The oral delivery of amphotericin B

The oral delivery of amphotericin Bby

Dolores R. Serrano1, Ijeoma F. Uchegbu2& Juan J. Torrado1,*

1 Farmacia y Tecnología Farmacéutica

Facultad de Farmacia

Universidad Complutense de Madrid

Plaza Ramon y Cajal

28040 Madrid, Spain

Tel: 34 91 3941620

Fax: 34 91 3941736

Email: serrano_lopez85@hotmail.com

torrado1@farm.ucm.es

2 Department of Pharmaceutics

UCL School of Pharmacy

29 - 39 Brunswick Square

London WC1N 1AX, U.K.

Tel: +44 207 753 5997

Fax: +44 207 753 5942

Email: ijeoma.uchegbu@ucl.ac.uk

* To whom correspondence should be addressed

Keywords: Amphotericin B, oral delivery, absorption, oral

bioavailability, lymphatic uptake, biodistribution,

efficacy, toxicity.

1

Photo:

Juan J. Torrado

Ijeoma F. Uchegbu

Dolores R. Serrano

2

3

Amphotericin B (AmB) is a broad spectrum antifungal agent

with a low incidence of clinical resistances. AmB is also

indicated in the treatment of leishmaniasis. AmB is a

zwitterionic amphiphilic molecule characterized by its low

oral bioavailability. Therefore, in clinical practice AmB

oral administration is restricted to situations in which

local action is desired, i.e. in order to prevent

nosocomial infections in the oesophageol and

gastrointestinal tract. With oral dosing the systemic

toxicity of AmB is usually avoided due to its low oral

absorption. After oral administration of AmB lozenges at a

dose of 40 mg per day or following oral rinsing with

suspensions at a dose of 500 mg q.i.d., a maximum AmB

plasma concentration of between 0.136 and 0.5 µg mL-1 has

been reported [1,2]. AmB concentrations lower than 0.5 µg

mL-1 are considered subtherapeutic [3]. The fraction of AmB

orally absorbed is estimated to be between 0.2 - 0.9% [2].

However, in some circumstances when low AmB doses are

administered, the fraction absorbed may increase to 9% [2].

To date, the treatment of systemic fungal infections and

visceral leishmaniasis with AmB has been restricted to

dosing via the parenteral route. The biodistribution of AmB

is dependent on the type of formulation used and there is a

poor correlation between the plasma levels of the drug and

organ levels [4]. For this reason AmB plasma levels must be

used in combination with target tissue pharmacokinetics to

inform pharmacodynamic studies. Thus, conventional studies

of oral bioavailability based only on plasma drug

4

concentration are not a reliable method of assessing

various AmB formulations.

In this manuscript, we explore the oral delivery systems

that have been thus far developed and examine the

possibility of a clinically useful oral AmB formulation

emerging.

New AmB oral delivery formulations

AmB poor oral bioavailability is due to its low solubility

(< 1 mg L-1 at physiological– pH = 6 - 7 [4]), its

tendency to self-aggregate in aqueous media [4] and its low

permeability (molecular weight of 924 Da, log P=0.95). To

overcome these limitations, different strategies have been

developed which can be classified as:

Nanosuspensions

Nanoparticles

Carbon nanotubes

Lipid-based systems

1. Nanosuspensions of AmB

To enhance adhesion to the gastrointestinal mucosa, an AmB

nanosuspension (0.4% AmB: 0.5% Tween 80: 0.25% Pluronic

F68: 0.05% sodium cholate, w/w) with a particle size of 528

nm was prepared by high pressure homogenization. After oral

administration of 5 mg kg-1 for 4 - 5 days, this new system

was more active than micronised AmB in model of visceral

leishmaniasis but only a 28.6% reduction in parasite load

in the liver was achieved [5].

5

2. Nanoparticles

AmB was encapsulated within PLGA nanoparticles of 165 nm

and administered orally at a dose of 10 mg kg-1. The

relative oral bioavailability was increased 8 fold compared

with orally administered Fungizone® reaching a peak plasma

concentration of 176 ng mL-1 at the 24 h time point [6]. In

later experiments after oral administration of 5 mg kg-1 of

AmB for 4 days, these nanoparticles exhibited a comparable

efficacy ( ̴ 99% reduction) to an intravenously administered

AmBisome® formulation when tested against a model of

pulmonary aspergillosis infection [7].

3. Carbon nanotubes

AmB, attached to functionalised carbon nanotubes and orally

administered at a dose of 15 mg kg-1 for 5 days, exhibited

superior antileishmanial activity when compared to an oral

dosed Miltefosine® and a similar efficacy to the

intraperitoneal administration of AmBisome® ( ̴ 99%

reduction). However, the micrometer size of the carbon

nanotubes continues to pose a toxicological problem and

there are issues associated with the biodegradability of

carbon nanotubes [8].

4. Lipid-based systems

Lipid formulations are the most studied vehicles for oral

AmB administration because the toxicity of AmB may be

reduced by lipids [4]. Lipid based strategies include:

6

Solid lipid nanoparticles

AmB cochleates

Other lipid-based formulations

4.1. Solid lipid nanoparticles

Solid lipid nanoparticles (SLNs) combine the advantages of

lipid emulsions and polymeric nanoparticle systems. SLNs

consist of a mixture of glyceride dilaurate and

phosphatidylcholine obtained by nanoprecipitation. SLNs

have a particle size of between 200 - 250 nm and exhibit a

greater oral relative bioavailability (216%) than AmB

solubilised in DMSO/methanol, with a peak plasma

concentration of 125 ng mL1 at 9 h, arising from a dose of

200 mg kg-1 [9].

4.2. AmB cochleates

Cochleates are defined as lipid-based delivery vehicles

consisting of crystalline phospholipid-calcium structures

that form spiral sheets to which AmB is bound [10]. AmB is

readily released from the 400 nm cochleates once the

cochleates interact with the target cells. Cochleates have

been orally administered at an AmB dose of up to 40 mg kg-1

per day and have been proven to be tolerable and as

effective as intraperitoneally administered deoxycholate

AmB for the treatment of both aspergillosis [11] and

systemic candidiasis [10].

4.3. Other lipid-based formulations

7

Self-emulsifying drug delivery systems (SEDDS) have been

used because of their ability to solubilise AmB. AmB

incorporated in 100% Peceol® (mixture of mono- and

diglycerides of oleic acid) exhibited an extremely high

peak plasma concentration in rodents of 1.5 µg mL-1, at the

4 h time point and after a single oral dose (50 mg kg-1).

Peceol® seems to increase the gastrointestinal absorption

of AmB by both enhancing the lymphatic transport (being

optimum for particle sizes of between 200 and 400 nm) and

decreasing the pre-systemic transporter-mediated drug

efflux pump [12]. In order to increase the stability of the

system, lipids with a melting point above ambient

temperature have been incorporated into the formulation

(e.g. lipids such as mono- and diglycerides with a mixture

of glycerol and pegylated esters of long fatty acids [12-

14]). AmB biodistribution depends on the final lipid

composition of the vehicle [15,16]. No toxicity was

observed even in multi-dose regime (20 mg kg-1 twice a day

for 5 days) [15]. These oral lipid formulations have also

proven to be highly effective against murine visceral

leishmaniasis [14], aspergillosis [17] and candidiasis

[13], indicating that there is adequate tissue distribution

as well as the high plasma levels.

General considerations related to oral versus parenteral

AmB administration

Successful oral AmB absorption is usually related to three

factors:

8

(i) Enhancement of AmB dissolution, but also protection

from the acidic gastric environment,

(ii) Increase in intestinal permeability using delivery

systems with an appropriate size to enhance

lymphatic uptake or carriers able to cross the

gastrointestinal barrier,

(iii) Prolongation of gastrointestinal transit time by

using bioadhesive systems.

Interestingly, the systemic AmB concentrations obtained

after oral administration with these delivery systems are

lower but more sustained than those obtained with the

parenteral formulations. One positive aspect is that these

systems seem not to be accompanied by toxic effects,

especially gastro- and nephrotoxicity [18]. However, it

must be stated that regulatory toxicity studies have not

been reported with most of the formulations. Higher doses

in oral formulations may be used than in parenteral

formulations. Doses up to 40 mg kg1 have been orally

administered, but doses of 5 mg kg-1 are usually preferred

as there is no a proportional relationship between dose and

absorption and more successful results have been obtained

with multiple low dose regimes (lower doses administered

once, twice and even three times a day for several days)

instead of a high single dose. Using multiple low doses

leads to the accumulation of AmB in target organs and

therapeutic organ levels which are effective for visceral

leishmaniasis and systemic fungal infections. Perhaps, one

9

of the most important criticisms to be still addressed is

that most of the experiments are based on rodent models,

especially important as rodent AmB pharmacokinetics is

different to that of humans [16,19]. For instance,

Gershkovich et al., [16] has pointed out that since rats do

not have a gall bladder, they are unable to digest rapidly

and efficiently larger volumes of lipids. It should be

remembered that AmB can be chemically considered to be a

lipid itself and some of the proposed lipid systems

incorporate high amounts of lipid excipients as well as the

drug. This physiological difference between rodents and

humans may prolong the absorption phase and even be the

reason for a flip-flop effect (absorption constant slower

than elimination constant) in rodents [16]. Moreover, when

comparing AmB distribution in different animals [19], it

was observed that there was a higher tissue accumulation in

rats and mice when compared to humans. Therefore, more

experiments in other animal species are required to

validate any new oral AmB delivery systems proposed.

In conclusion AmB is a challenging drug to deliver orally

due to its poor water solubility at pH 6 – 7 and its low

permeation across the gastrointestinal epithelium. A

number of oral formulations have been trialled, some of

them offering little advantages when compared to the

parenteral dosage form and others such as SEDDS and

cochleates progressing to clinical trial after preclinical

studies demonstrated efficacy in rodent models of visceral

leishmaniasis. An oral formulation of AmB will be of

10

benefit to neutropenic patients and patients suffering from

visceral leishmaniasis.

11

References

1. Epstein JB, Truelove EL, Hanson-Huggins K et al. Topicalpolyene antifungals in hematopoietic cell transplantpatients: tolerability and efficacy. Support Care Cancer,12(7), 517-525 (2004).

2. Ching MS, Raymond K, Bury RW, Mashford ML, Morgan DJ.Absorption of orally administered amphotericin B lozenges.Br J Clin Pharmacol, 16(1), 106-108 (1983).

3. Pendleton RA, Holmes JHt. Systemic absorption ofamphotericin B with topical 5% mafenideacetate/amphotericin B solution for grafted burn wounds:is it clinically relevant? Burns, 36(1), 38-41 (2010).

4. Torrado JJ, Espada R, Ballesteros MP, Torrado-Santiago S.Amphotericin B formulations and drug targeting. J Pharm Sci,97(7), 2405-2425 (2008).

5. Kayser O, Olbrich C, Yardley V, Kiderlen AF, Croft SL.Formulation of amphotericin B as nanosuspension for oraladministration. Int J Pharm, 254(1), 73-75 (2003).

6. Italia JL, Yahya MM, Singh D, Ravi Kumar MN. Biodegradablenanoparticles improve oral bioavailability of amphotericinB and show reduced nephrotoxicity compared to intravenousFungizone. Pharm Res, 26(6), 1324-1331 (2009).

7. Italia JL, Sharp A, Carter KC, Warn P, Kumar MN. Peroralamphotericin B polymer nanoparticles lead to comparable orsuperior in vivo antifungal activity to that ofintravenous Ambisome(R) or Fungizone. PLoS One, 6(10),e25744 (2011).

8. Prajapati VK, Awasthi K, Yadav TP, Rai M, Srivastava ON,Sundar S. An oral formulation of amphotericin B attachedto functionalized carbon nanotubes is an effectivetreatment for experimental visceral leishmaniasis. J InfectDis, 205(2), 333-336 (2012).

9. Patel PA, Patravale VB. AmbiOnp: solid lipid nanoparticlesof amphotericin B for oral administration. J BiomedNanotechnol, 7(5), 632-639 (2011).

10. Santangelo R, Paderu P, Delmas G et al. Efficacy of oralcochleate-amphotericin B in a mouse model of systemiccandidiasis. Antimicrob Agents Chemother, 44(9), 2356-2360(2000).

11. Delmas G, Park S, Chen ZW et al. Efficacy of orally deliveredcochleates containing amphotericin B in a murine model ofaspergillosis. Antimicrob Agents Chemother, 46(8), 2704-2707(2002).

12

12. Sachs-Barrable K, Lee SD, Wasan EK, Thornton SJ, Wasan KM.Enhancing drug absorption using lipids: a case studypresenting the development and pharmacological evaluationof a novel lipid-based oral amphotericin B formulation forthe treatment of systemic fungal infections. Adv Drug DelivRev, 60(6), 692-701 (2008).

13. Ibrahim F, Gershkovich P, Sivak O, Wasan EK, Bartlett K,Wasan KM. Efficacy and toxicity of a tropically stablelipid-based formulation of amphotericin B (iCo-010) in arat model of invasive candidiasis. Int J Pharm, 436(1-2),318-323 (2012).

14. Wasan EK, Gershkovich P, Zhao J et al. A novel tropicallystable oral amphotericin B formulation (iCo-010) exhibitsefficacy against visceral Leishmaniasis in a murine model.PLoS Negl Trop Dis, 4(12), e913 (2010).

15. Gershkovich P, Sivak O, Wasan EK et al. Biodistribution andtissue toxicity of amphotericin B in mice followingmultiple dose administration of a novel oral lipid-basedformulation (iCo-009). J Antimicrob Chemother, 65(12), 2610-2613 (2010).

16. Gershkovich P, Wasan EK, Lin M et al. Pharmacokinetics andbiodistribution of amphotericin B in rats following oraladministration in a novel lipid-based formulation. JAntimicrob Chemother, 64(1), 101-108 (2009).

17. Risovic V, Rosland M, Sivak O, Wasan KM, Bartlett K.Assessing the antifungal activity of a new oral lipid-based amphotericin B formulation following administrationto rats infected with Aspergillus fumigatus. Drug Dev IndPharm, 33(7), 703-707 (2007).

18. Sivak O, Gershkovich P, Lin M et al. Tropically stable noveloral lipid formulation of amphotericin B (iCo-010):biodistribution and toxicity in a mouse model. Lipids HealthDis, 10, 135 (2011).

19. Robbie G, Chiou WL. Elucidation of human amphotericin Bpharmacokinetics: identification of a new potential factoraffecting interspecies pharmacokinetic scaling. Pharm Res,15(10), 1630-1636 (1998).

13