FORMULATION DEVELOPMENT

2. FORMULATION DEVELOPMENT

2.1. Overview The initial formulation given to our development group was

relatively simple: 50 g PFC plus 2.4 g purified EYP per 100 mL emulsion product. PFC emulsions are produced using

a standard procedure (4) outlined in Fig. 2 . Briefly, EYP is first dispersed in hot water for injection by means of a high shear overhead mixer (UltraTurrax); nitrogen-sparged PFC is then added at a controlled rate through a narrow orifice while continuing high shear mixing to form a coarse pre- emulsion or ‘‘premix.’’ Next, this dispersion is transferred to

a high-pressure homogenizer (e.g., APV Gaulin, Inc, Model M3) for emulsification at about 10,000 psig and 35–40 C under continuous nitrogen protection. The finished product is then filtered through a 10 mm stainless steel mesh into washed, quarter-liter, borosilicate glass bottles. These bottles are protected with nitrogen headspace gas, stoppered, secured with aluminum overseals, and then terminally heat sterilized in a rotating steam autoclave with air overpressure. Using this procedure, a sterile product typically exhibits a mean drop size of about 200–300 nm as measured by photon correlation spectroscopy (Coulter N4).

2.2. Biological Screening As part of an initial biological screening for this experimental

product, we conducted incubations with heparinized whole blood to evaluate emulsion effects on erythrocyte morphology ex vivo. Normal morphology is necessary for proper distribution of blood flow in the microcirculation. Deformation or crenation (5) is the result of membrane damage and can serve as a marker for hemo-incompatibility. Briefly, whole blood is diluted with fresh plasma (1:1) which is mixed with

Case Study: Injectable Perfluorocarbon Emulsion 375

Figure 2 Production of a perfluorocarbon emulsion. Liquid PFC is added slowly to a hot aqueous dispersion of EYP with continuous high speed mixing to form a pre-emulsion (or premix). High-pres- sure homogenization is required to reduce the drop size distribution to the submicron range. During production, all steps are performed in a closed system under a protective blanket of nitrogen gas.

376 Lyons

Figure 3 Erythrocyte crenation. Deformation of erythrocytes is a result of subtle membrane damage. Incubating test formulations with whole blood ex vivo serves as a sensitive biological screening tool to predict unacceptable cytotoxicity.

test emulsion (1:1) and then incubated for 10 min at 37 C. Erythrocyte morphology is evaluated by microscopy under

As shown in Fig. 3, crenated erythrocytes exhibit prominent spoke-like projections called spicules. Addition of 3.5 mM oleic acid or 0.3 mM lysolecithin to these incubations will result in virtually 100% crenated red blood cells. At higher concentra- tions, these surface-active agents will cause total hemolysis.

While crenation testing evaluates effects of emulsion pro- duct on blood cells, a second useful biological screen ex vivo involves evaluating effects of blood plasma on emulsion integrity. Fresh or frozen plasma, anticoagulated with either citrate or EDTA, is incubated in varying proportions with test emulsion for 30 min at 37

C. Again using phase-contrast microscopy, we evaluate relative resistance of the emulsion to flocculation (6,7). Floccules appear as irregular-shaped, loose aggregates of emulsified oil droplets. These range in size from small ‘‘grape clusters’’ (2–10 mm) to massive ‘‘ice floes’’ of 100 mm or larger. An approved parenteral fat emulsion such as Intralipid Õ 20% (soybean oil emulsified with egg phospholipids) may be used as

Case Study: Injectable Perfluorocarbon Emulsion 377

Figure 4 Emulsion flocculation. Serial dilutions of test emulsions are incubated ex vivo with human blood plasma. Microscopic evidence of flocculation predicts hemo-incompatibility for the new formulation.

a negative control for this test. A pronounced tendency to flocculate in plasma is predictive of poor rheological properties, undesirable product deposition in organs such as liver, spleen, and lungs, and elevated systemic toxicity. As shown in Fig. 4, initial samples of this PFC emulsion formulation showed a high tendency to flocculate when incubated as described.

378 Lyons Table 1 Effect of Excipients on Emulsion Flocculation and

Erythrocyte Crenation Test

Flocculation RBC crenation Relative emulsion

Excipient

score

(% of total) flow rates a

50% PFC None

0.28 M l-alanine

0.05 M phosphate 0 0 0.98 50% PFC

75% 0.33 (ADMIXED) Intralipid 20% 2.25% glycerin

0.05 M phosphate

0 0 0.93 (0) ¼ none, (þ) ¼ trace, (þþ) ¼ moderate, (þþþ) ¼ heavy, (þþþþ) ¼ severe.

a Normalized to blood:saline (3:1 v =v).

Relative effects of crenation and =or flocculation on blood rheology may be estimated by means of a glass flow visc- ometer (8). Flow times for blood:emulsion (3:1 v =v) mixtures are expressed as ratios to times for control blood–saline mix- tures. Compared to Intralipid–blood mixtures, we observed significantly increased viscosities with PFC emulsion sam- ples. In order to address this problem, a series of small-scale (400 mL) alternative emulsions were made. Test excipients were added prior to steam sterilization, and the sterile product was tested in blood mixtures for resistance to flocculation, ability to induce erythrocyte crenation, as well as for relative flow viscosity. A summary of some of these experiments is shown in Table 1.

We observed that added glycerin had no effect on either crenation or flocculation, while a neutral amino acid such as alanine had some beneficial action. However, addition of sodium phosphate (adjusted to pH 7.4) was very effective in preventing these effects and preserving low flow viscosity. Surprisingly, phosphate added (admixed) to previously steri- lized emulsion was ineffective in this regard. Commercial Intralipid 20% (soybean oil emulsion) resisted both crenation and flocculation under these test conditions. As a result of a series of such studies, a modified formulation was adopted that includes 0.05 M sodium phosphate (pH 7.4) added prior to terminal steam sterilization (9).

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Flocculation also occurs in vivo. In a typical experiment, Sprague–Dawley rats were infused with 20 mL =kg body weight (bw) of PFC emulsion via the tail vein. Blood samples were collected, anticoagulated with EDTA, and scored for floc- culation by phase-contrast microscopy as described above. Under these conditions, large floccules were observed for at least 4 h post-infusion with the original formulation, while phosphate-containing emulsion was much more resistant over this time period.