Doxorubicin

Introduction

Liposomes are vesicles consisting of one or more phospholipids bilayers encasing an aqueous compartment as seen in Figure 1 (1-3). By allowing encapsulation of drugs, DNA or proteins within the compartment, it can facilitate drug movement to particular targets while eliciting varying pharmacokinetic characteristics that enhances the therapeutic effect of delivered contents (2, 3). Due to their ease of preparation, versatility and biologically non-toxic nature, liposomes have been a focus of medical research in hopes of improving therapeutic outcomes for drugs with unfavourable pharmacokinetics (1).

One such aforementioned drug is Doxorubicin, a widely used chemotherapeutic agent used in the treatment of cancer conditions such

as breast cancer, Kaposi’s sarcoma, multiple myeloma and many others (1, 6-8). It is classed as an anthracycline and is associated with multiple side effects, most notably cardiotoxicity (1, 6-8). Being one of the earliest drugs to be associated with liposomal formulations, doxorubicin is currently available in two forms being the conventional (Doxorubicin/Adriamycin®) and pegylated liposomal (Caelyx®) forms within Australia (8).
Liposome Formulation and Formulational Issues

Figure 2. Basic Outline of the thin layer hydration method of liposome preparation (5)
Liposomal formulations are typically prepared using modified forms of the original method of thin layer hydration as summarized in Figure 2 (5). The thin layer hydration method being the most simple and commonly adapted method in major liposomal studies was first described by Bangham et al. (1965) (10) . The method involves the formation of a multi-lamellar lipid film also known as a lipid cake on the surface of a container by means of evaporation or freeze dry methods (5, 10). An aqueous solution is then added to the container to hydrate the lipid layers to swell and become fluid (5). Once agitated, the hydrated lipid sheets would detach itself from the lipid cake or container wall (5). As detached sheets of lipid reassemble to prevent water interactions with the non-polar tails, the lipid sheets would encapsulate the surrounding aqueous solution (2, 5). Formed liposomes could then undergo extrusion, sonication or homogenization processes to purify liposomes as desired (2, 5).
The thin layer hydration method not only allows for formulations to include both lipid or water soluble drug compounds, but also allow for a variety of particular sizes and lamellarity as noted in Figure 3 to be obtained adding to methodology versatility (2-5, 7). However, due to its extensively variable nature of liposome formation subject to conditions such as composition and surface charges, liposome sizes and structure formed vary and may be unstable resulting in low drug encapsulation percentages especially after being extruded for specific formulation requirements (2, 5).
As low encapsulation efficacies are most often the primary drawback in formulation of liposomal drug delivery, numerous customized methods have been developed to overturn the issue with varying degrees of success (2, 5). Among the more successful of these methods is the remote loading technique in which vesicles were initially prepared in a fixed solution pH (2). The external media of the vesicles are then exchanged by gel-exclusion chromatography with a different pH solution containing uncharged drug molecules that may diffuse into the vesicle before becoming protonated and subsequently entrapped within the aqueous compartment (2). A novel variation of the remote loading technique by Haran et al. (1993) with doxorubicin led to the development of Doxil®, the first nano-drug with FDA approval in 1995 (2, 11).
Pharmaceutical significance and Pharmacokinetics of Liposomal Formulations
Advent use of liposomal doxorubicin over its conventional intravenous counterpart is due to the conferred enhanced therapeutic effect (6, 12). The enhancement is resultant from the superior pharmacokinetics of the liposome carriers facilitating increased amounts of active ingredient doxorubicin deposition within the sites of action, both within the tumour interstitial fluid or cells as seen in Figure 4 (6, 9). The comparison not only touts liposomal doxorubicin as a more effective form of medication but also as a more efficient medication in which less active ingredient is wasted upon administration that may either adversely affect healthy cells or be rapidly eliminated by the mononuclear phagocyte system (6).

Superior pharmacokinetics attributed to liposomes can be elaborated as a targeted effect on the particular tissues (2, 4, 13).

Doxorubicin is an anti-cancer agent which should only be affecting cancerous tissues but conventional doxorubicin lacks the ability to differentiate normal from cancerous cells and affects all tissues based on its systemic circulation (2, 4). Liposomal doxorubicin as nanocarriers size within the nano to micro ranges being larger than conventional small drug molecules allows manipulation of a phenomenon called the enhanced penetration and retention (EPR) (2, 4). EPR is a passive targeting effect in which the molecule takes advantage of tumour pathophysiological characteristics that extravasate macromolecules into cancerous tissues while depleting lymphatic drainages to prevent macromolecule clearance as seen Figure 5 (4).

Furthermore, current marketed liposomal doxorubicin features pegylation, in which liposomal doxorubicin is pegylated or otherwise known as coated with polyethylene glycol (13). The polyethylene glycol impedes detection by the mononuclear phagocyte system allowing it extended circulation periods without elimination (7, 13). Increases in circulation period compound aid to liposomes EPR effect in accumulating within cancerous

tissues.
Conclusion
Table 1. Table of marketed liposomal and lipid-based products (3)

With many innovations to improving liposomal drug delivery, such as active targeting, multifunctioning, magnetic, triggered release and many other liposomes types on the rise; liposomes have a vast potential for improving our healthcare systems allowing for more effective and safe delivery of drugs. Some said liposomal drugs that have already been approved for market can even be noted in Table 1 (3).
References
1. Kroon J, Metselaar JM, Storm G, van der Pluijm G. Liposomal nanomedicines in the treatment of prostate cancer. Cancer treatment reviews. 2014 May;40(4):578-84. PubMed PMID: 24216226. Epub 2013/11/13. eng.
2. Eloy JO, Claro de Souza M, Petrilli R, Barcellos JP, Lee RJ, Marchetti JM. Liposomes as carriers of hydrophilic small molecule drugs: Strategies to enhance encapsulation and delivery. Colloids and surfaces B, Biointerfaces. 2014 Sep 22. PubMed PMID: 25280609. Epub 2014/10/05. Eng.
3. Kraft JC, Freeling JP, Wang Z, Ho RJ. Emerging research and clinical development trends of liposome and lipid nanoparticle drug delivery systems. Journal of pharmaceutical sciences. 2014 Jan;103(1):29-52. PubMed PMID: 24338748. Pubmed Central PMCID: PMC4074410. Epub 2013/12/18. eng.
4. Matsumura Y. The drug discovery by nanomedicine and its clinical experience. Japanese journal of clinical oncology. 2014 Jun;44(6):515-25. PubMed PMID: 24755547. Epub 2014/04/24. eng.
5. Lasic DD. Liposomes in Gene Delivery: Taylor & Francis; 1997.
6. Barenholz Y. Doxil® — The first FDA-approved nano-drug: Lessons learned. Journal of Controlled Release. 2012 6/10/;160(2):117-34.
7.

Blanco E, Ferrari M. Emerging nanotherapeutic strategies in breast cancer. Breast (Edinburgh, Scotland). 2014 Feb;23(1):10-8. PubMed PMID: 24215984. Epub 2013/11/13. eng.

8. Handbook AM, Buckley N, Australia PSo. Australian Medicines Handbook 2014: Australian Medicines Handbook Pty. Limited; 2014.

9. Lasic DD, Martin FJ. Stealth Liposomes: Taylor & Francis; 1995.

10. Bangham AD, Standish MM, Watkins JC. Diffusion of univalent ions across the lamellae of swollen phospholipids. Journal of Molecular Biology. 1965 8//;13(1):238-IN27.

11. Haran G, Cohen R, Bar LK, Barenholz Y. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochimica et Biophysica Acta (BBA) - Biomembranes. 1993 9/19/;1151(2):201-15.

12. Accardo A, Aloj L, Aurilio M, Morelli G, Tesauro D. Receptor binding peptides for target-selective delivery of nanoparticles encapsulated drugs. International journal of nanomedicine. 2014;9:1537-57. PubMed PMID: 24741304. Pubmed Central PMCID: PMC3970945. Epub 2014/04/18. eng.

13. Allen TM, Hansen C, Martin F, Redemann C, Yau-Young A. Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochimica et Biophysica Acta (BBA) - Biomembranes. 1991

7/1/;1066(1):29-36.