FACTORS INFLUENCING DRUG ABSORPTION

4. FACTORS INFLUENCING DRUG ABSORPTION

4.1. Influence of Injection Volume and Drug Concentration

4.1.1. Conventional Drug Formulations Hydrophilic compounds such as atropine, sodium chloride,

sugars, and polyols such as mannitol and sorbitol, are reported to be absorbed rapidly when the compounds are administered in relatively small injection volumes (44–46). The faster absorption rate of hydrophilic drugs from smaller injection volumes can be explained by higher concentrations of the drug in the aqueous vehicle which result in a higher diffusion rate into tissue fluid.

In contrast to the volume-dependent absorption rates of hydrophilic drugs in solution, hydrophilic drugs in suspension are found to be absorbed slowly from small injection volumes. In suspensions, the drug concentration does not depend on the volume and consequently the drug diffusion rate into tissue fluid is constant. Hirano et al.(47–50) studied the kinetic beh- avior of suspensions after i.m. and s.c. injection. They found that increasing concentrations and decreasing volumes lead to increased sustained release properties of their suspended model compounds and thus slower absorption. This phenom- enon is explained by the aggregation of the separate particles by physicochemical forces and tissue tension. In 1958, Ober et al. stated that aggregation in concentrated particulate sys- tems often gives rise to enlarged viscosity, specific rheological features and as a result a diminished dissolution rate after injection (51). In addition, it is known that viscosity and spe- cific rheological features such as (pseudo) plastic behavior in suspensions and emulsions increase with increasing concen- tration and with decreasing particle size. Increased viscosity hampers diffusion of the drug out of an aggregated clot.

The influence of injection volume on the rate and extent of bioavailability of solutions of relatively lipophilic drugs in aqueous vehicles has been studied in rats (52). The study was performed to find an explanation for the incomplete absorption

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of the more lipophilic b-blocking agents as described in the for- mer section of this chapter. Propranolol was used as a model compound. The rate and extent of absorption 8 hr after injection appeared to increase with increasing injection volume. This was explained by the observation that with increasing vehicle volume, the residence time of the vehicle at the injection site increased. Consequently, the drug was dissolved over a longer time-period resulting in a faster release rate compared to when the drug is present in the solid state (Fig. 6). Moreover a larger volume may increase the vehicle flow, including the drug in solution, from the depot. This effect explains the higher absorp- tion rate during the initial phase.

The influence of injection volume on the absorption of drugs in solution was studied by determining the absorption of testosterone and some other lipophilic model drugs from different volumes of several oily vehicles (53). Lipophilic com- pounds are faster absorbed when they are dosed in smaller

Figure 6 Individual release rate constants after i.m. injection of 3 mg propranolol HCl in 50, 100, and 200 ml aqueous solution in rats. Lines connect the individual values of the same rat (n = 6). (From Ref. 52.)

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volumes of the oily vehicle. This effect is the same as occurs for solutions of hydrophilic drugs in aqueous vehicles, where the diffusion potential is dominating the absorption rate.

4.1.2. Drug Carrier Systems In line with findings on drug absorption from hydrophilic sus-

pensions, drug release rates from drug carrier formulations, are found to be slower when smaller volumes are injected and when higher dosages are used (54–59). The most likely explana- tion is that the observed effect is related to the formation of a pressure-induced aggregate at the injection site as discussed above. Smaller injection volumes result in a smaller and more compact depot from which drug release will be slower than from

a larger depot. Studies with i.m. and s.c. injections of liposomal chloroquine in mice have confirmed that an increased volume at

a fixed concentration of liposomes results in an increased absorption rate, whereas an increased dose or liposome concen- tration results in a decreased absorption rate. Hence, the absorption rate constants showed a positive correlation with injection volume after both routes of administration (60).

The finding that smaller volumes and higher concentra- tions in dispersed systems formulations may decrease the rate and extent of drug absorption has important clinical implications. Increasing the injected dose is often thought to lead to a proportional drug concentration increase in the blood circulation. However, dose increase in dispersed systems such as suspensions or liposomal dispersions often leads to a decrease in the absorption rate due to aggregation. This may cause even lower drug plasma concentrations and longer residence times at the site of injection. In general, in the case of dispersed injections, higher plasma concentrations can only

be reached with multiple injections.

4.2. Injection Depth Injection depth appears to be a major factor determining the

absorption profile of lipophilic drugs. Generally, for i.m. injec- tion a needle length larger than the s.c. fat layer is needed.

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However, one should realize that the thickness of the s.c. fat layer varies greatly. For example, in the gluteal region, the s.c. layer shows a large interindividual variation. Moreover, large differences are found between males and females. Cock- shott et al. investigated the thickness of the gluteal s.c. fat layer and found a large difference in mean skin to muscle dis- tance between the investigated 63 males and 60 females (61). The skin to muscle distance was about 3–9 cm within the group of females whereas 1 cm to about 7 cm was measured in males. In line with these observations, large differences in absorption rates were found between males and females after injection of cefradine in the gluteal region (62). Differ- ences in absorption rate between deep i.m. injections and shallow s.c. or intra-adipose injections are shown in Fig. 3 . The absorption rate of the hydrophilic atenolol after i.m. injection is considerably faster than after s.c. administration. Atenolol was already completely absorbed within 8 hr after i.m. injection whereas 24 hr after s.c. or intra-adipose injec- tion absorption still occurred (15).

Generally, absorption from the i.m. injection site is in most cases much faster than from the adipose s.c. fat layer which can be explained by structural and physiological differences in the tissue. The fatty connective tissues and adipose layers at the s.c. injection site are more lipomatous and much less perfused than muscular tissues (2). Lipophilic drugs will be more easily absorbed from hydrophilic tissue than from lipophilic tissue. Absorption of hydrophilic drugs from aqueous solutions is less dependent on injection depth.

These observations have important clinical implications. In 1974, a letter appeared in the Lancet (63) in which diaze- pam concentrations in plasma, 90 min after i.m. injection (administered by either 3 or 4 cm long needles) in the gluteal region were measured in females. With the 3-cm needle injec- tions, plasma diazepam concentrations appeared very low and were therapeutically inadequate. In contrast, injections given with 4 cm needles resulted in much, therapeutically relevant diazepam concentrations. This indicates that the 3-cm injec- tion was too shallow and that the injection was very likely placed in the s.c. fat layer instead of the intended gluteus

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maximus. Additionally, a too shallow injection might have great clinical impact in disease prophylaxis by vaccination. For example, despite timely post-exposure treatment with rabies vaccine in the gluteal region, a patient died from rabies encephalitis (64), probably due to too shallow injection.

In contrast to fast absorption from the i.m. injection site, s.c. injection generally results in slow absorption. Therefore, sustained release injections are better placed in the s.c. or lipo- matous tissues. This has been demonstrated by Modderman et al. (65) and Pieters et al. (66). They found marked differ- ences in dapsone absorption profile between males and females. Males showed high peak concentrations in the first week and a shorter mean residence time. These differences were not found if injections were given at two-third of the indi- vidually measured skin to muscle distance, which is a guaran- tee for s.c. injection. Additionally, the sustained release characteristics were much better after intra-adipose (s.c. in the gluteal region) than after i.m. injection. Release times up to a month were obtained with dapsone and up to 3 months with its more lipophilic derivative monoacetyldapsone (67).

4.3. Anatomical Site of Injection The anatomical site of injection may be another important

factor determining the absorption rate of drug carriers after s.c. administration. This was clearly demonstrated when small (0.1 mm) liposomes were administered at different sites of the body of rats. After s.c. injection into the flank of rats, disap- pearance from the site of injection was much lower than after injection in the footpad or into the dorsal side of the foot. In fact, after s.c. injection into the flank of rats, the injected lipo- somes remain to a large, almost complete extent, at the site of injection, whereas about 60% of the injected dose reaches the blood circulation after injection into the foot ( Fig. 7 ). The observed site-dependent disposition is attributed to differences in the structural organization of the s.c. tissue at the different sites of injection (68). These results demonstrate that the ana- tomical site of s.c. injection should be considered carefully when designing injectable dispersions for local administration.

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Figure 7 Recovery of liposomal label from blood after s.c. admin- istration of liposomes at three different sites of injection. A single dose of radiolabeled liposomes (EPC:EPG:Chol, molar ratio 10:1:4; mean diameter, 0.1 mm, 2 mmol total lipid) was injected s.c. into the flank, into the dorsal side of the foot and into the footpad of rats. Values represent the mean percentage of injected dose circulating in the total blood volume

5. CARRIER KINETICS AND TARGETING The i.v. route is the preferable route of administration

to study carrier kinetics. Here, we will discuss mainly the biodistribution and pharmacokinetics of liposomes after i.v. administration as an example. Other particulate systems such as microspheres behave similarly to large liposomes because size and surface characteristics are the major determinants.

Studies on the in vivo fate or elimination of i.v. adminis- tered liposomes also provide information on other routes of injection, since liposomes, which are injected by other routes

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than the i.v. route, usually reach the circulation to some extent (23). Upon i.v. injection or intact absorption from local injection sites, liposomes and other particulate systems are mainly eliminated by accumulation in organs rich in cells belonging to the mononuclear phagocyte system (MPS). Due to rich blood supplies and the abundance of phagocytic MPS cells, the major sites of accumulation are the liver and spleen.

A dummy dose of a particulate system, or other phagocytotic depressant, can decrease the uptake of a dispersed system by the MPS through presaturation of the system. Dose- dependent kinetics follow logically from these observations and have been demonstrated for liposomes (69–72).

A number of studies have illustrated that when lipo- somes are exposed to serum or plasma, they rapidly acquire

a coating of proteinaceous molecules (73–76). Protein binding can be demonstrated by separating liposomes from serum or plasma incubations and then analyzing the liposome-protein complexes by sodium dodecylsulfate–polyacrylamide gel elec- trophoresis. Protein binding differs considerably in amount and pattern depending on the dose, size, lipid composition, bilayer rigidity, and surface characteristics (such as charge and hydrophilicity) of the vesicles. These liposome–blood pro- tein interactions have a number of important consequences for the subsequent pharmacokinetic behavior of the vesicles in vivo and for their use as drug carrier systems. Generally, small liposomes are cleared more slowly than large liposomes, partly due to less mechanical obstruction but also resulting from a lower affinity of serum proteins (opsonines) which are involved with the liver uptake by phagocytic cells (77). Opsonic activity also seems to depend on the lipophilicity of the particle surface, and on the presence of divalent cations which probably play a role in conformational changes in the opsonins (78). In addition, the net surface charge influences the opsonization process. Positively charged and neutral liposomes of similar size appear to circulate longer than nega- tively charged liposomes (79). Increasing the negative charge results in a dramatic acceleration of liposome clearance (80). Additionally, bilayer fluidity, which can be influenced by the

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use of phospholipids with long and saturated acyl chains or by the incorporation of cholesterol in the liposomal bilayer, determines stability in plasma and thus blood circulation times of liposomes.

Liposomes are relatively rapidly cleared from the blood circulation by the MPS. To avoid this rapid clearance, sur- face-modified liposomes which escape from MPS uptake and achieve prolonged circulation half-times in vivo have been extensively investigated during the last decade. Steric hinder- ing of negative charge by surface modification, hydrophiliza- tion, leads to increased circulation times and half-life by preventing interaction with opsonins. Increase of carrier half- life can be realized by the addition of carbohydrate moieties or the introduction of poly(ethyleneglycol) (PEG) derivatives absorbed or covalently bound to particulate materials or mem- brane lipids of liposomes. The most popular means to obtain sterically stabilized liposomes is to incorporate PEG conju- gated to distearoyl-phosphatidyl-ethanolamine (DSPE-PEG) into the liposomal bilayer (28,81–86).

Prolonged circulation times of sterically stabilized lipo- somes result in increased amounts of liposomes extravasating in areas where the permeability of the endothelial barrier is increased, specifically infected tissue and tumor tissue. There- fore, sterically stabilized liposomes can be used to target drugs to these tissues (passive targeting) (87–89). An approach to site specific delivery of liposomes is to conjugate them with homing devices such as monoclonal or polyclonal antibodies (active targeting). These immunoliposomes demonstrate high selectiv- ity of drug delivery in vitro (90–92). However, coupling of antibodies to liposomes increases MPS uptake. As a result, cir- culation times of immunoliposomes in blood are generally shorter than circulation times of conventional liposomes. Therefore, sterically stabilized immunoliposomes are currently under investigation (93–98). Results demonstrate that these liposomes combine the advantages of both systems, long circu- lation times, and site, specific drug delivery. To date, only a few reports on the therapeutic applications of long circulating immunoliposomes have appeared (99–102).

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