VERSATILITY OF DRUGS DELIVERED USING LIPOSOMES

3. VERSATILITY OF DRUGS DELIVERED USING LIPOSOMES

Since their discovery in the mid-1960s, liposomes have been extensively studied as parenteral delivery systems for a wide range of pharmaceutical therapeutic drug candidates that include traditional low molecular weight compounds, biotechnology-derived proteins and peptides as well as

252 Patil and Burgess

DNA-based therapeutics. Liposomal delivery for low molecular weight drugs is one of the earliest and conse- quently one of the most clinically advanced amongst all other therapeutics.

3.1. Low Molecular Weight Drugs Low molecular weight pharmaceutical compounds that have

been investigated for parenteral delivery in liposome systems include antifungals (10,11), antibiotics (12,13), and anti cancer drugs (14). Liposomal delivery of these drugs can be used to alter drug pharmacokinetics, to assure adequate levels in tissues of interest and to reduce =avoid toxic side effects (for details, refer Sec. 4 ). Some of the approved low molecular weight liquid parenteral liposomal formulations include: AmBisome Õ , which contains amphotericin B and is indicated for systemic fungal infections (11) and visceral

leishmaniasis; Amphotec Õ (branded as Amphocil outside United States) and Abelcet Õ which also contain amphotericin

B (15–17) and are indicated for systemic fungal infections (18); DaunoXome Õ , which contains duanorubicin and is indi-

cated for first-line treatment of advanced Kaposi’s sarcoma; DepoDur Õ , which contains morphine and is indicated for pain

management following major surgery; DepoCyt Õ , which con- tains cytarabine and is indicated for lymphomatous meningi-

tis; Doxil Õ (branded as Caelyx outside United States), and

Myocet Õ (formerly known as Evacet ), both of which contain doxorubicin and, whereas, Doxil is indicated for metastatic

ovarian cancer and Kaposi’s sarcoma, Myocet is being used for treating breast cancer; MiKasome Õ , which contains the

aminoglycoside antibiotic amikacin and is indicated for severe infections; and Visudyne Õ , which contains the photosensitizer

dye verteporfin and is indicated for age-related macular degeneration (19). The reader is referred to the case studies in this book on the development of Doxil by Martin and on the development of AmBisome by Adler-Moore. Currently, sev- eral liposome formulations containing low molecular weight drugs are in clinical trials for a range of pharmacologic effects (such as antibacterial, anticancer, antifungal, and anti-HIV activities) (20,21). See Table 1 for additional information on

Liposomes:

Table 1 Marketed Liposomal Products Design

Marketed formulation—Company

Liposome composition

Indication and

AmBisome—Gilead-Fujisawa

Systemic fungal Manufacturing Healthcare

Amphotericin B

HSPC, cholesterol,

and DSPG

infections and visceral leishmaniasis

Amphotec =Amphocil a,b —InterMune

Cholesteryl sulfate a Aspergillosis Abelcet—Elan

Amphotericin B

Fungal infections DaunoXome—NeXstar Pharmaceuticals

Amphotericin B

DMPC and DMPG

Daunorubicin

Cholesterol and DSPC

Kaposi’s sarcoma

citrate

Doxil =Caelyx b,c —Ortho Biotech

Doxorubicin

MPEG-DSPE, HSPC,

Metastatic ovarian

hydrochloride

and Cholesterol

cancer and Kaposi’s sarcoma

DepoDur—SkyePharma—Endo

Morphine sulfate

Cholesterol, DOPC,

Pain following major

surgery DepoCyt—SkyePharma—Enzon

and DPPG

Cytarabine

Cholesterol, DOPC,

Lymphomatous

meningitis Myocet (formerly known and

and DPPG

Breast cancer Evacet)—Elan

Doxorubicin

Egg PC and cholesterol

hydrochloride

MiKasome—NeXstar

Amikacin

HSPC, cholesterol,

Bacterial infections

and DSPG

253 (Continued)

© 2005 by Taylor & Francis Group, LLC

Table 1 254 Marketed Liposomal Products (Continued ) Marketed formulation—Company

Pharmaceutical

name

Indication Visudyne—Novartis

drug

Liposome composition

Age-related macular degeneration Epaxal d —Berna Biotech

Verteporfin

Egg PG and DMPC

Hepatitis A

Hemagglutinin,

Hepatitis A

antigen

neuraminidase, and lecithin

Inflexal V d —Berna Biotech

Influenza Pevaryl Lipogel e —Cilag

Dermatomycosis and gynecological mycosis

Topical anesthetic as ELA-Max)—

L.M.X.4 e (formerly known

Lidocaine

Cholesterol

and hydrogenated

Ferndale Laboratories

lecithin

MPEG-DSPE, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt; HSPC, hydrogenated soy phosphatidylcholine; DSPG, distearoyl phosphatidylglycerol; DMPC, dimyristoyl phosphatidylcholine; DMPG, dimyr- istoyl phosphatidylglycerol; DOPC, dioleoyl phosphatidylcholine; DPPG, dipalmitoyl phosphatidylglycerol; DSPC, distearoyl phosphati- dylcholine; Egg PC, egg phosphatidylcholine; Egg PG, egg phosphatidylglycerol.

a Amphotec b Brand name of the product marketed outside United States. =Amphocil is a amphotericin B cholesteryl sulfate complex for injection. Patil

c Sterically stabilized (Stealth) liposomes. d Immunopotentiating reconstituted influenza virosomes.

and e Formulation for transdermal application. Burgess

© 2005 by Taylor & Francis Group, LLC © 2005 by Taylor & Francis Group, LLC

3.2. Protein Therapeutics Liposomes have been used as vaccine adjuvants for recombi-

nant protein-based vaccines. Epaxal and Inflexal V are licensed liposomal protein vaccines that were globally approved in the mid-1990s for hepatitis A and influenza, respectively. Epaxal and Inflexal are immunopotentiating unilamellar vesicles known as virosomes since they contain phospholipids and pro- teins derived from the influenza virus (22,23). Virosome formu- lations are comprised of vaccine-specific antigens of interest embedded in the liposomal bilayer composed of the following two components: (i) natural and synthetic phospholipids (lecithin, cephalin, and envelope phospholipids from the influ- enza virus); and (ii) influenza surface glycoproteins: hemagglu- tinin and neuraminidase. For the Epaxal formulation (24), the antigen of interest is the hepatitis A virion, whereas, the Inflexal V (25) formulation contains a combination of surface antigens of three currently circulating strains of the influenza

mal products for the delivery of proteins.

3.3. DNA-Based Therapeutics In addition to the commercial licensing of a range of liposomal

formulations, recent years have witnessed significant research efforts directed toward the development of liposomes as delivery vectors for DNA-based therapeutics (26–29). In vivo delivery of DNA-based therapeutics such as plasmids for gene expression, antisense oligonucleotides, siRNAs, ribozymes, etc., typically requires assistance of delivery vectors (29). Currently used gene delivery vectors are attenuated replica- tion-defective viruses such as adenoviruses, adeno-associated viruses, polyomavirus, and retroviruses (26,30–33). Due to their natural mechanism of targeted introduction of DNA into cells, these vectors have high efficiency in DNA transfer (30). However, a number of potential problems are associated with viral vectors, including deletion of sequences during replication, recombination with endogenous sequences to pro-

Liposomes: Design and Manufacturing 255

virus. See Table 1 for additional information on approved liposo-

256 Patil and Burgess

duce infectious recombinant viruses, activation of cellular oncogenes, introduction of viral oncogenes, inactivation of host genes, development of toxic immune responses and inflamma- tion that has been demonstrated to be lethal (26,34–36). Phar- maceutical formulations of viruses are expensive and difficult to manufacture, have a very low shelf life, and can lose potency upon storage (37,38). Thus, despite their superlative efficiency, many of these vectors are unsuitable for clinical use. As a con- sequence of the problems associated with viral gene delivery vectors, several alternative non-viral gene delivery systems are under development (29,39–41). Liposomal delivery vectors are emerging to be the most popular non-viral alternative to viral vectors for gene delivery (26,40,41). Liposomal formula- tions of DNA-based therapeutics protect these molecules from enzymatic inactivation in the plasma by degradative endo- and exo-nucleases, facilitate the entry of DNA into the cell cyto- plasm, are relatively easy and inexpensive to manufacture, and are non-immunogenic (26). Both anionic and cationic lipo- somes have been utilized for gene delivery (26,42,43). DNA typically is entrapped inside anionic liposomes and is sur- face-complexed with cationic liposomes. These liposomal for- mulations have achieved tremendous success in introducing DNA-based gene therapeutics into a wide range of tissues and cells in animal as well as cell culture models. The first clin- ical trial for gene therapy for melanoma using cationic lipo- somes was conducted in 1992 by Nabel et al. (44,45). Currently, although several cationic liposomes are in advanced clinical trials for gene therapy of cystic fibrosis, and cancer, there is no commercially licensed formulation (45). In addition to their use in protein delivery, as described in detail in Sec. 3.2, virosomes have also been employed for gene transfer (46,47).