Sink conditions

The performance of the dmg dehvery system needs to be characterized. The rate of dmg release and the total amount of dmg loaded into a dmg dehvery system can be deterrnined in a dissolution apparatus or in a diffusion ceU. Typically, the dmg is released from the dmg dehvery system into a large volume of solvent, such as water or a buffer solution, that is maintained at constant temperature. The receiver solution is weU stirred to provide sink conditions. Samples from the dissolution bath are assayed periodically. The cumulative amount released is then plotted vs time. The release rate is the slope of this curve. The total dmg released is the value of the cumulative amount released that no longer changes with time. 

The mass transfer equation applicable to the transport-limited extraction of a solute from an aqueous solution to an organic phase (sink conditions), was derived ... 

In a typical experimental situation a membrane is used between two compartments, one containing a drug solution ( donor compartment) and the other sink conditions (i.e., zero concentration receiver compartment). For a homogeneous barrier membrane of thickness h, Pick s First Law may be written as... 

Perfect sink conditions externally with finite mass transfer boundary layers. 

Finite mass transfer into the bulk environment which approximates sink conditions ... 

Figure 3 illustrates a situation in which this may not be true. When 250 mL of water was taken with erythromycin tablets, the extent of absorption was much greater than when the tablets were taken with only 20 mL of water. In the latter case, dissolution probably did not occur under sink conditions. Hence, the dissolution rate decreased, and it appears that not all of the erythromycin had a chance to dissolve in the GIT. Note than the dissolution was not, however, the ratedetermining step in absorption, since the time to reach the peak concentration was the same in all situations. 

If a small polydisperse sample of powder is dissolved under sink conditions, then the dimensions, b, of the particle will decrease linearly with respect to time [33-37] ... 

Strictly speaking, sink conditions are when the amount dissolved plotted versus time yields a line which, within experimental error, is linear. When the surface area, A, is constant, then this corresponds to 15% dissolved. When the surface area changes (e.g., during particulate dissolution), then this number may be smaller. 

Equation (1) predicts that the rate of release can be constant only if the following parameters are constant (a) surface area, (b) diffusion coefficient, (c) diffusion layer thickness, and (d) concentration difference. These parameters, however, are not easily maintained constant, especially surface area. For spherical particles, the change in surface area can be related to the weight of the particle that is, under the assumption of sink conditions, Eq. (1) can be rewritten as the cube-root dissolution equation ... 

Dissolution test data will be required in all cases (and for all strengths of product) for development and routine control and should be based on the most suitable discriminatory conditions. The method should discriminate between acceptable and unacceptable batches based on in vivo performance. Wherever possible Ph Eur test methods should be used (or alternatives justified). Test media and other conditions (e.g., flow through rate or rate of rotation) should be stated and justified. Aqueous media should be used where possible and sink conditions should be maintained. A small amount of surfactant may be added where necessary to control surface tension or for active ingredients of very low solubility. Buffer solutions should be used to span the physiologically relevant range—the current advice is over pH 1 6.8 or perhaps up to pH 8 if necessary. Ionic strength of media should be reported. The test procedure should employ six dosage forms (individually) with the mean data and a measure of variability reported. 

The relevance of Eq. (2.2) (which predicts how quickly molecules pass through simple membranes) to solubility comes in the concentration terms. Consider sink conditions, where Ca is essentially zero. Equation (2.2) reduces to the following flux equation... 

The survey of over 50 artificial lipid membrane models (pION) in this chapter reveals a new and very promising in vitro GIT model, based on the use of levels of lecithin membrane components higher than those previously reported, the use of negatively charged phospholipid membrane components, pH gradients, and artificial sink conditions. Also, a novel direction is suggested in the search for an ideal in vitro BBB model, based on the salient differences between the properties of the GIT and the BBB. 

The equations used to calculate permeability coefficients depend on the design of the in vitro assay to measure the transport of molecules across membrane barriers. It is important to take into account factors such as pH conditions (e.g., pH gradients), buffer capacity, acceptor sink conditions (physical or chemical), any precipitate of the solute in the donor well, presence of cosolvent in the donor compartment, geometry of the compartments, stirring speeds, filter thickness, porosity, pore size, and tortuosity. 

For ionizable sample molecules, it is possible to create an effective sink condition in PAMPA by selecting buffers of different pH in the donor and acceptor compartments. For example, consider salicylic acid (v>Ka 2.88 see Table 3.1). According to the pH partition hypothesis, only the free acid is expected to permeate lipophilic membranes. If the donor pH < 2 and the acceptor pH is 7.4, then as soon as the free acid reaches the acceptor compartment, the molecule ionizes, and the concentration of the free acid becomes effectively zero, even though the total concentration of the species in the acceptor compartment may be relatively high. This situation may be called an ionization-maintained sink. 

In this chapter we use the term sink to mean any process that can significantly lower the concentration of the neutral form of the sample molecule in the acceptor compartment. Under the right conditions, the ionization and the binding sinks serve the same purpose as the physically maintained sink often used in Caco-2 measurements. We will develop several transport models to cover these chemical sink conditions. When both of the chemical sink conditions (ionization and binding) are imposed, we will use the term double sink in this chapter. 

We define this permeability as apparent, to emphasize that there are important but hidden assumptions made in its derivation. This equation is popularly (if not nearly exclusively) used in culture cell in vitro models, such as Caco-2. The sink condition is maintained by periodically moving a detachable donor well to successive acceptor wells over time. At the end of the total permeation time f, the mass of solute is determined in each of the acceptor wells, and the mole sum mA (t) is used in Eq. (7.10). Another variant of this analysis is based on evaluating the slope in the early part of the appearance curve (e.g., solid curves in Fig. 7.14) ... 

It is important to remember that Eqs. (7.10) and (7.11) are both based on assumptions that (1) sink conditions are maintained, (2) data are taken early in the transport process (to further assure sink condition), and (3) there is no membrane retention of solute. In discovery settings where Caco-2 assays are used, the validity of assumption 3 is often untested. 

In Section 7.7.5.4, we discuss the effects of additives in the acceptor wells that create a sink condition, by strongly binding lipophilic molecules that permeate across the membrane. As a result of the binding in the acceptor compartment, the transported molecule has a reduced active (unbound) concentration in the acceptor compartment, cA(t), denoted by the lowercase letter c. The permeability equations in the preceding section, which describe the nonsink process, are inappropriate for this condition. In the present case, we assume that the reverse transport is effectively nil that is, CA(t) in Eq. (7.1) may be taken as cA(t) 0. As a result, the permeability equation is greatly simplified ... 

When the pH is different on the two sides of the membrane, the transport of ioniz-able molecules can be dramatically altered. In effect, sink conditions can be created by pH gradients. Assay improvements can be achieved using such gradients between the donor and acceptor compartments of the permeation cell. A three-compartment diffusion differential equation can be derived that takes into account gradient pH conditions and membrane retention of the drug molecule (which clearly still exists—albeit lessened—in spite of the sink condition created). As before, one begins with two flux equations... 

Figure 7.17 shows the asymmetry ratios of a series of compounds (acids, bases, and neutrals) determined at iso-pH 7.4, under the influence of sink conditions created not by pH, but by anionic surfactant added to the acceptor wells (discuss later in the chapter). The membrane barrier was constructed from 20% soy lecithin in dodecane. All molecules show an upward dependence on lipophilicity, as estimated by octanol-water apparent partition coefficients, log KdaA). The bases are extensively cationic at pH 7.4, as well as being lipophilic, and so display the highest responses to the sink condition. They are driven to interact with the surfactant by both hydrophobic and electrostatic forces. The anionic acids are largely indifferent... 

Membrane Retention (under Iso-pH and in the Absence of Sink Condition)... 

Two-Component Anionic Lipid Models with Sink Condition in the Acceptor Compartment... 

Since there would be increased overall lipid concentration in the dodecane solution, we decided to create a sink condition in the acceptor wells, to lower the membrane retention. We discovered that the pH 7.4 buffer saturated with sodium laurel sulfate serves as an excellent artificial sink-forming medium. Since the new PAMPA membranes would possess substantial negative charge, the negatively charged micellar system was not expected to act as an aggressive detergent to the two-component artificial membrane infused in the microfilter. 

Six two-component models were tested under sink conditions (models 5.1-10.1 in Table 7.3), employing three negatively charged lipids (dodecylcarboxylic acid, phosphatidic acid, and phosphatidylglycerol). These models were also tested in the absence of the sink condition (models 5.0-10.0 in Table 7.3). 

Tables 7.6-7.8 list the Pe, SD, and %R of the 32 probe molecules in the thirteen new PAMPA lipid models, one of which is 2% DOPC assayed under sink conditions (model 1.1). The latter model served as a benchmark for assessing the effects of negative membrane charge.

Furthermore, the membrane retentions of the lipophilic probe molecules are dramatically decreased in the presence of the sink condition in the acceptor wells, as shown in Fig. 7.27. All molecules show R < 35%, with progesterone and phenazo-pyridine showing the highest values, 34% and 26%, respectively. 

The combination of increased Pe and decreased %R allowed the permeation time to be lowered to 4 h, in comparison to the originally specified time of 15 h [547,550], a considerable improvement for high-throughput applications. The quality of the measurements of the low-permeability molecules did not substantially improve with sink conditions or the reduced assay times. 

DOPC with Dodecyicarboxyiic Acid under Sink Conditions... 

---