term “clodronate” interchangeably with “clodronate liposomes” throughout the paper. While clodronate is the active ingredient in clodronate liposomes, this entire website and thousands of publications have resulted from the differences in biological activities between a solution of clodronate and a suspension of clodronate liposomes. Given the discussion above on the potential complications generated by a mixture of free clodronate and encapsulated clodronate within a single suspension, it is imperitive that “clodronate”, or better, “free clodronate”, only be used to describe a clodronate solution and “clodronate liposomes”, “liposomal clodronate” or “encapsulated clodronate” (or Clodrosome, if appropriate) be used whenever liposomally encapsulated clodronate is being described.

 

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