madman
Super Moderator
Abstract
Oil depots are parenteral drug formulations meant for sustained release of lipophilic compounds. Until now, a comprehensive understanding of the mechanism of drug absorption from oil depots is lacking. The aim of this paper was to fill this gap. A clinical study with healthy volunteers was conducted. An oil depot with nandrolone decanoate and benzyl alcohol was subcutaneously administered in the upper arm of female volunteers. Pharmacokinetic profiles of both substances were related to each other and to literature data. Benzyl alcohol absorbs much more rapidly than nandrolone. In detail, it appears that benzyl alcohol enters the central compartment directly, while nandrolone decanoate is recovered in serum after a lag time. This lag time is also seen in literature data, although not reported explicitly. The absorption of nandrolone is enhanced by the presence of benzyl alcohol. This is most likely an effect of altered oil viscosity and partition coefficient between the oil and aqueous phase. The absorption rate constant of compounds is found to be related to the logP of the solubilized prodrug. The absorption rate is however not only determined by the physicochemical properties of the formulation but also by the tissue properties. Here, it is argued that lymphatic flow must be considered as a relevant parameter.
1. Introduction
Oily solutions of lipophilic compounds are widely used as a sustained release formulation. Although this pharmaceutical approach has been applied for several decades already, relatively little research has been published on the fundamental parameters that determine the absorption characteristics. Generally, the formulation of an oil depot contains arachis or sesame oil as well as an amount of benzyl alcohol (BOH) which increases the solubility of the (pro)drug in the oil. In addition to these excipients, the formulation contains the active compound, most often as the esterified substance.
Theoretically, there are a number of factors that determine drug absorption from a parenteral oil depot:
1) The drug dissolved in the oil is released as a result of the concentration gradient. Relevant parameters are a) the concentration in the oil, b) the thickness of the diffusion layer as well as the diffusion coefficient in the oil, c) the surface area of the depot, d) the partition coefficient (P) between oil and tissue fluid and finally e) the thickness of the diffusion layer in the aqueous phase as well as the diffusivity in this compartment. Basically, this represents the rate at which the drug is transported through the tissue (Kadir et al., 1990, 1992; Minto et al., 1997; Tanaka et al., 1974; Zuidema et al., 1994, 1988). A simplification of the real vivo situation is depicted in Fig. 1. The oil liquid is not injected directly into the bloodstream, while yet the absorption is normally measured in this central compartment (Cserum). Therefore, a membrane should be included in this model representing the tissue that is situated in between the oil and the central circulation.
2) BOH exhibits not only a significant solubility in the oil phase, but does also dissolves in the aqueous phase. Consequently, it will also be released out of the depot. Because of the different physicochemical properties, it can be expected that BOH shows a completely different release profile than the prodrug. BOH in turn has a significant influence on the solubility of the active compound in both the oil and the aqueous phase (Rowe et al., 2009). Therefore, it is obvious that the partition coefficient is not a constant value during the release from the depot. This is presented as the partition coefficient between the concentrations at the interface in Fig. 1.
3) In most cases a lipophilic ester is used as a prodrug. After release out of the oil, this ester has to be hydrolyzed to the parent drug. The prodrug exhibits a significantly higher logP than the parent compound. As a consequence, the transport through the tissue can be considerably different; highly lipophilic drugs show both retardations by tissue absorption effects and lymphatic transport whereas less lipophilic compounds diffuse directly to the central circulation. Hence, the speed and the place at which (enzymatic) hydrolysis occurs may have an impact on the rate of absorption.
Other factors that may also contribute to the absorption rate are: injection depth (Ronald et al., 1993), site of injection (Soni et al., 1988; Vukovich et al., 1975), lymphatic absorption (Zuidema et al., 1994), massage before injection (Soni et al., 1988) and muscle activity (Soni et al., 1988). Although these suggested and obvious factors could lead to a complete understanding of drug absorption from oil depots, no studies have been published on this topic so far. This article makes a distinction between release and absorption kinetics: substance release from the depot can be translated from the mass flux from oil towards the aqueous phase, while absorption represents the entire process in which the substance enters in de central compartment. Hence, absorption includes the release out of the oil and the subsequent transfer through the tissue to the bloodstream.
The current study started with a clinical trial in which an oil depot containing nandrolone decanoate was used. Frequent sampling of volunteers enabled us to monitor the absorption phase in detail. The aim of this paper is to create a further understanding of the fundamental mechanisms that determine the drug absorption from a parenteral oil depot. Second, this paper elucidates the effect of BOH on nandrolone absorption. The observations are compared with results reported in the literature and put into perspective with the pharmacokinetic profile of BOH that has been published separately [Kalicharan, BOH article].
2. Materials and methods
2.1 Experimental design
Drug product was manufactured under current Good Manufacturing Practice conditions in the hospital pharmacy at the University Medical Center Utrecht, The Netherlands. Each 1.0 ml of the solution contained 117 μmol nandrolone decanoate (ND), 28,000 IU cholecalciferol, 926 μmol (10% (m/v)) BOH and ad 1.0 mL sesame oil. In this study, 0.5 ml of the solution was subcutaneously (s.c.) injected in the upper arm. Fourteen female volunteers participated in this study. Fully informed written consent was obtained from the volunteers, conforming to the Declaration of Helsinki. Inclusion criteria were: good physically and mentally healthy Caucasian females with an age between 65–80 years old and a body mass index (BMI) between 20–30 kg/m2. Volunteers were excluded when using any drug, food, or beverages that influence the metabolism of ND from 2 weeks or 5 half-lives of the medication (whichever is longer) prior to drug administration. Smoking was allowed, provided no more than 4 cigarettes or equivalents were used per day.
A validated LC–MS/MS bioassay was used to determine serum nandrolone concentrations (LoQ = 0.12 pmol/mL). Blood samples were taken directly after injection (0 h) and 2, 4, 8, 12, 15, 22, 24, 36, 48, 72, 96, 168, 216, 264, 360, 456, 552, 648 and 840 h after injection.
2.2. Pharmacokinetic analysis
Pharmacokinetic parameters were examined using Microsoft Excel 2010. The following parameters were determined: maximum serum concentration (Cmax) and time to reach this concentration (Tmax); the area under the serum concentration-time curve (AUC) was calculated using the linear trapezoidal rule. All data are expressed as mean ± standard error of the mean (SEM).
3. Results and discussion
The baseline characteristics of the fourteen females were (mean ± standard error): Age = 70.7 ± 4.3 year, length = 1.65 ± 0.06 m, weight = 68.0 ± 9.0 kg and BMI = 24.8 ± 2.3 kg/m2 . All subjects were included in the following analysis. No adverse reactions were reported during or after the study.
3.1. Pharmacokinetic profile of nandrolone
The serum profile of nandrolone is presented in Fig. 2. The pharmacokinetic parameters are summarized in Table 1. Remarkably, Fig. 2 shows two distinct Cmax values; there is a peak at 22 h while a second maximum is reached between 9–15 days post-injection. Surprisingly, literature data (Table 2) show these double peaks in all nandrolone oil depot studies, although it is not mentioned explicitly. In clinical papers about oil depots containing antipsychotics, the occurrence of two peaks has been noted, however (Jann et al., 1985). Here, the first peak was thought to be associated with the presence of a certain quantity of parent compound, i.e. the substance without the ester moiety. In our clinical study, no additional peaks were obtained during the quality control analysis of the investigational medicinal product (IMP). This indicates no significant amount of parent compound in the IMP, which may cause the first nandrolone peak. This phenomenon will be discussed further below.
Fig. 2 also shows the serum profile of benzyl alcohol. This has been reported earlier [Kalicharan et al., BOH article], in a paper which also reports the development of a bioassay for BOH. As can be seen, BOH shows a completely different pharmacokinetic profile compared to the nandrolone profile; BOH is present in the central circulation at the earliest moment samples were taken, i.e. only a few minutes after the moment of injection. This suggests principally different mechanisms of absorption compared to nandrolone; BOH appears to absorb fairly rapidly, whereas nandrolone exhibits a slow absorption. Another observation is that the first nandrolone peak at 22 h post-injection coincides with a peak in BOH level, after which both substances show a decline in serum concentration (Fig. 2A). Furthermore, the absorption of nandrolone is slower after 36 h post-injection compared to the period directly after injection. BOH was only recovered during the first 36 h post-injection, which may suggest that the initially increased absorption of nandrolone is related to the quick release of BOH; the obvious explanation can be that this will lead to a decrease in the concentration of the solubilizing compounds in the oil phase while it will have the reverse effect in the aqueous phase at the same time. Basically, this means that the partition coefficient between oily and aqueous phase changes during the release of BOH until there is a full depletion. In addition, also the viscosity of the oil depot changes during BOH release (Fig. 3). As expressed by the Stokes-Einstein law, the diffusion coefficient decreases upon increasing viscosity.
After the depletion of BOH, nandrolone clearly absorbs more slowly, resulting in a second prolonged serum peak, representing a steady-state situation. Fig. 4A depicts the cumulative absorption profile of nandrolone. These results were obtained by converting the serum level (Fig. 2) to the amount of absorbed nandrolone (Fig. 4) using the Wagner–Nelson method. Also, the cumulative release of BOH is given. This is in this case expressed as area under the curve instead of the percentage absorbed because the Wagner–Nelson method could not be applied; the kinetic parameters elimination constant and the distribution volume are unknown for BOH. After 52 h post-injection, no BOH was detected in serum anymore, which implies that the depot at this point is depleted of BOH. Therefore, we assume a nearly 100% recovery of BOH from the depot.
3.2. Lag time
As can be seen in Fig. 4B, the absorption phase for BOH is quick and complete. In contrast, the absorption of nandrolone is principally different; the exposure is not instantaneous. There appears to be a lag time before nandrolone enters the central circulation. From a physical perspective, it is obvious that the compound release starts immediately after injection. This has been confirmed in in vitro studies (Larsen et al., 2006; Thing et al., 2012). It must be noted that these in vitro models did not include a membrane. BOH seems to behave according to these vitro models, but knowing that there is no direct contact between blood and oil, it should be concluded that the lag time for BOH is extremely short. Hence, the transport through the tissue layer occurs very rapidly. The transport of ND from the oil to the central circulation appears to be slower, however. Obviously, there is a retarding factor in the tissue and therefore it is clear that this lag time period reflects the mechanism of transport in the tissue. There were no relationships found between the volunteers' baseline characteristics (e.g. BMI, weight, length or age) and the lag time. In this study, nandrolone injected subcutaneously as decanoate, exhibits a lag time of about eight hours (Fig. 4B).
The literature demonstrates that this phenomenon is not unique to a specific oil depot. A lag time is seen in all parenteral oil depot formulations (Table 2). This is not a constant interval, however. Several endogenous factors may contribute to this delayed absorption: (temporary) cell membrane adsorption, cell absorption, esterase activity, interstitial and/or lymph flow, and/or alternative pathways. After injection of the depot into the tissue, the prodrug starts to release and enters the interstitial space, in which the depot is injected. These prodrugs, being ester compounds, can spontaneously be hydrolyzed with water or actively be hydrolyzed by esterases. Van der Vies (1970) published half-life hydrolysis of 4 min for 2.5 ∗ 10–3 μmol/mL (1 μg/mL) nandrolone phenylpropionate in rat plasma. Although no hydrolysis data on nandrolone decanoate in human fluids have been published yet, Wijnand et al. (1985) assumed that the time needed for hydrolysis of 2.3 ∗ 10–5 μmol/mL (=0.01 μg/mL) nandrolone decanoate in serum is presumably below one hour. They based their assumptions on data published by Van Der Vies in 1970. In the present study, released nandrolone decanoate will then be hydrolyzed within one hour in plasma, because all nandrolone serum concentrations were below 1.0 ∗ 10– 5 μmol/mL (Fig. 2B). Yet a delayed absorption is seen, indicating that ester hydrolysis is not occurring in the interstitial fluid, but probably only in the central circulation. When hydrolysis is not immediate, the prodrug may flow towards lymph vessels, whether or not linked to proteins such as globulins. Interstitial space consists of reticular and collagenous fibers (Olszewski, 1985), to which molecules could be adsorbed. The absorption rate into the lymph depends on the rate of diffusion through the interstitium (O'Hagan et al., 1992) and pressure gradients (Swartz and Fleury, 2007). It has already been suggested by Zuidema and colleagues that the interstitial space can be compared to reversed-phase chromatography (Zuidema et al., 1988): the interstitial fluid acts as the mobile phase, whereas the cells and fibers represent the stationary phase. The lag time is probably a combination of the mentioned factors above.
The nearly immediate absorption of BOH suggests a fast pathway to the central compartment (Fig. 1). Oil depots injected into a muscle or subcutaneous tissues are surrounded by (micro) blood vessels. It is well known that blood vessels are natural barriers because they are constructed out of a concatenation of endothelial cells. Porter and Charman showed that small molecules (b2000 molecular weight) with a logP (octanol/water) under five enter the systemic circulation directly after oral ingestion (Porter et al., 2007). These substances can pass capillary walls which result in instantaneous absorption into the systemic circulation (Porter and Charman, 2000). Analogous to oral absorption, parent compounds can immediately enter the bloodstream when they meet the two chemical properties molecular weight and logP.
It is interesting to realize that drug absorption from an oil depot is not well described by a simple two-phase mass transfer model. There is obviously a considerable resistance towards mass transfer in the tissue surrounding the depot, which is given in Fig. 1 as a tissue membrane. The presence of this membrane fits with the finding of a substantial lag time for nandrolone. The difficulty with this theoretical presentation is that this membrane represents body tissue where not only diffusion takes place, but where also lymphatic transport and hydrolysis may play a role.
3.3. Absorption
After the lag time period, a steady absorption is established (Fig. 4), resulting in a plateau as seen in Fig. 2B. In general, the obvious parameters that have an influence on drug release can be understood from Fig. 1; the higher the concentration in the depot, the higher the driving force for release (Bagchus et al., 2005). The same holds for the surface area. Minto et al. (1997) have shown that a simultaneous increase in volume and a proportional decrease in concentration does not change the absorption significantly (Table 2). It has been postulated (Tanaka et al., 1974; Weng Larsen and Larsen, 2009) that the digestion of oil would play a role in the rate at which compounds are absorbed, but that is not confirmed by our data.
After approximately two weeks, the plasma profile declines, which in fact reflects the depletion of the depot; the concentration gradient gradually decreases because of the amount of drug that already has been released. This is generally referred to as ‘flip-flop’ pharmacokinetics; what might be perceived as the elimination rate constant basically equals the absorption rate constant (ka) from the formulation (Wijnand et al., 1985; Yáñez et al., 2011). In the case of the oil depots studied, a higher ka reflects a faster depletion of the depot, which actually means that the resistance towards mass transfer in the surrounding tissue is relatively low. As discussed above, this mass transfer is determined by e.g. the diffusion through the interstitial fluid and the flow of the lymphatic system into the circulation.
Fig. 5 shows that the absorption of ingredients from depots injected in the gluteal muscle is determined by the partition coefficient of the prodrugs. The role of logP is twofold; it determines the concentration Ctissue i , which means that a high logP will yield a low Ctissue i and subsequently a low driving force for mass transfer in the aqueous phase (Fig. 1). At the same time, a high logP results in increased absorptive and adsorptive interactions with tissue components by which the permeability decreases. Similar relationships have been published for intramuscularly injected beta-blockers (aqueous solutions) in pigs (Kadir et al., 1990) where the absorption rate appeared also to be affected by the lipophilicity. Remarkably, there is no such relationship as depicted in Fig. 5 for the parent compound, which seems to confirm our conclusion that it is the prodrug that is transported through the tissue and that conversion to the parent compound takes place in the central compartment.
Absorption rate constants of different compounds were compared after administration in the gluteal muscle (Fig. 5). In contrast, Fig. 6 shows the normalized profiles of equal formulations injected in different muscles: As can be seen, the site of injection appears to have a considerable influence on the absorption. For comparison, also the subcutaneous injection of the present study is given. It must be noted however that the used oil, as well as the volume of injection, were different. Our own experiments (based on Andrés et al., 2015, procedure 2) showed that the type of oil does not affect the partition coefficient of nandrolone decanoate: logP (sesame oil: serum) = 2.36 and logP (Arachis oil: serum) = 2.44 (both mixed with 10% BOH), which emphasizes the low absorption from the subcutaneous injection.
The i.m. depot formulations were equal, i.e. they contained the same compound at the same concentration in the same volume. This limits the number of explanations for these differences. First, it can be speculated that the surface areas of the depots were different in the three muscles because of differences in spreading. The larger the surface, the larger the total mass flux (Φ″). Unfortunately, the surface areas of injected oil depots are unknown. This has been a reason for us to start a study to visualize the fate of oil when injected in muscles, the results of which will be reported later. The role of surface area may also be relevant for the s.c. injection; the absorption was significantly lower, which can be attributed to the smaller surface area of the depot as it is likely that the shear forces in subcutaneous tissue are substantially lower than those in muscles. In addition, the way the compound is being transported through different tissues may be different. In this respect, the lymphatic flow rate may therefore be interesting to study as well.
Fig. 6 shows that absorption from the deltoid muscle is much lower compared to that from gluteal and vastus lateralis muscles. This is counterintuitive since blood flow in the deltoid muscle is higher than the flow in gluteal and vastus lateralis (Evans et al., 1975). However, as has been argued, it is not the blood perfusion but the lymphatic drainage that determines drug absorption in this case. Table 2 shows that the ND absorption rate constant from the deltoid muscle is lower compared to the gluteal and vastus lateris muscle. There appears to be a relationship between the absorption rate constant and the lag time within one muscle group (Fig. 7). The reasonable explanation for this must be that both parameters may be a result of the same variable. For example, when the differences in the release are due to different surface areas, the lag time would be the same, since the way the compound is transferred to the bloodstream would not be changed. Therefore, it is more likely that the differences in transport through tissue, such as lymphatic transport may determine the absorption phase. This would also explain the considerably deviating results of the s.c. injection; the low release rate may be attributed to a low surface area, whereas the relatively short lag time may be a result of relatively high lymphatic flow or shorter lymph vessels. Unfortunately, little is known about the human lymphatic flow rate in the studied muscles, although one article reported a lymph flow rate of 0.25–0.41 mL/h in the superficial lymph vessel of the leg (Olszewski and Engeset, 1980). In rats and rabbits, however, skin lymph flow (measured as albumin clearance rate) is shown to be higher compared to the muscle lymph flow (Bach and Lewis, 1973; Renkin and Wiig, 1994). Of course, this does not necessarily mean that this also applies to the human situation.
Another approach to verify the suggested mechanism would be to change the lymphatic flow. Two studies report a raised lymph flow (measured as albumin clearance rate) in the lymphatic vessels of the vastus lateralis muscle during exercise (Havas et al., 1997) or during massage (rabbits) (Ikomi et al., 2014). However, Soni et al. (1988) described no increased absorption of fluphenazine after exercise or massage from a fluphenazine decanoate sesame oil depot, injected in the tight and buttock skeletal muscle. Clearly, there is no unambiguous explanation for the observed differences at this moment. Future studies on this subject should provide clarity.
4. Conclusions
This study discusses critical parameters that determine the release and absorption mechanisms of (active) substances from oil depots. It is shown that small molecules (e.g. BOH) are directly and fully absorbed, while larger, more lipophilic substances (e.g. prodrugs) exhibit an incomplete and slow absorption pattern. A lag time is seen, which is a critical parameter for absorption into the systemic circulation. This means that the absorption of compounds from a depot is significantly affected by the mass transfer in the tissue. The more lipophilic the compound, the more this plays a role. It is suggested that the concentration of the compound in the oil, the in situ surface area of the depot as well as the partition coefficient of the compound are the most important formulation parameters. The mass transfer is mainly determined by the lipophilicity of the compound, while also the lymphatic flow is suggested to be relevant for drug absorption. In oil depots, BOH is often used as an excipient. It appeared that the absorption of nandrolone is enhanced by the presence of benzyl alcohol in the first few days. Subsequently, upon BOH depletion, a change in absorption of nandrolone is seen. Injections of equivalent formulation in different muscles demonstrate that the mass transfer through these tissues is not the same. It is argued that this may be due to differences in lymphatic transport.
Oil depots are parenteral drug formulations meant for sustained release of lipophilic compounds. Until now, a comprehensive understanding of the mechanism of drug absorption from oil depots is lacking. The aim of this paper was to fill this gap. A clinical study with healthy volunteers was conducted. An oil depot with nandrolone decanoate and benzyl alcohol was subcutaneously administered in the upper arm of female volunteers. Pharmacokinetic profiles of both substances were related to each other and to literature data. Benzyl alcohol absorbs much more rapidly than nandrolone. In detail, it appears that benzyl alcohol enters the central compartment directly, while nandrolone decanoate is recovered in serum after a lag time. This lag time is also seen in literature data, although not reported explicitly. The absorption of nandrolone is enhanced by the presence of benzyl alcohol. This is most likely an effect of altered oil viscosity and partition coefficient between the oil and aqueous phase. The absorption rate constant of compounds is found to be related to the logP of the solubilized prodrug. The absorption rate is however not only determined by the physicochemical properties of the formulation but also by the tissue properties. Here, it is argued that lymphatic flow must be considered as a relevant parameter.
1. Introduction
Oily solutions of lipophilic compounds are widely used as a sustained release formulation. Although this pharmaceutical approach has been applied for several decades already, relatively little research has been published on the fundamental parameters that determine the absorption characteristics. Generally, the formulation of an oil depot contains arachis or sesame oil as well as an amount of benzyl alcohol (BOH) which increases the solubility of the (pro)drug in the oil. In addition to these excipients, the formulation contains the active compound, most often as the esterified substance.
Theoretically, there are a number of factors that determine drug absorption from a parenteral oil depot:
1) The drug dissolved in the oil is released as a result of the concentration gradient. Relevant parameters are a) the concentration in the oil, b) the thickness of the diffusion layer as well as the diffusion coefficient in the oil, c) the surface area of the depot, d) the partition coefficient (P) between oil and tissue fluid and finally e) the thickness of the diffusion layer in the aqueous phase as well as the diffusivity in this compartment. Basically, this represents the rate at which the drug is transported through the tissue (Kadir et al., 1990, 1992; Minto et al., 1997; Tanaka et al., 1974; Zuidema et al., 1994, 1988). A simplification of the real vivo situation is depicted in Fig. 1. The oil liquid is not injected directly into the bloodstream, while yet the absorption is normally measured in this central compartment (Cserum). Therefore, a membrane should be included in this model representing the tissue that is situated in between the oil and the central circulation.
2) BOH exhibits not only a significant solubility in the oil phase, but does also dissolves in the aqueous phase. Consequently, it will also be released out of the depot. Because of the different physicochemical properties, it can be expected that BOH shows a completely different release profile than the prodrug. BOH in turn has a significant influence on the solubility of the active compound in both the oil and the aqueous phase (Rowe et al., 2009). Therefore, it is obvious that the partition coefficient is not a constant value during the release from the depot. This is presented as the partition coefficient between the concentrations at the interface in Fig. 1.
3) In most cases a lipophilic ester is used as a prodrug. After release out of the oil, this ester has to be hydrolyzed to the parent drug. The prodrug exhibits a significantly higher logP than the parent compound. As a consequence, the transport through the tissue can be considerably different; highly lipophilic drugs show both retardations by tissue absorption effects and lymphatic transport whereas less lipophilic compounds diffuse directly to the central circulation. Hence, the speed and the place at which (enzymatic) hydrolysis occurs may have an impact on the rate of absorption.
Other factors that may also contribute to the absorption rate are: injection depth (Ronald et al., 1993), site of injection (Soni et al., 1988; Vukovich et al., 1975), lymphatic absorption (Zuidema et al., 1994), massage before injection (Soni et al., 1988) and muscle activity (Soni et al., 1988). Although these suggested and obvious factors could lead to a complete understanding of drug absorption from oil depots, no studies have been published on this topic so far. This article makes a distinction between release and absorption kinetics: substance release from the depot can be translated from the mass flux from oil towards the aqueous phase, while absorption represents the entire process in which the substance enters in de central compartment. Hence, absorption includes the release out of the oil and the subsequent transfer through the tissue to the bloodstream.
The current study started with a clinical trial in which an oil depot containing nandrolone decanoate was used. Frequent sampling of volunteers enabled us to monitor the absorption phase in detail. The aim of this paper is to create a further understanding of the fundamental mechanisms that determine the drug absorption from a parenteral oil depot. Second, this paper elucidates the effect of BOH on nandrolone absorption. The observations are compared with results reported in the literature and put into perspective with the pharmacokinetic profile of BOH that has been published separately [Kalicharan, BOH article].
2. Materials and methods
2.1 Experimental design
Drug product was manufactured under current Good Manufacturing Practice conditions in the hospital pharmacy at the University Medical Center Utrecht, The Netherlands. Each 1.0 ml of the solution contained 117 μmol nandrolone decanoate (ND), 28,000 IU cholecalciferol, 926 μmol (10% (m/v)) BOH and ad 1.0 mL sesame oil. In this study, 0.5 ml of the solution was subcutaneously (s.c.) injected in the upper arm. Fourteen female volunteers participated in this study. Fully informed written consent was obtained from the volunteers, conforming to the Declaration of Helsinki. Inclusion criteria were: good physically and mentally healthy Caucasian females with an age between 65–80 years old and a body mass index (BMI) between 20–30 kg/m2. Volunteers were excluded when using any drug, food, or beverages that influence the metabolism of ND from 2 weeks or 5 half-lives of the medication (whichever is longer) prior to drug administration. Smoking was allowed, provided no more than 4 cigarettes or equivalents were used per day.
A validated LC–MS/MS bioassay was used to determine serum nandrolone concentrations (LoQ = 0.12 pmol/mL). Blood samples were taken directly after injection (0 h) and 2, 4, 8, 12, 15, 22, 24, 36, 48, 72, 96, 168, 216, 264, 360, 456, 552, 648 and 840 h after injection.
2.2. Pharmacokinetic analysis
Pharmacokinetic parameters were examined using Microsoft Excel 2010. The following parameters were determined: maximum serum concentration (Cmax) and time to reach this concentration (Tmax); the area under the serum concentration-time curve (AUC) was calculated using the linear trapezoidal rule. All data are expressed as mean ± standard error of the mean (SEM).
3. Results and discussion
The baseline characteristics of the fourteen females were (mean ± standard error): Age = 70.7 ± 4.3 year, length = 1.65 ± 0.06 m, weight = 68.0 ± 9.0 kg and BMI = 24.8 ± 2.3 kg/m2 . All subjects were included in the following analysis. No adverse reactions were reported during or after the study.
3.1. Pharmacokinetic profile of nandrolone
The serum profile of nandrolone is presented in Fig. 2. The pharmacokinetic parameters are summarized in Table 1. Remarkably, Fig. 2 shows two distinct Cmax values; there is a peak at 22 h while a second maximum is reached between 9–15 days post-injection. Surprisingly, literature data (Table 2) show these double peaks in all nandrolone oil depot studies, although it is not mentioned explicitly. In clinical papers about oil depots containing antipsychotics, the occurrence of two peaks has been noted, however (Jann et al., 1985). Here, the first peak was thought to be associated with the presence of a certain quantity of parent compound, i.e. the substance without the ester moiety. In our clinical study, no additional peaks were obtained during the quality control analysis of the investigational medicinal product (IMP). This indicates no significant amount of parent compound in the IMP, which may cause the first nandrolone peak. This phenomenon will be discussed further below.
Fig. 2 also shows the serum profile of benzyl alcohol. This has been reported earlier [Kalicharan et al., BOH article], in a paper which also reports the development of a bioassay for BOH. As can be seen, BOH shows a completely different pharmacokinetic profile compared to the nandrolone profile; BOH is present in the central circulation at the earliest moment samples were taken, i.e. only a few minutes after the moment of injection. This suggests principally different mechanisms of absorption compared to nandrolone; BOH appears to absorb fairly rapidly, whereas nandrolone exhibits a slow absorption. Another observation is that the first nandrolone peak at 22 h post-injection coincides with a peak in BOH level, after which both substances show a decline in serum concentration (Fig. 2A). Furthermore, the absorption of nandrolone is slower after 36 h post-injection compared to the period directly after injection. BOH was only recovered during the first 36 h post-injection, which may suggest that the initially increased absorption of nandrolone is related to the quick release of BOH; the obvious explanation can be that this will lead to a decrease in the concentration of the solubilizing compounds in the oil phase while it will have the reverse effect in the aqueous phase at the same time. Basically, this means that the partition coefficient between oily and aqueous phase changes during the release of BOH until there is a full depletion. In addition, also the viscosity of the oil depot changes during BOH release (Fig. 3). As expressed by the Stokes-Einstein law, the diffusion coefficient decreases upon increasing viscosity.
After the depletion of BOH, nandrolone clearly absorbs more slowly, resulting in a second prolonged serum peak, representing a steady-state situation. Fig. 4A depicts the cumulative absorption profile of nandrolone. These results were obtained by converting the serum level (Fig. 2) to the amount of absorbed nandrolone (Fig. 4) using the Wagner–Nelson method. Also, the cumulative release of BOH is given. This is in this case expressed as area under the curve instead of the percentage absorbed because the Wagner–Nelson method could not be applied; the kinetic parameters elimination constant and the distribution volume are unknown for BOH. After 52 h post-injection, no BOH was detected in serum anymore, which implies that the depot at this point is depleted of BOH. Therefore, we assume a nearly 100% recovery of BOH from the depot.
3.2. Lag time
As can be seen in Fig. 4B, the absorption phase for BOH is quick and complete. In contrast, the absorption of nandrolone is principally different; the exposure is not instantaneous. There appears to be a lag time before nandrolone enters the central circulation. From a physical perspective, it is obvious that the compound release starts immediately after injection. This has been confirmed in in vitro studies (Larsen et al., 2006; Thing et al., 2012). It must be noted that these in vitro models did not include a membrane. BOH seems to behave according to these vitro models, but knowing that there is no direct contact between blood and oil, it should be concluded that the lag time for BOH is extremely short. Hence, the transport through the tissue layer occurs very rapidly. The transport of ND from the oil to the central circulation appears to be slower, however. Obviously, there is a retarding factor in the tissue and therefore it is clear that this lag time period reflects the mechanism of transport in the tissue. There were no relationships found between the volunteers' baseline characteristics (e.g. BMI, weight, length or age) and the lag time. In this study, nandrolone injected subcutaneously as decanoate, exhibits a lag time of about eight hours (Fig. 4B).
The literature demonstrates that this phenomenon is not unique to a specific oil depot. A lag time is seen in all parenteral oil depot formulations (Table 2). This is not a constant interval, however. Several endogenous factors may contribute to this delayed absorption: (temporary) cell membrane adsorption, cell absorption, esterase activity, interstitial and/or lymph flow, and/or alternative pathways. After injection of the depot into the tissue, the prodrug starts to release and enters the interstitial space, in which the depot is injected. These prodrugs, being ester compounds, can spontaneously be hydrolyzed with water or actively be hydrolyzed by esterases. Van der Vies (1970) published half-life hydrolysis of 4 min for 2.5 ∗ 10–3 μmol/mL (1 μg/mL) nandrolone phenylpropionate in rat plasma. Although no hydrolysis data on nandrolone decanoate in human fluids have been published yet, Wijnand et al. (1985) assumed that the time needed for hydrolysis of 2.3 ∗ 10–5 μmol/mL (=0.01 μg/mL) nandrolone decanoate in serum is presumably below one hour. They based their assumptions on data published by Van Der Vies in 1970. In the present study, released nandrolone decanoate will then be hydrolyzed within one hour in plasma, because all nandrolone serum concentrations were below 1.0 ∗ 10– 5 μmol/mL (Fig. 2B). Yet a delayed absorption is seen, indicating that ester hydrolysis is not occurring in the interstitial fluid, but probably only in the central circulation. When hydrolysis is not immediate, the prodrug may flow towards lymph vessels, whether or not linked to proteins such as globulins. Interstitial space consists of reticular and collagenous fibers (Olszewski, 1985), to which molecules could be adsorbed. The absorption rate into the lymph depends on the rate of diffusion through the interstitium (O'Hagan et al., 1992) and pressure gradients (Swartz and Fleury, 2007). It has already been suggested by Zuidema and colleagues that the interstitial space can be compared to reversed-phase chromatography (Zuidema et al., 1988): the interstitial fluid acts as the mobile phase, whereas the cells and fibers represent the stationary phase. The lag time is probably a combination of the mentioned factors above.
The nearly immediate absorption of BOH suggests a fast pathway to the central compartment (Fig. 1). Oil depots injected into a muscle or subcutaneous tissues are surrounded by (micro) blood vessels. It is well known that blood vessels are natural barriers because they are constructed out of a concatenation of endothelial cells. Porter and Charman showed that small molecules (b2000 molecular weight) with a logP (octanol/water) under five enter the systemic circulation directly after oral ingestion (Porter et al., 2007). These substances can pass capillary walls which result in instantaneous absorption into the systemic circulation (Porter and Charman, 2000). Analogous to oral absorption, parent compounds can immediately enter the bloodstream when they meet the two chemical properties molecular weight and logP.
It is interesting to realize that drug absorption from an oil depot is not well described by a simple two-phase mass transfer model. There is obviously a considerable resistance towards mass transfer in the tissue surrounding the depot, which is given in Fig. 1 as a tissue membrane. The presence of this membrane fits with the finding of a substantial lag time for nandrolone. The difficulty with this theoretical presentation is that this membrane represents body tissue where not only diffusion takes place, but where also lymphatic transport and hydrolysis may play a role.
3.3. Absorption
After the lag time period, a steady absorption is established (Fig. 4), resulting in a plateau as seen in Fig. 2B. In general, the obvious parameters that have an influence on drug release can be understood from Fig. 1; the higher the concentration in the depot, the higher the driving force for release (Bagchus et al., 2005). The same holds for the surface area. Minto et al. (1997) have shown that a simultaneous increase in volume and a proportional decrease in concentration does not change the absorption significantly (Table 2). It has been postulated (Tanaka et al., 1974; Weng Larsen and Larsen, 2009) that the digestion of oil would play a role in the rate at which compounds are absorbed, but that is not confirmed by our data.
After approximately two weeks, the plasma profile declines, which in fact reflects the depletion of the depot; the concentration gradient gradually decreases because of the amount of drug that already has been released. This is generally referred to as ‘flip-flop’ pharmacokinetics; what might be perceived as the elimination rate constant basically equals the absorption rate constant (ka) from the formulation (Wijnand et al., 1985; Yáñez et al., 2011). In the case of the oil depots studied, a higher ka reflects a faster depletion of the depot, which actually means that the resistance towards mass transfer in the surrounding tissue is relatively low. As discussed above, this mass transfer is determined by e.g. the diffusion through the interstitial fluid and the flow of the lymphatic system into the circulation.
Fig. 5 shows that the absorption of ingredients from depots injected in the gluteal muscle is determined by the partition coefficient of the prodrugs. The role of logP is twofold; it determines the concentration Ctissue i , which means that a high logP will yield a low Ctissue i and subsequently a low driving force for mass transfer in the aqueous phase (Fig. 1). At the same time, a high logP results in increased absorptive and adsorptive interactions with tissue components by which the permeability decreases. Similar relationships have been published for intramuscularly injected beta-blockers (aqueous solutions) in pigs (Kadir et al., 1990) where the absorption rate appeared also to be affected by the lipophilicity. Remarkably, there is no such relationship as depicted in Fig. 5 for the parent compound, which seems to confirm our conclusion that it is the prodrug that is transported through the tissue and that conversion to the parent compound takes place in the central compartment.
Absorption rate constants of different compounds were compared after administration in the gluteal muscle (Fig. 5). In contrast, Fig. 6 shows the normalized profiles of equal formulations injected in different muscles: As can be seen, the site of injection appears to have a considerable influence on the absorption. For comparison, also the subcutaneous injection of the present study is given. It must be noted however that the used oil, as well as the volume of injection, were different. Our own experiments (based on Andrés et al., 2015, procedure 2) showed that the type of oil does not affect the partition coefficient of nandrolone decanoate: logP (sesame oil: serum) = 2.36 and logP (Arachis oil: serum) = 2.44 (both mixed with 10% BOH), which emphasizes the low absorption from the subcutaneous injection.
The i.m. depot formulations were equal, i.e. they contained the same compound at the same concentration in the same volume. This limits the number of explanations for these differences. First, it can be speculated that the surface areas of the depots were different in the three muscles because of differences in spreading. The larger the surface, the larger the total mass flux (Φ″). Unfortunately, the surface areas of injected oil depots are unknown. This has been a reason for us to start a study to visualize the fate of oil when injected in muscles, the results of which will be reported later. The role of surface area may also be relevant for the s.c. injection; the absorption was significantly lower, which can be attributed to the smaller surface area of the depot as it is likely that the shear forces in subcutaneous tissue are substantially lower than those in muscles. In addition, the way the compound is being transported through different tissues may be different. In this respect, the lymphatic flow rate may therefore be interesting to study as well.
Fig. 6 shows that absorption from the deltoid muscle is much lower compared to that from gluteal and vastus lateralis muscles. This is counterintuitive since blood flow in the deltoid muscle is higher than the flow in gluteal and vastus lateralis (Evans et al., 1975). However, as has been argued, it is not the blood perfusion but the lymphatic drainage that determines drug absorption in this case. Table 2 shows that the ND absorption rate constant from the deltoid muscle is lower compared to the gluteal and vastus lateris muscle. There appears to be a relationship between the absorption rate constant and the lag time within one muscle group (Fig. 7). The reasonable explanation for this must be that both parameters may be a result of the same variable. For example, when the differences in the release are due to different surface areas, the lag time would be the same, since the way the compound is transferred to the bloodstream would not be changed. Therefore, it is more likely that the differences in transport through tissue, such as lymphatic transport may determine the absorption phase. This would also explain the considerably deviating results of the s.c. injection; the low release rate may be attributed to a low surface area, whereas the relatively short lag time may be a result of relatively high lymphatic flow or shorter lymph vessels. Unfortunately, little is known about the human lymphatic flow rate in the studied muscles, although one article reported a lymph flow rate of 0.25–0.41 mL/h in the superficial lymph vessel of the leg (Olszewski and Engeset, 1980). In rats and rabbits, however, skin lymph flow (measured as albumin clearance rate) is shown to be higher compared to the muscle lymph flow (Bach and Lewis, 1973; Renkin and Wiig, 1994). Of course, this does not necessarily mean that this also applies to the human situation.
Another approach to verify the suggested mechanism would be to change the lymphatic flow. Two studies report a raised lymph flow (measured as albumin clearance rate) in the lymphatic vessels of the vastus lateralis muscle during exercise (Havas et al., 1997) or during massage (rabbits) (Ikomi et al., 2014). However, Soni et al. (1988) described no increased absorption of fluphenazine after exercise or massage from a fluphenazine decanoate sesame oil depot, injected in the tight and buttock skeletal muscle. Clearly, there is no unambiguous explanation for the observed differences at this moment. Future studies on this subject should provide clarity.
4. Conclusions
This study discusses critical parameters that determine the release and absorption mechanisms of (active) substances from oil depots. It is shown that small molecules (e.g. BOH) are directly and fully absorbed, while larger, more lipophilic substances (e.g. prodrugs) exhibit an incomplete and slow absorption pattern. A lag time is seen, which is a critical parameter for absorption into the systemic circulation. This means that the absorption of compounds from a depot is significantly affected by the mass transfer in the tissue. The more lipophilic the compound, the more this plays a role. It is suggested that the concentration of the compound in the oil, the in situ surface area of the depot as well as the partition coefficient of the compound are the most important formulation parameters. The mass transfer is mainly determined by the lipophilicity of the compound, while also the lymphatic flow is suggested to be relevant for drug absorption. In oil depots, BOH is often used as an excipient. It appeared that the absorption of nandrolone is enhanced by the presence of benzyl alcohol in the first few days. Subsequently, upon BOH depletion, a change in absorption of nandrolone is seen. Injections of equivalent formulation in different muscles demonstrate that the mass transfer through these tissues is not the same. It is argued that this may be due to differences in lymphatic transport.
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