PRODUCTION ENVIRONMENT

4. PRODUCTION ENVIRONMENT

4.1. Engineering Penetration and Distribution Studies

Perform triplicate studies with thermocouples penetrating the product containers as well as thermocouples distributed outside the product containers in a production sterilizer at nominal operating process parameters.

4.2. Sterilization Cycles There are two types of batch sterilizers that can be used for the

sterilization of lipid emulsions, the shaking cycle and the rotary cycle. The filled and sealed containers are sterilized

406 Berger

by exposure to circulated hot water spray or saturated steam at a specific temperature for a specified F 0 . Air overpressure is used during heating, sterilizing, and cooling. The time, temperature, and pressure requirements are set to pre- determined values to assure that the product will receive a

thermal input equivalent to an F 0 minimum of, e.g., 8.0. Criti- cal and key process parameters specified are to be controlled, monitored, and recorded. The target temperature of the circu- lating sterilizing medium during the peak dwell portion of the cycle must be maintained in a specific range, e.g., 121–123. For the shaking cycle, the product must be agitated lying on its side providing oscillatory movement along the container cen- troidal axis. The agitation frequency maintained throughout

product must be agitated throughout the sterilization process. The containers shall be horizontally positioned in rack(s) in a

4.3. Sterilizer Microbial Emulsion SubProcess Validation

Table 7 shows the microbial emulsion validation conducted at subprocess conditions in a fully bulked load in a production sterilizer. The acceptance criteria of 6 spore logarithmic reduc- tion (SLR) must be achieved for the BI, C. sporogenes and a 3 SLR for the more moist heat resistant BI, B. stearothermophi- lus. Each emulsion (20 containers) is inoculated with the

6 2 for

C. sporogenes and B. stearothermophilus, respectively. The

20 inoculated containers are distributed throughout the pro- duction sterilizer for sterilization at subprocess conditions.

4.4. Sterilizer Microbial Closure SubProcess Validation

Table 8 shows the microbial closure validation at subprocess conditions in a fully bulked production sterilizer. The BIs used were C. sporogenes and B. subtilis. Acceptance criteria of 3 SLR must be achieved for the moist heat BI (C. sporogenes) and the dry heat BI (B. subtilis). The R&D sterilization

Case Study:

Microbial Fraction Negative (F Table 7 A =N) Analysis Following Exposure in a Moist Heat Cycle with Lipid Agitation, C. sporogenes vs. B. stearothermophilus. Lipid Emulsion Microbial Solution Validation.

Sterilization Validation of IV Lipid Emulsion (Inoculated in Oil Phase) in the 200 mL Abbovac Bottle with Emulsion—Sterilization Fraction Negative Method

Positive

Positive

Avg. # spores =

#Test Organism

#Positive

#Negative

samples SLR C. sporogenes

0 =20 > 7.0 C. sporogenes

0 =20 > 7.1 B. stearo.

0 =20 > 3.2 B. stearo.

F 0 Range: (c) 5.8–-7.6 Agitation: 67–73. Temperature range: 120–125 C.

© 2005 by Taylor & Francis Group, LLC

Table 8 Microbial Fraction Negative (F =N) Analysis Following Exposure in a Moist Heat Cycle with Agitation, C. sporogenes vs. B. stearothermophilus. Lipid Emulsion Lipid Emulsion Microbial Closure Validation Sterilization Validation of 200 mL Abbovac Bottle Inoculated Closure Surface Coated with IV Fat Emulsion in Cycle with Agitation

#Positive

Test Microorganism

Initial population =

#Positive

#Negative

samples SLR C. sporogenes

0 =20 > 5.2 B. subtilis

0 =20 > 5.8 F 0 range: 6.3–6.4.

Agitation: 67–73 cpm. Temperature range: 120–125 C.

Berger

© 2005 by Taylor & Francis Group, LLC

Case Study: A Lipid Emulsion—Sterilization 409

validation of the inoculated closure system coated with the

IV fat emulsion in a 200 mL Abbovac container is shown in Table 8 . The surface of the stopper that comes into direct contact with the sidewall of the bottle was inoculated with the appropriate BI, dried and then a few drops of emulsion were placed over the inoculum to simulate manufacturing conditions.

The Halvorson and Ziegler equation is used to calculate the SLKR value as follows (1):

a. Positive for the indicator microorganism.

b. SLR ¼ log a – log b, where a ¼ initial population of spores; b ¼ 2.303 log(N =q) ¼ in(N=q), where N ¼ total number of units tested, q ¼ number of sterile units. When N ¼ q, assume one (1) positive for the purpose of calculating an SLR.

c. F 0 ¼ integrated lethality or equivalent minutes at 121.1

C for the hottest and coldest thermocouple containers.

4.5. Production Environment Bioburden Screening Program

Refer to the flow diagram in Fig. 4 . A negative heat shock at

10 min exposure would indicate the bioburden’s resistance as aD 121 C of less than 0.079 min (a 0.0079F 0 =min is accumu- lated at 100 C). A positive heat shock at 30 min exposure sig- nifies that the surviving organisms should have a more detailed moist heat analysis conducted (e.g. 121, 118, and 112

C exposures).

5. REGULATORY SUBMISSION The following checklists pertain to the sterilization portion of

documents required in support of an FDA submission for aseptically processed and terminally sterilized products (10).

If a parenteral formulation can tolerate heat, then the moist heat sterilization process is the method of choice when compared to aseptic processing (11).

410 Berger

Figure 4 Heat shock is a method used for screening thermally resistant microorganisms. The application of a known amount of moist heat (approximately 10 or 30 min if required at less than 100

C) allows the isolation of bioburden microorganisms that poten- tially have moist heat resistance from microorganisms that have no moist heat resistance.

The following summarize the documents required to sup- port a New Drug Application for product formulations that are aseptically processed or terminally sterilized.

5.1. Aseptic Processing Microbiological sterilization and depyrogenation: Depyrogenation validation of glass =stopper.

Microbiological sterilization validation of the stop- per(s) (steam sterilizer). Microbiological sterilization validation of representa- tive items for the family category of setups (filling line items, e.g., filling line needles, stoppers, etc.) in the steam sterilizer. Microbiological sterilization validation of representa- tive items for the family category of filters in the steam sterilizer.

Case Study: A Lipid Emulsion—Sterilization 411

Stability of BIs: Engineering information: Performance qualification for equipment, e.g.,

sterilizers, ovens, etc. Thermocouple diagram during PQs.

Procedures and specifications for media and environ- mental data. Sterility testing methods and release criteria:

Bacteriostasis, fungistasis. Sterility testing.

Bacterial endotoxin test product validation data.

5.2. Terminal Sterilization The sterilization process: the operation and control of the production auto-

clave; the autoclave process and performance specifica- tions; specification of the sterilization cycle.

Autoclave loading patterns: description =diagram of representative autoclave

loading patterns. Thermal qualification of the cycle: heat distribution in the production autoclave;

heat penetration in the production autoclave. Depyrogenation validation of container =closure prior

to sterilization. Microbiological efficacy of the cycle:

identification; resistance; stability of BIs.

412 Berger

Information and data concerning the identification, resistance, and stability of BIs used in the biological validation of the cycle should be provided. Include ATCC number, stock, resistance value and test date in each microbial validation report.

The resistance of the BI relative to that of the bio- burden microbiological challenge studies:

microbial challenge of emulsion in a production vessel; microbial challenge of closure in a production vessel.

Demonstrate container integrity following maximum processing exposure:

Maintenance of sterility BET validation data: LAL compatibility worksheet;

bulk drug and final product inhibition = enhancement data.

Sterility testing methods and release criteria:

bacteriostasis, fungistasis sterility testing. Preservative efficacy at time zero, 3 months acceler-

ated stability and time expire.

REFERENCES 1. Young RF. In: Morrissey RF, et al., eds. Sterilization with

Steam Under Pressure, Sterilization Technology. New York: Van Nostrand Reinhold, 1993:120.

2. Owens JE. In: Morrissey RF, et al., eds. Sterilization of LVP’s and SVP’s, Sterilization Technology. New York: Van Nostrand Reinhold, 1993:254.

3. Association for the Advancement of Medical Instrumentation. Resistometers used for characterizing the performance

Case Study: A Lipid Emulsion—Sterilization 413 of biological and chemical indicators, Vol. 1.2. Arlington,

VA: Association for the Advancement of Medical Instrumenta- tion, 2003:1.

4. Berger TJ, Nelson PA. The effect of formulation of parenteral emulsions on microbial growth-measurement of D- and z-values. PDA J Pharm Sci Tech 1995; 49:32.

5. Feldsine PT, Shechtman AJ, Korczzynski MS. Survivor kinetics of bacterial spores in various steam-heated parenteral emulsions. Develop Industr Microbiol 1970; 18.

6. Caputo RA, Odlaug TE, Wilkinson RL, Mascoli CC. J Parent Drug Assoc 1979; 33:214.

7. Berger TJ, Chavez C, Tew RD, Navasca FT, Ostrow DH. Biological indicator comparative analysis in various product for- mulations and closure sites. PDA J Pharm Sci Tech 2000; 54:101.

8. Pflug IJ, Holcomb RG. In: Block SS. ed. Principles of Thermal Destruction of Microorganisms Disinfection, Sterilization and Preservation, 3rd ed. Philadelphia: Lea and Febiger, 1983:759.

9. Berger TJ, May TB, Nelson PA, Rogers GB, Korczynski MS. The effect of closure processing on the microbial inactivation of biological indicators at the closure-container interface. PDA J Pharm Sci Tech 1998; 52:70.

10. FDA guideline for submitting documentation for sterilization process validation in applications for human and veterinary drug products. Federal Register, 58, No. 231, Friday, December

3, 1993, Notices, p. 63996. 11. Cooney PH. Aseptic fill vs. terminal sterilization. Presented at

the Pharmaceutical Technology Conference, Cherry Hill, NJ, September 16–18, 1986.

Case Study: Formulation of an Intravenous Fat Emulsion

BERNIE MIKRUT Pharmaceutical Research

& Development, Hospira, Inc., Lake Forest, Illinois, U.S.A.

1. INTRODUCTION The history of i.v. fat emulsions can be traced as far back as

1873 when Holder infused milk in cholera patients. In the 1920s, Yamakawa (1) in Japan produced a product called ‘‘Janol’’ from caster oil, butter, fish oil, and lecithin which had many side effects. It was not until 1945 that Stare et al. (2) produced the first relatively non-toxic emulsion using purified soy phospholipids. This product was further refined by Schuberth and Wretlind (3) in 1961, who made 1506 infu- sions in 422 patients using a soybean oil emulsion made with purified egg phospholipids with no untoward reactions

416 Mikrut

in humans. This led to the product, Intralipid Õ , which was approved in Sweden in 1961. Intralipid was approved in the

US in 1975 and Liposyn Õ was approved in the US in 1979.

2. FORMULATION i.v. fat emulsions are oil-in-water emulsions of soybean or a

1:1 soybean =safflower oil mixture emulsified using purified egg phosphatide. Tonicity is adjusted with glycerin and pH is adjusted with sodium hydroxide. i.v. fat emulsions with fat contents of 10%, 20%, and 30% are commercially available.

2.1. Oil The current products available in the US use either a 1:1 com-

bination of safflower oil and soybean oil or soybean oil exclu- sively. Worldwide, other products are available which contain medium chain triglyceride (MCT) oil in combination with soybean oil. Both safflower and soybean oils are listed in USP 23 and their respective fatty acid profiles are summar- ized in Table 1.

Safflower and soybean oils are highly unsaturated and prone to oxidation through initial peroxide formation. Therefore, they must be maintained under nitrogen gas protection during storage and handling. Both oils contain some saturated waxes and sterols which must be removed by the standard oil industry practice of ‘‘winterization.’’ In this process, the oils are refrigerated for a length of time dur- ing which the waxes and sterols crystallize out and settle to the bottom of the drum. The oils are then quickly cold filtered

Table 1 Fatty Acid Composition Safflower oil, USP

Soybean oil, USP Palmitic acid 2–10%

Palmitic acid 7–14% Stearic acid 1–10%

Stearic acid 1–6% Oleic acid 7–42%

Oleic acid 19–30% Linoleic acid 72–84%

Linoleic acid 44–62% Linolenic acid 4–11%

Case Study: An Intravenous Fat Emulsion 417

to remove these unwanted components and stored under nitrogen gas protection prior to their use in emulsion manu- facture. The oils must be food-grade oils and of high chemical purity, pyrogen-free and free of herbicides and pesticides. The FDA requires these oils to be tested to show the absence of herbicides and pesticides (4).

2.2. Emulsifier Highly purified egg lecithin is used as the emulsifier in all

commercial i.v. fat emulsions. Historically, soy phosphatides were used; however, they were rejected due to untoward clin- ical effects. Pluronic F68 was investigated, but was discarded because of toxic effects (5,6). The main components of egg phospholipid are phosphatidylcholine (PC) and phosphatidy- lethanolamine (PE), along with minor components. Pure PC and PE make poor emulsions. The minor components of lecithin are necessary to produce a stable emulsion (7). The components of a typical egg phospholipid used in the manufacture of i.v. fat emulsions are presented in Table 2.

2.3. Tonicity Adjuster The tonicity adjuster of choice is glycerin at a concentration of

2.25% (Intralipid) or 2.5% (Liposyn II =III). Glycerin was used by Schuberth and Wretlind (3) in their classic work in 1961 and is still used today. Dextrose is not used since it has been reported to interact with egg phospholipid to produce brown discoloration upon autoclaving and storage (8,9).

Table 2 Typical Egg Phospholipid Composition PC

73.0% Lysophosphatidylcholine (LPC)

5.8% PE

15.0% Lysophosphatidylethanolamine (LPE)

2.1% Phosphatidylinositol (PI)

0.6% Sphingomylin (SP)

2.5% (Adapted from Ref. 10.)

418 Mikrut

2.4. Others

2.4.1. pH Small amounts of sodium hydroxide are used to adjust the pH

to approximately 9.5 during manufacture. This pH level has two effects: it causes the ionization of the acidic phospholipids present in the egg phospholipid mixture, creating a net nega- tive charge for droplet repulsion; it also forms some free fatty acids. These fatty acids form sodium soaps and further stabi- lize the emulsion by acting as auxiliary emulsifiers. The ionization characteristics of the individual phospholipids are summarized in Table 3 (10).

2.4.2. Preservatives No preservatives are used in i.v. fat emulsions. Small quanti-

ties of vitamin E and BHA =BHT are present since these occur in the original soybean or safflower oils. i.v. fat emulsions have been shown to be a good growth medium for microbial growth (11) and therefore are designed as a single-dose product.

Table 3 Ionization Characteristics of Phospholipids Ionic

Ionization Phosphatide

species characteristics Phosphatidic

Primary Strong acid acid (PA)

phosphate, PO 2 4 (pK a 3.8, 8.6) PC, LPC

Secondary Isoelectric over a

phosphate-choline,

wide range of pH

PO –NMe þ 4 3

PE Secondary Negative at pH 7

phosphate-amine,

(pK a 4.1, 7.8)

PO 4 –NH 3 þ

Phosphatidylserine Secondary Negative at pH 7.5 (PS)

phosphate-carboxyl-amine, (pK a 4.2, 9.4)

PO 4 –COO –NH 3 þ

Phosphatidylinositol Secondary Negative above (PI)

phosphate-sugar,

pH 4 (pK a 4.1)

PO 4 –sugar

(From Ref. 10.)

Case Study: An Intravenous Fat Emulsion 419

3. PROCESSING Several methods of emulsion manufacture can and have been

used, but the equipment of choice for i.v. fat emulsions is the standard high-pressure homogenizer. The egg phospholi- pid is first dispersed in a portion of the water for injection (WFI) or dissolved in the oil. Both of these methods have been used successfully to manufacture acceptable emulsions. Abbott currently disperses egg phosphatide in WFI, the gly- cerin is added and the mixture homogenized to a fine disper- sion. This dispersion is filtered through a 0.45 mm membrane and more WFI added. Oil is then added with agitation to form

a crude emulsion, which is homogenized further to form the finished emulsion. pH is adjusted with sodium hydroxide at several points during the process so that the final emulsion has a pH of approximately 9.0 prior to autoclaving. Two dif- ferent methods of homogenization have been used. The tank-to-tank method homogenizes the emulsion alternately from one tank to another until the desired globule size is attained. The other, the recirculation method, uses only one tank and continuously recirculates the emulsion through the homogenizer and back to the tank until the desired glo- bule size is attained. The graphical representation ( Fig. 1 ) from the homogenizer manufacturer correlates the efficiency of the two methods. The entire process must be oxygen-free as much as possible. All WFI is degassed and nitrogen gas purging =flushing is used throughout the process. A typical manufacturing scheme is outlined in Fig. 2 . Since the final emulsion product has a mean globule size very close to the usual 0.45 mm filtration of intravenous fluids, emulsion stability would be compromised if the final product was fil- tered through a 0.45 mm membrane. All i.v. fluids are filtered through at least a 0.45 mm filter to reduce particulates. In this case, it was decided to filter each of the ingredients through a

0.45 mm filter prior to homogenization. The oil is also filtered through a 0.45 mm filter prior to homogenization. The 0.8 mm filtration is after the final homogenization to reduce the particulates introduced during the manufacturing procedure. Any breakdown of the emulsion which may have been caused

420 Mikrut

Figure 1 Comparison of tank-to-tank homogenization vs. continuous recirculation. (From APV Gaulin bulletin TB-71.)

Case Study: An Intravenous Fat Emulsion 421

Figure 2 Typical manufacturing scheme for i.v. fat emulsions.

422 Mikrut

by the filtration at this point is repaired by the final homo- genization pass. The final 5.0 mm filtration is a regimesh stain- less steel filter just prior to the emulsion going to the filling line.

4. FILLING/PACKAGING The currently marketed i.v. fat emulsions are available in 50,

100 =200, 200, 500 and 1000 mL glass containers which are evacuated and flushed with nitrogen gas. The use of nitrogen gas reduces the degradation of the emulsion by oxidation as well as the formation of toxic by-products. Unpublished work in Abbott Laboratories to evaluate the packaging of i.v. fat emulsions in flexible containers using a full aluminum foil over- wrap has shown that a crack =break or pin hole in the aluminum foil layer will let in oxygen and result in by-products which have been shown to be toxic to mice. Both Type I and Type II glass containers are approved for packaging of i.v. fat emulsions.

5. STABILITY EVALUATION The main stability-indicating parameters used in the stability

evaluation of i.v. fat emulsions are pH, free fatty acids, extraneous particulates, visual evaluation, and globule size distribution.

5.1. pH and Free Fatty Acids The pH of i.v. fat emulsions before autoclaving is approxi-

mately 9.0. Subsequent to terminal heat sterilization, the pH drops to approximately 8.5. This drop in pH is normal and is the result of hydrolysis of the PC and PE to their respective lyso compounds and the subsequent formation of free fatty acids. Since the emulsions have no buffering capacity, the pH drops upon autoclaving and also on aging. The Abbott pH specifications are 6.0–9.0 over the 24-month shelf life. i.v. fat emulsions with a pH of less than 5.0 have significantly decreased zeta potential values and are subject to coalescence and globule size growth as noted by Davis (10).

Case Study: An Intravenous Fat Emulsion 423

5.2. Particulate Particulate evaluation of i.v. fat emulsions is determined by

the conductance of a microscopic particle counting method, wherein the sample is filtered through a 0.8 mm gray, gridded membrane to isolate solid extraneous particles. The light- obscuration method is unsuitable because of the composition of the sample, i.e., liquid micro-droplets of oil in a water-based vehicle. These droplets are counted as particles by the light- obscuration sensor and erroneous data are produced. When the microscopic method is attempted, emulsion droplets pass through or are absorbed by the filter and are not observed on the filter membrane. Particulate contamination can be created by stainless steel wear of the homogenizer or mixer parts and is observed as fine black particulates. Viton rubber or teflon wear particles can occur if the homogenizer plunger

packings use these materials. Also, Ca þþ or Mg contamina- tion may result in particles originating from the precipitation

of salts of the free fatty acids which are formed by hydrolysis during processing, autoclaving, and storage.

5.3. Visual Evaluation Visual evaluation is very important since no instrumental

globule-sizing technique can detect free oil droplets floating on the emulsion surface. One technique of visualizing these droplets is to view the emulsion surface at an angle using an inspection lamp in a darkened room. The oil droplets show up as shiny specks on the dull emulsion surface. This techni- que requires practice in order to see beyond the glass surface and focus on the emulsion surface.

Creaming of i.v. fat emulsions is normal and the emul- sion is easily re-dispersed with gentle agitation. Creaming is observed in the bottle as a whitish layer in the top portion of the emulsion and a darker, less opaque layer toward the bottom. This is a result of the low-density oil droplets slowly rising to the surface as a result of gravitational forces. Upon inversion of the container, the emulsion should be uniform in color and opacity.

424 Mikrut

5.4. Globule Size Globule size and distribution are the most important factors

in i.v. fat emulsion stability. Measurement of globule size and distribution must be evaluated using several instru- mental techniques since no one technique can measure the entire size range. In addition to the instrumental techniques described here, visual evaluation should always be conducted.

5.5. Accelerated Stability Testing

5.5.1. Temperature i.v. fat emulsions have been shown to be stable for up to 6

months at 40

C. This corresponds to approximately 24 months at 25–30

C results in a signif- icant increase in free fatty acid formation due to phosphlipid hydrolysis and a concomitant decrease in pH. Abbott normally tests i.v. fat emulsions at 25, 30, and 40 C.

C. Longer storage at 40

5.5.2. Freeze–Thaw Cycling Cycles that comprise slow freezing at –20

C, followed by undis- turbed thawing at room temperature can be used to evaluate i.v. fat emulsion stability. Unpublished data at Abbott have shown that one cannot correlate this to shelf life at room temperature, but it can be useful for rank-order stability evaluation of various emulsion formulations.

5.5.3. Stress Shake Horizontal shaking at approximately 200 cycles per minute

can also cause emulsion breakdown and is useful for rank- order stability evaluation of various emulsion formulations.

REFERENCES 1. Yamakawa S. J Jpn Soc Intern Med 1920; 17:1.

2. Stare FJ, et al. Studies on fat emulsions for intravenous alimentation. J Lab Clin Med 1945; 30:488.

Case Study: An Intravenous Fat Emulsion 425 3. Schuberth O, Wretlind A. Intravenous infusion of fat emul-

sions, phosphatides and emulsifying agents. Acta Chirgurgica Scandinavica 1961; (suppl 278).

4. FDA Deficiency Letter, dated November 29, 1991, for Liposyn Õ III 30%.

5. Pelham D. Rational use of intravenous fat emulsions. Am J Hosp Pharm 1981; 38:198–208.

6. Geyer RP, Mann GV, Young J, et al. J Lab Clin Med 1948; 33: 163.

7. Scharr PE. Cancer Res 1969; 29:258. 8. Hansrani PK, Davis SS, Groves MJ. The preparation and prop-

erties of sterile intravenous emulsions. J Paren Sci Tech 1983; 37(4):145.

9. Tayeau F, Neuzil E. Bordeaus Med 1972; 10:1117. 10. Davis SS. The stability of fat emulsions for intravenous admin-

istration. In: Proceedings of the Second International Sympo- sium on Advanced Clinical Nutrition, 1983, pp. 213–239.

11. Unpublished data from Abbott Laboratories.

Case Study: DOXIL, the Development of Pegylated Liposomal Doxorubicin

FRANK J. MARTIN ALZA Corporation, Mountain View,

California, U.S.A.

1. INTRODUCTION Over the past 30 years, liposomes have been proposed as a

vehicle for improving the delivery (and thereby the therapeu- tic utility) of dozens of drugs. The cytotoxic anthracycline antibiotics doxorubicin and daunorubicin and the polyene antibiotics amphotericin B and nystatin are perhaps the most often cited examples. The vast majority of these publications originated from academic laboratories and thus do not gener- ally address the pharmaceutical attributes required for the regulatory approval of a commercially viable product.

428 Martin

Development of a liposomal product in most respects para- llels that of any other ethical pharmaceutical product. A medical need must be identified and the product must be shown in well- controlled clinical trials to meet that need and to do so safely. Moreover, it must have at least comparable activity to other drug products approved for the same clinical indication. Implicit in the ability to conduct clinical trials is the availability of the drug product in sufficient quantity to supply clinical investigators. It must also be of proper quality to meet regulatory requirements.

A commercially successful product must also be cost-effective, reproducibly made in large scale and stable enough to be sup- plied through the normal channels of distribution.

Four liposomal products meet these requirements and are approved in the US and

=or Europe, DOXIL TM (pegylated liposomal doxorubicin, also known as Caelyx TM in Europe),

DaunoXome TM (liposomal daunorubicin), Ambisome TM (lipo- somal amphotericin B), and Myocet TM (liposomal doxorubicin). The case study reported here examines the formulation design, clinical evaluation, and regulatory strategy used in the deve- lopment and registration of DOXIL. The case study following this one ( Chapter 15 of this book) focuses on the formulation design, clinical evaluation, and regulatory strategy used in the development and registration of AmBisome.

2. BACKGROUND Early liposome formulations of doxorubicin were shown to sig-

nificantly reduce cardiotoxicity and acute lethality in animals but on a dose-equivalent basis, were no more active than the unencapsulated drug (1). Why was there no improvement of anti-tumor activity? In retrospect, two related biological responses provoked by the injection of liposomes appear to

be to blame. Firstly, liposomes released a proportion (up to 50%) of their encapsulated doxorubicin as a consequence of opsonization by components of blood (lipoproteins, albumin, complement components, formed elements) (2). Obviously, drug that is lost in this way is not available to be delivered to a tumor in encapsulated form. Secondly, the liposomes that

DOXIL 429

survive destabilization in blood are rapidly removed from circulation by fixed macrophages in the liver and spleen (the mononuclear phagocyte system or MPS; also known by an older designation as the reticuloendothelial system or RES) (3). Once internalized by macrophages, the liposome matrix is digested by lysosomal lipases and the drug is released intracellularly. This combination of leakage and MPS uptake virtually eliminates any opportunity for ‘‘true’’ targeting, as drug loaded liposomes never reach the tumor.