Microencapsulation in Yeast Cells and ... - ACS Publications

Chapter 19

Microencapsulation in Yeast Cells and Applications in Drug Delivery *

G. Nelson , S. C. Duckham, and M. E. D. Crothers

Downloaded by COLUMBIA UNIV on July 6, 2012 | http://pubs.acs.org Publication Date: February 16, 2006 | doi: 10.1021/bk-2006-0923.ch019

Micap plc, No.1, The Parks, Lodge Lane, Newton-Le-Willows, WA12 0JQ, United Kingdom *Corresponding author: [email protected]

Yeast cells as preformed microcapsules can be used to improve the bioavailability of poorly soluble drugs in the gastrointestinal tract. Microorganisms have been recognised as potential preformed natural microcapsules since the early 1070s, when Swift and Co., USA, patented a technique using specifically prepared yeast containing high concentrations of lipid, greater than 40% by weight. Using commercially available yeast strains, such as Saccharomyces cerevisiae, from the baking and brewing industry, this paper describes the encapsulation of peppermint, fenofibrate and econazole nitrate and explores the release of peppermint in the mouth of human volunteers and fenofibrate in the duodenum of beagle dogs.

Introduction Microorganisms have been recognised as potential preformed natural microcapsules since the early 1070s, when Swift and Co., USA, patented a technique using specifically prepared yeast, containing high concentrations of lipid, greater than 40% by weight (J). Synthetic polymeric carrier materials and polymers extracted from natural materials have been investigated for drug 268

© 2006 American Chemical Society

In Polymeric Drug Delivery I; Svenson, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.


269 delivery applications over the last few decades (2). However, as an alternative, in recent years microorganisms have been considered as novel vectors for the delivery of recombinant vaccines and other bioactive proteins and peptides via the gastrointestinal tract (3-5). Working particlualry with live lactobacillus species and common baker's yeast {Saccharomyces cerevisiae), researchers have promoted the use of these organisms for targeting site-specific areas throughout the digestive tract. The live organism would potentially manufacture the beneficial compounds in-situ, either expressing on the surface or secreting the compound in response to the digestive environment or as a result of cell lysis (68). However, many would consider the use of live organisms for drug delivery difficult to accept and have approached drug delivery using non-viable bacteria (9), yeast and yeast cell walls, where much of the site-specific targeting remains possible without problematic issues in controlling the activity of the microorganism. This paper will concentrate on the roll of non-viable yeast cells as a drug delivery vehicle. Yeast are unicellular fungi and must be considered as one of the most commercially important classes of microorganism with applications in brewing, (beer and wine production), ethanol production, baking, and production of recombinant proteins and biopharmaceuticals (10). The cell mass is used to produce yeast extract for food and fermentation media applications, and whole yeast cells are incorporated into human food and animal feed (11). In the health food industry, yeast cells and the vitamins they contain are utilized widely (12). Although there are many species of yeast which reproduce by budding or fission, by far the most commercially important belong to the genus Saccharomyces, probably the best-characterized of the yeast genera. The ascomycetous yeast Saccharomyces cerevisiae is a key microorganism in traditional biotechnological processes as well as a tool of choice to understand the eukaryotic cell. An average Saccharomyces cell is approximately 5 micrometers in dia meter, although other species of yeast can be as much as 20 micrometers in diameter. The ovoid shape is defined by the cell wall, a physically rigid struc ture which primarily protects the yeast internal membrane and organelles from the environment (13). The cell wall is composed of complex and highly crosslinked glucan, mannan and, to a lesser degree, chitin which is associated with the bud scars remaining after a daughter cell has budded from the mother cell. The wall is approximately 100-200 nm thick, comprising 15-25% of the dry mass of the cell. The cell wall surrounds the much thinner (18 hours) prior to the administration of the test formulations. All studies were conducted at a fixed dose of 30 mg/kg and food was allowed 2 hours post dose administration. The animals in group 1 (n=2) received a single oral admini stration of Tricor® capsules (Abbott Laboratories) and the animals in groups 2-3 (n=5 each group), which received the yeast capsule formulations (dose volume 1 mL/kg), were anesthetized with Telazol (5mg/kg, 1M). Isoflurane delivered via a oxygen vaporiser with a nose cone was used as needed. An endotracheal tube was placed and general anaesthesia maintained with isoflurane in oxygen. A flexible endoscope was passed down the oesophagus through the stomach and into the duodenum. A catheter was passed down the working channel and the test formulations delivered. The endoscope and catheter were withdrawn and the endotracheal tube was removed when the animal regained its swallowing reflex. The animals were monitored for normal recovery from anaesthesia. Blood samples were collected (2mL whole blood) via the jugular vein or other appropriate vessels, placed in tubes containing EDTA, and stored on ice until centrifiiged. Sample intervals were prior to dosing and approximately 0.5, 1, 2, 3, 4, 6, 8 and 24 hours after dosing. After centrifugation, lOOuL of the plasma sample was transferred into a 2.0-mL microcentrifuge tube and 20 mL methanol added. The sample was centrifiiged for 10 minutes, and the supernatant was transferred directly into an HPLC vial. Plasma concentrations of fenofibric acid were determined using a Waters Series HPLC system consisting of a Waters 2695 pump, Waters 2695 autosampler, diode array UV detector (287nm), Phenomenex Luna C-18 column and Waters Millennium data collection software (ver. 4.0). The mobile phase used was 55:45 acetonitrile:0.2% phosphoric acid



275 solution (v/v) with a flow rate of 1.2 mL per minute and an injection volume of 20 uL. Two model low-molecular weight lipophilic drug entities, econazole nitrate (drug A) and fenofibrate (drug B), were chosen for trial encapsulation in yeast cell capsules, based on availability of the drugs, molecular size and partition coefficient, the latter being properties known to effect successful loading of lipophilic flavor molecules in yeast capsules. Three yeast varieties were used to determine whether the encapsulation was yeast strain specific. The encapsula tions proved successful for both drugs in all three yeast varieties. The loadings of each drug with yeast variety are detailed in Figure 3. The particle size of the spray-dried products, approximately 30 micrometers in diameter, resulted from agglomerates of yeast cell capsules (Figure 4). The drug loading levels were found to be in the range 100-200 mg/g yeast (10-20% w/w). Further develop ment work on the encapsulation process suggests that this is typical for a wide range of pharmaceutical actives. Successful encapsulation can be achieved with low molecular weight drugs with masses in the range 200-1000 g/mol and octanol-water partition coefficients of greater than 2.0 - 6.0. The yeast variety influences the loading level for each drug. For example, with fenofibrate the loading varies between 100 and 180mg/g yeast between the three yeast varieties. The difference in loading levels observed for different yeast types may be attributed to a number of structural characteristics of the yeast cells such as the membrane and cell wall composition or to the pre processing and manufacturing history of the yeast strain used, for example whether the yeast had been produced in a batch or continuous process or whether the yeast had been dried, or even the type of drying used (e.g. roller drying, fluidized-bed drying or spray drying).

Results and Discussion Flavor release in the mouth was initiated by contact with the mucous mem brane surface, when a peppermint-flavored chewing gum was prepared and the flavor release monitored using real time breath-by-breath analysis (Figure 1). However, when placed in a mechanical chewing device, the peppermint flavor did not release from the chewing gum by mechanical action or by the presence of saliva, unless SDS was present, as monitored by gas chromato graphy (Figure 2). On analysis by light microscopy, the yeast cell capsules were seen to be intact.



276

Figure 1. Real time breath-by-breath analysis of menthol, the main component of peppermint, during chewing of chewing gum samples containing peppermint oil (left) and yeast encapsulated peppermint oil (right).

— - .0.52% P e p * SDS

/

/

/ /

/'

/

0.5% Pep 1.93% Micap+ SDS

10

15 20 25 Time (minutes)

30

35

40

Figure 2. The release of menthol as an indication ofpeppermint flavor released from a chewing gum during mechanical chewing (peppermint was added to 0.52% w/w), andfrom yeast capsules (1.93% w/w, the equivalent flavor loading). SDS was added to the dissolution phase at 1% w/v.



277

Figure 3. Plot showing encapsulation levels of two model drugs in three different yeast types (drug A - econazole nitrate), drug B - fenofibrate).

Figure 4. A typical agglomerate ofyeast capsules produced by spray-drying after encapsulation offenofibrate.

It is well known from previous work with lipophilic flavor molecules in yeast cell capsules that the active ingredients, once encapsulated, remain inside the yeast cells in the presence of water and are not released unless the cell membrane is disrupted, for example by the use of surface active agents. This phenomenon was also found with encapsulated drugs (Figure 5). The dissolution profile indicates that SDS must be present above the critical micelle concentration for release to take place. With flavor release ,on the surface of the


278 tongue, the direct contact with the cell surface also initiated release. The pharmacokinetic profiling work was designed to determine if a lipophilic active could be released from the yeast cell without the use of surface active agents, and ultimately enter the bloodstream.


Dissolution profile

Time (min)

Figure 5. The in-vitro release profile offenofibrate in the presence of sodium dodecyl sulphate. The two yeast varieties containing fenofibrate were administered directly to the duodenum of 5 beagle dogs, and the level of fenofibrate acid in the plasma quantified over a 24-hour period using HPLC analysis. For comparison, a commercially available fenofibrate preparation (Tricor®) was administered to 2 beagle dogs orally. The results, detailed in Figure 5, indicate that the feno fibrate was successfully released from the yeast cell capsules into the systemic circulation. Both yeast cell capsule formulations and Tricor® reached the maximum drug concentration in the systemic circulation within the same time period, although the yeast formulation 2 reached its maximum concentration slightly earlier than the other two formulations. All three formulations have a similar absorption profile, with the profile of yeast formulation 1 being most similar to that of the commercial formulation, although yeast formulation 1 shows a much more controlled release of fenofibrate from the formulation than Tricor®, and the initial burst of drug into the systemic circulation was reduced. There appears to be more fenofibrate absorbed from the commercial Tricor® formulation. However, the area under the curve provides a better indication of total amount of drug absorbed from the formulations, which in turn is a better indication of the potential bioavailability. The areas under the curve calculated


279 from Figure 6 above are plotted in Figure 7. From this plot it is clear that most drug was absorbed into the systemic circulation from yeast formulation 1, com pared to the commercial product and yeast formulation 2. It therefore appears that with careful choice of yeast variety and successful encapsulation, the bioavailability of lipophilic drugs can be improved. The potential improvement may be large, considered that no formulation development had taken place during the yeast cell capsule preparations.


Tricor

Yeast Form. 1 ^ 15

20

^

^ Yeast Form. 2 25

30

Time (hours)

Figure 6. Time-dependent concentration offenofibric acid in plasma for a commercial formulation, Trico®r and two yeast formulations (yeast I Saccharomyces cerevisiae and yeast 2 -Saccharomyces boulardi).

20 E CUD

s

U

10

< e

*

c 0 Tricor

Yeast Form. 1

Yeast Form. 2

Figure 7. The mean area under the curve (A UC) (pg hr/ml) for yeast encapsulatedfenofibrate compared to the commercial formulation Tricor® (yeast 1 - Saccharomyces cerevisiae and yeast 2 -Saccharomyces boulardi).


280

Conclusions Yeast cells can be utilized as microcapsules for the encapsulation of lipo philic drug molecules. The drug remains stable within the capsule until release is initiated by addition of a surfactant or by contact with a mucous membrane. When administered directly into the duodenum, the lipophilic drug is released from the cell and enters the blood stream with a reduced burst effect and pro longed release profile.


Acknowledgements The authors would like to acknowledge the help and advice of Juerg Lange of SkyPharma, Muttenz, Switzerland, in preparing the protocols for this development.

References 1. Swift and Company; US patent specification 40001480, 1977. 2. Langer R. Drug delivery and targeting. Nature, 1998, 392, Suppl. 5-10. 3. Fichetti, V.A.; Medaglini, D.; Pozzi, G. Gram-positive commensal bacteria for mucosal vaccine delivery. Curr Opin Biotechnol; 1996, 7, 659-666. 4. Blanquet, S., Antonelli, R., Laforet, L., Denis, S., Marol-Bonnin, S., Alric, M . Living recombinant Saccharomyces cerevisiae secreting proteins or peptides as a new drug delivery system in the gut. J. Biotechnol., 2004,110,37-49. 5. Stubbs, A.C., Martin, K. S., Coeshott, C., Skaates, S.V., Kuritzkes, D. R., Bellgrau, D., Franzusoff, A., Duke, R.C., Wilson, C.C. Whole recombinant yeast vaccine activates dendritic cells and elicits pro tective cell-mediated immunity. Nature Medicine, 2001, 7, 625-629. 6. Drouault, S., Corthier, G., Ehrlich, S.D., Renault, P. Survival, physio logy, and lysis of Lactococcus lactis in the digestive tract. Appl. Environ Microbiol, 1999, 65, 4881-4886. 7. Schreuder, M.P., Deen, C., Boersma, W.J.A., Pouwels, P.H., Klis, F.M. Yeast expressing hepatitis B virus surface antigen determinants on its surface: Implications for a possible oral vaccine. Vaccine, 1996, 14, 383-388. 8. Steidler, L., Hans, W., Schotte, L., Neirynck, S., Obermeier, F., Falk, W., Fiers, W., Remaut, E. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science, 2000, 289, 1352-1355.


281 9.

10. 11. 12.


13. 14. 15. 16. 17. 18. 19. 20. 21.

Paukner, S., Kohl, G., Jlava, K., Lubitz, W. Sealed bacterial ghosts Novel targeting vehicles for advanced drug delivery of water-soluble substances. J. Drug Targeting, 2003, 111, 151-161. Biology of Microorganisms, Brock, T.D.; Madigan, M.T.; PrenticeHall, Englewood Cliffs, New Jersey, 1988, 381. Dziezak, J.D. Yeasts and yeast derivatives-Definitions, characteristics, and processing. Food Technology, 1987, 104-108. Biotechnology in Food Processing; Trivedi, N.; Noyes Publications, Park Ridge, 1986, 115. Yeast, Physiology and Biotechnology, Walker, G.M.; Wiley: Chichester, 1998, 17. Overton, E. Naturforsch. Ges. Zuerich, 1899, 44, 88. Scherrer, R.; Louden, L.; Gerhardt, P. Porosity of yeast-cell wall and membrane. J Bacteriol, 1974, 118, 534-540. Serozym Laboratories; French patent specification 2179528, 1973. AD2 (now owned by Micap plc); European patent specification, 0242135, 1987. British Textile Technology Group; European patent specification, 0511258, 1991 Bishop, J.R.P.; Nelson, G.; Lamb, J. Microencapsulation in yeast cells. J. Microencapsulation 1998, 15, 761-773. Nelson, G. Application of microencapsulation in textiles. Int. J. Pharm. 2002, 242, 55-62. Anandaraman, S.; Reinceccius, G.A. Perfumer and Flavorist, 1987, 12, 33.


Microencapsulation in Yeast Cells and ... - ACS Publications

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