madman
Super Moderator
Key points here!
*the prodrug is neither hydrolysed in the interstitial fluid nor is able to diffuse in a significant way into the tissue.
*hydrolysis does not take place in the tissue around the site of injection, but rather occurs in the central circulation or in the liver.
*ND hydrolysis only occurred in human whole blood. The hydrolysis did not start immediately, but after a lag time.
* This lag time is most likely caused by the postponed ND hydrolysis which would mean that hydrolysis does not take place in the tissue around the site of injection, but rather occurs in the central circulation or in the liver. This research elucidates whether the hydrolysis of N occurs at the injection site, or in the central circulation.
*In literature, a half-life time hydrolysis of 4 min for 2.5 * 103mmol/mL (1mg/mL) nandrolone phenyl propionate in rat plasma has been reported (14). Based on these data, the duration time to hydrolyse 2.3 * 105mmol/mL (=0.01mg/mL) ND in HS was proposed by Wijnand et al. (Wijnand et al., 1985) to be below one hour. However, as reported by Li et al. and Rudakova et al. (Li et al., 2005; Rudakova et al., 2011), no carboxylesterases are present in HP. This implies that no enzymatical hydrolysis of ND to N can occur in HP and HS, which is confirmed in this current study: In human serum and plasma, no hydrolysis of ND occurred during 5 h of incubation. Also, no chemical hydrolysis of ND occurred in these media.
*Moreover, this entails that ND hydrolysis does not occur in interstitial fluid since interstitial fluid originates from blood plasma. It contains water and molecules less than approximately 40 kD molecular weight (Charman and Stella, 1992; Wiig et al., 2012). This means that even when carboxylesterases would be present in human blood plasma, they would not appear in the interstitial fluid as the molecular weight of human carboxylesterases is approximately 60 kD (Imai and Ohura, 2010).
*This paper demonstrates that ND hydrolysis does occur in human bl Apparently human blood contains carboxylesterases. The appearance of N was observed after an average lag time of 34.9 2.5 min. Noticeable was the absence of Michaelis-Menten behaviour. Instead of nonlinear enzyme kinetics, a linear rate of hydrolysis was seen (Fig. 4). These observations suggest that the carboxylesterases, involved in ND hydrolysis, must be present in the blood cells since the distinction between whole blood and plasma is the presence of cells. Hydrolysis can occur either on the blood cell membrane, or occurs intracellularly. Because a lag time of N appearance was noticed, carboxylesterase activity on the cell membrane is apparently not very pronounced. Otherwise, the N recovery should be seen instantaneously. It can therefore be concluded that hydrolysis occurs after membrane diffusion and, subsequently intracellularly in erythrocytes cytosol (Quon and Stampfli, 1985) and probably in leukocytes. This is in line with literature, where it is reported that carboxylesterases are present in the endoplasmic reticulum and cytosol in human cells.
*After injection of the oil depot, the released ND molecules appear in the interstitial tissue. Due to the high partition coefficient (log P = 8.1 (““ChemSpider,” 2015)) of ND, it is likely that ND adheres to small proteins ( < 40 kD) in the interstitial fluid. As argued above, the prodrug is neither hydrolysed in the interstitial fluid nor is able to diffuse in a significant way into the tissue. The interstitial fluid is drained via the lymph vessels, and therefore ND absorption into the central circulation via the lymphatic system is in fact the only likely route. Therefore, it can be reasoned that ND is hydrolysed when entering the central circulation. Alternatively, it is possible that the cellular components in the lymphatic system take care of (a part of) the hydrolysis.
Abstract
Long-term therapy of nandrolone (N) is recommended to increase mineral density and muscle strength. Using a parenteral sustained release drug formulation with nandrolone decanoate (ND), therapeutic N levels can be achieved and maintained. Until now, it is unknown if hydrolysis of ND into N occurs in tissue at the injection site or after systemic absorption. Therefore, hydrolysis studies were conducted to investigate the location and rate of ND hydrolysis after its release from the oil depot.
ND hydrolysis was studied in porcine tissues, to mimic the human muscular and subcutaneous tissues. Additionally, the ND hydrolysis was studied in human whole blood, plasma and serum at a concentration range of 23.3–233.3mM.
ND hydrolysis only occurred in human whole blood. The hydrolysis did not start immediately, but after a lag time. The mean lag time for all studied concentrations was 34.9 2.5 min. Because of a slow penetration into tissue, hydrolysis of ND is found to be very low in surrounding tissue. Therefore the local generation of the active compound is clinically irrelevant.
It is argued that after injection of the oil depot, ND molecules will be transported via the lymphatic system towards lymph nodes. From here, it will enter the central circulation and within half an hour it will hydrolyse to the active N compound.
1. Introduction
Androgens can be used to increase bone mineral density and muscle strength (Crawford et al., 2003; Erdtsieck et al., 1994; Frisoli et al., 2005; Notelovitz, 2002). For this purpose, long-term therapy of nandrolone is recommended. Therapeutic nandrolone levels in blood can be maintained using a parenteral sustained release drug formulation (Bagchus et al., 2005; Kalicharan et al., 2016c; Minto et al., 1997; Wijnand et al., 1985). An example of such a parenteral drug formulation is an oil depot. In general, slow release from oil depots is a result of the high partition coefficient of lipophilic compounds; The release rate decreases when the compound is more lipophilic. Increased lipophilicity can be accomplished through esterification with a fatty acid. For nandrolone, the decanoate has been selected as the appropriate moiety. In contrast to nandrolone (the active parent compound), nandrolone decanoate (ND) is an inactive prodrug. Oil depots with ND have been applied in several clinical studies, in which they were administrated by intramuscular (i.m.) (Bagchus et al., 2005; Minto et al., 1997; Wijnand et al., 1985) or subcutaneous (s.c.) routes (Kalicharan et al., 2016c).
Although pharmacokinetic profiles of nandrolone depots have been published, the fundamental mechanisms of drug release and absorption into the central circulation have hardly been studied. In theory, ND is released from the oil depot into the interstitial (tissue) fluid. The rate at which this occurs is largely determined by the compound concentration in the oil formulation and its partition coefficient. Subsequently, ND is hydrolysed into nandrolone. Until now, it is generally assumed that ND is hydrolysed in serum and not in the tissue (fluid) at the site of injection (Wijnand et al., 1985)
Recently, we have demonstrated that there exists a delay (lag time) in the appearance of nandrolone in the central circulation (Kalicharan et al., 2016c). Since nandrolone has a log P of 3.0 (“ChemSpider,” 2015) and therefore will be absorbed relatively rapidly, this observed lag time indicates that immediate hydrolyses of ND does not occur. The lag time can be affected by several factors, such as diffusion in tissue fluid, cell membrane adsorption and cell absorption (Kalicharan et al., 2016c). On the one hand, at the site of injection, interstitial fluid transport is slower than blood flow. On the other hand, this transport is faster than diffusion. Another factor, cell membrane adsorption is relevant, because lipophilic prodrugs have high affinity with lipophilic cell structures such as cell membranes and membrane proteins. Due to the adherence to the cell membrane, cell absorption seems a logical consequence. Once absorbed, the lipophilic prodrug can be hydrolysed by esterases (if present) localized in cytosol and microsomes (Jewell et al., 2007; Li et al., 2005; Prusakiewicz et al., 2006). After hydrolysis, efflux of the active parent compound out of the cell must occur in order to reach the central circulation. All these factors may contribute to a prolonged residence time of the lipophilic prodrug in tissues and fluids around the injection site.
Hydrolysis can occur via chemical processes or by carboxylesterases (Imai and Ohura, 2010; Jewell et al., 2007; Prusakiewicz et al., 2006). These enzymes hydrolyse a different ester prodrug, haloperidol decanoate (Nambu et al., 1987; Oh-E et al., 1987). Because the ester bond in this prodrug is similar to the ester bond in ND, it is likely that ND hydrolysis also occurs by carboxylesterases. To our knowledge, this has however never been published yet and will be studied in this paper.
Interestingly, carboxylesterases are inhibited by benzil (Hatfield and Potter, 2011). This compound shows great similarity on molecular structure with a commonly added oil depot additive: benzyl alcohol (BOH). Although BOH is processed in a significant quantity in oil depots of 1–10% (m/v) (Bagchus et al., 2005; Kalicharan et al., 2016c; Minto et al., 1997; Van Weringh et al., 1994; Wijnand et al., 1985), any inhibitory effect of BOH on carboxylesterases is yet unknown but can be clinically relevant if it inhibits carboxylesterases.
The time period between ND release from the oil depot and metabolism in the liver may account for the complete lag time, but it is also well possible that hydrolysis occurs earlier in the absorption phase. Until now, this has never been unambiguously demonstrated.
4. Discussion
This research aimed to study whether locally injected ND as an oil depot is hydrolysed at the site of injection. Drug absorption from a locally injected oil depot into the central circulation exhibits a delayed absorption pattern, which is defined as lag time (Kalicharan et al., 2016c). About 8 h after a s.c. ND oil depot injection, N is observed in the central circulation (Kalicharan et al., 2016c). Remarkably, this lag time is much shorter when the oil depot is injected in the vastus lateralis muscle (2.7 h), about the same when injected in the gluteal muscle (11–12 h) and much longer when injected in the deltoid muscle (26.4 h) (Kalicharan et al., 2016c). This phenomenon of various lag times will be discusses in another article. This lag time is most likely caused by the postponed ND hydrolysis which would mean that hydrolysis does not take place in the tissue around the site of injection, but rather occurs in the central circulation or in the liver. This research elucidates whether the hydrolysis of N occurs at the injection site, or in the central circulation.
In literature, a half-life time hydrolysis of 4 min for 2.5 * 103mmol/mL (1mg/mL) nandrolone phenyl propionate in rat plasma has been reported (14). Based on these data, the duration time to hydrolyse 2.3 * 105mmol/mL (=0.01mg/mL) ND in HS was proposed by Wijnand et al. (Wijnand et al., 1985) to be below one hour. However, as reported by Li et al. and Rudakova et al. (Li et al., 2005; Rudakova et al., 2011), no carboxylesterases are present in HP. This implies that no enzymatical hydrolysis of ND to N can occur in HP and HS, which is confirmed in this current study: In human serum and plasma, no hydrolysis of ND occurred during 5 h of incubation. Also, no chemical hydrolysis of ND occurred in these media. Moreover, this entails that ND hydrolysis does not occur in interstitial fluid since interstitial fluid originates from blood plasma. It contains water and molecules less than approximately 40 kD molecular weight (Charman and Stella, 1992; Wiig et al., 2012). This means that even when carboxylesterases would be present in human blood plasma, they would not appear in the interstitial fluid as the molecular weight of human carboxylesterases is approximately 60 kD (Imai and Ohura, 2010).
*This paper demonstrates that ND hydrolysis does occur in human blood. Apparently human blood contains carboxylesterases. The appearance of N was observed after an average lag time of 34.9 2.5 min. Noticeable was the absence of Michaelis-Menten behaviour. Instead of nonlinear enzyme kinetics, a linear rate of hydrolysis was seen (Fig. 4). These observations suggest that the carboxylesterases, involved in ND hydrolysis, must be present in the blood cells since the distinction between whole blood and plasma is the presence of cells. Hydrolysis can occur either on the blood cell membrane, or occurs intracellularly. Because a lag time of N appearance was noticed, carboxylesterase activity on the cell membrane is apparently not very pronounced. Otherwise, the N recovery should be seen instantaneously. It can therefore be concluded that hydrolysis occurs after membrane diffusion and, subsequently intracellularly in erythrocytes cytosol (Quon and Stampfli, 1985) and probably in leukocytes. This is in line with literature, where it is reported that carboxylesterases are present in the endoplasmic reticulum and cytosol in human cells.
In porcine muscle and subcutaneous tissues, little hydrolysis of ND occurred in porcine muscle tissue during 5 h of incubation (Fig. 5). The tissues were chosen to be originated from a pig instead of other species (such as rats) to prevent false-positive results. Similar to human tissues, interstitial fluid between porcine tissue cells does not contain carboxylesterases (Li et al., 2005). Because the penetration depth of lipophilic molecules into tissue is very low (Lerner et al., 2006), it is relevant to study the carboxylesterase activity in cells that are in the direct proximity of the oil depot. The results with tissue conclude that hydrolysis in porcine muscle and subcutaneous cells occurs slowly, indicating that only little ND has reached an enzyme. In line with hydrolysis in HB, this also suggests that in vivo hydrolysis only occurs intracellularly, because immediate (within 30 min) N appearance was absent
Before intracellular hydrolysis can occur, ND must diffuse through the cell membrane. As can be seen in Fig. 4, the enzymatic conversion is proportional to the concentration of the substrate.
This suggests that there must be another process that plays a role. This process is the diffusion of ND through the cell membrane which is the rate-limiting step. Otherwise, Michaelis-Menten kinetics would have been observed and a lag time absent. As ND is more lipophilic (log P = 8.1 (“ChemSpider,” 2015)) than N (log P = 3.0 (“ChemSpider,” 2015)), we assume that the limiting step in mass transfer is the cell influx of ND rather than the cell efflux of N. Therefore, the N efflux will be neglected in the following estimation. With the value of lag time being roughly 2100 s and the literature value of the erythrocyte membrane thickness (h) (7*107 cm (Changjun Liu et al., 2003)), the diffusion coefficient (D) of ND through this membrane can be estimated to be 3.9*1017 cm2 /s according to the following equation:
This calculated value of the diffusion coefficient is much lower than other steroid diffusion coefficients. For example, the D of testosterone in percutaneous absorption has been reported to be 1.95*1011 cm2 /s (Scheuplein et al., 1969). Calculated lag time for this testosterone would be 4.2*103 s (h was kept constant), which gives a negligible corrected (for the N efflux) diffusion coefficient for ND in current study (D remains 3.9*1017 cm2 /s). There is a significant difference between the values of ND and testosterone and this may be a source of some discussion. However, it is at least a reflection of the large difference that exists between a prodrug having a log P = 8 and a parent drug having a log P = 3. The conclusion of this observation is that ND hardly does permeate through tissue. This indicates that ND must be absorbed into the central circulation via other routes, as direct absorption is excluded due to the high partition coefficient. It is assumed that ND subsequently adheres to small proteins (<40 kD) and migrates into lymph vessels to be absorbed into the central circulation.
Recently, it was shown that the in situ surface of a 0.5 mL injected oil depot is 755.4 mm2 (Kalicharan et al., 2016a). As can be seen in Table 2, the amount of cells per 1000 mm2 tissue is around 105 –108 cells, which is negligible with the amount of cells in blood (1015 cells/mL). Another advantage of blood, is that it is continuously refreshed (sink conditions).
Benzyl alcohol (BOH) is commonly used in oil depots at a concentration of 1–10% (m/v) (Bagchus et al., 2005; Kalicharan et al., 2016c; Minto et al., 1997; Van Weringh et al., 1994; Wijnand et al., 1985). It is used as viscosity reducer, local anaesthetic and as co-solvent in oil depots (Rowe et al., 2009). Recently, BOH was reported to have a very different absorption profile than N (Kalicharan et al., 2016c). In contrast to N, BOH was detected in the bloodstream within minutes after injection. Furthermore, whereas N was measured for 5 weeks after injection, BOH was cleared form the central circulation within 36 h (Kalicharan et al., 2016b).
The BOH molecular structure shows great similarity to the one of benzil (Table 3), which is an inhibitor of carboxylesterases (Hatfield and Potter, 2011). The inhibition is due to steric interaction of the benzene ring of benzil with the pocket of human carboxylesterase 1 (hCE1) (Harada et al., 2009). This was the reason to study the influence of BOH on ND hydrolysis. As observed, the addition of 1.0% BOH significantly inhibits the ND hydrolysis in human blood.
Although not investigated, the effect of BOH on the carboxylesterases in porcine muscle and subcutaneous tissues can be predicted. This can be estimated by calculating the amount of BOH locally using the log P of BOH. The log P of BOH is 1.03 (““ChemSpider,” 2015) which indicates a distribution in the water phase (interstitial fluid) of approximately 10% of the total amount injected BOH. The processed amount BOH in oil depots is 10% (m/ v), which would result in a concentration less than 1% in the water phase. In the current study, 1.0% BOH significantly inhibited carboxylesterases in whole blood. Therefore, it is possible that this inhibition will also occurs in the proximity of the oil depot. This is only temporary however, since nearly all BOH in the oil depot is released within 36 h. Indicating a large amount of BOH that enter the interstitial fluid and diffuses through the tissues near the injection site, and, consequently, it will temporary inhibit the carboxylesterases in these tissues
This study reveals that ND is stable in NBCS during incubation for 90 min at 37 C (Fig. 1). This implies the absence of chemical and enzymatic degradation of ND in NBCS (Fig. 1). It also has been shown that carboxylesterases from porcine liver are active in tissues from other species than pigs since porcine carboxylesterase also induced hydrolysis in new born calf serum (NBCS) and in human blood. This experimental setup was therefore suitable for use with human intravascular fluids.
5. Conclusion: contribution of nandrolone decanoate hydrolysis to nandrolone absorption
After injection of the oil depot, the released ND molecules appear in the interstitial tissue. Due to the high partition coefficient (log P = 8.1 (““ChemSpider,” 2015)) of ND, it is likely that ND adheres to small proteins ( < 40 kD) in the interstitial fluid. As argued above, the prodrug is neither hydrolysed in the interstitial fluid nor is able to diffuse in a significant way into the tissue. The interstitial fluid is drained via the lymph vessels, and therefore ND absorption into the central circulation via the lymphatic system is in fact the only likely route. Therefore, it can be reasoned that ND is hydrolysed when entering the central circulation. Alternatively, it is possible that the cellular components in the lymphatic system take care of (a part of) the hydrolysis.
Once entered in the central compartment, it will take about half an hour before ND hydrolysis will occur. All these phenomena, transport through the lymph and diffusion through cell membranes, contribute to the lag time observed after the injection of an oil depot.
Although this article focusses on the location and rate of ND hydrolysis, the outcome is also applicable for other esterified prodrugs processed in oil depots and i.m. or s.c. injected, such as haloperidol decanoate (Van Weringh et al., 1994), fluphenazine decanoate (Soni et al., 1988) or testosterone undecanoate (Morgentaler et al., 2008).
*the prodrug is neither hydrolysed in the interstitial fluid nor is able to diffuse in a significant way into the tissue.
*hydrolysis does not take place in the tissue around the site of injection, but rather occurs in the central circulation or in the liver.
*ND hydrolysis only occurred in human whole blood. The hydrolysis did not start immediately, but after a lag time.
* This lag time is most likely caused by the postponed ND hydrolysis which would mean that hydrolysis does not take place in the tissue around the site of injection, but rather occurs in the central circulation or in the liver. This research elucidates whether the hydrolysis of N occurs at the injection site, or in the central circulation.
*In literature, a half-life time hydrolysis of 4 min for 2.5 * 103mmol/mL (1mg/mL) nandrolone phenyl propionate in rat plasma has been reported (14). Based on these data, the duration time to hydrolyse 2.3 * 105mmol/mL (=0.01mg/mL) ND in HS was proposed by Wijnand et al. (Wijnand et al., 1985) to be below one hour. However, as reported by Li et al. and Rudakova et al. (Li et al., 2005; Rudakova et al., 2011), no carboxylesterases are present in HP. This implies that no enzymatical hydrolysis of ND to N can occur in HP and HS, which is confirmed in this current study: In human serum and plasma, no hydrolysis of ND occurred during 5 h of incubation. Also, no chemical hydrolysis of ND occurred in these media.
*Moreover, this entails that ND hydrolysis does not occur in interstitial fluid since interstitial fluid originates from blood plasma. It contains water and molecules less than approximately 40 kD molecular weight (Charman and Stella, 1992; Wiig et al., 2012). This means that even when carboxylesterases would be present in human blood plasma, they would not appear in the interstitial fluid as the molecular weight of human carboxylesterases is approximately 60 kD (Imai and Ohura, 2010).
*This paper demonstrates that ND hydrolysis does occur in human bl Apparently human blood contains carboxylesterases. The appearance of N was observed after an average lag time of 34.9 2.5 min. Noticeable was the absence of Michaelis-Menten behaviour. Instead of nonlinear enzyme kinetics, a linear rate of hydrolysis was seen (Fig. 4). These observations suggest that the carboxylesterases, involved in ND hydrolysis, must be present in the blood cells since the distinction between whole blood and plasma is the presence of cells. Hydrolysis can occur either on the blood cell membrane, or occurs intracellularly. Because a lag time of N appearance was noticed, carboxylesterase activity on the cell membrane is apparently not very pronounced. Otherwise, the N recovery should be seen instantaneously. It can therefore be concluded that hydrolysis occurs after membrane diffusion and, subsequently intracellularly in erythrocytes cytosol (Quon and Stampfli, 1985) and probably in leukocytes. This is in line with literature, where it is reported that carboxylesterases are present in the endoplasmic reticulum and cytosol in human cells.
*After injection of the oil depot, the released ND molecules appear in the interstitial tissue. Due to the high partition coefficient (log P = 8.1 (““ChemSpider,” 2015)) of ND, it is likely that ND adheres to small proteins ( < 40 kD) in the interstitial fluid. As argued above, the prodrug is neither hydrolysed in the interstitial fluid nor is able to diffuse in a significant way into the tissue. The interstitial fluid is drained via the lymph vessels, and therefore ND absorption into the central circulation via the lymphatic system is in fact the only likely route. Therefore, it can be reasoned that ND is hydrolysed when entering the central circulation. Alternatively, it is possible that the cellular components in the lymphatic system take care of (a part of) the hydrolysis.
Abstract
Long-term therapy of nandrolone (N) is recommended to increase mineral density and muscle strength. Using a parenteral sustained release drug formulation with nandrolone decanoate (ND), therapeutic N levels can be achieved and maintained. Until now, it is unknown if hydrolysis of ND into N occurs in tissue at the injection site or after systemic absorption. Therefore, hydrolysis studies were conducted to investigate the location and rate of ND hydrolysis after its release from the oil depot.
ND hydrolysis was studied in porcine tissues, to mimic the human muscular and subcutaneous tissues. Additionally, the ND hydrolysis was studied in human whole blood, plasma and serum at a concentration range of 23.3–233.3mM.
ND hydrolysis only occurred in human whole blood. The hydrolysis did not start immediately, but after a lag time. The mean lag time for all studied concentrations was 34.9 2.5 min. Because of a slow penetration into tissue, hydrolysis of ND is found to be very low in surrounding tissue. Therefore the local generation of the active compound is clinically irrelevant.
It is argued that after injection of the oil depot, ND molecules will be transported via the lymphatic system towards lymph nodes. From here, it will enter the central circulation and within half an hour it will hydrolyse to the active N compound.
1. Introduction
Androgens can be used to increase bone mineral density and muscle strength (Crawford et al., 2003; Erdtsieck et al., 1994; Frisoli et al., 2005; Notelovitz, 2002). For this purpose, long-term therapy of nandrolone is recommended. Therapeutic nandrolone levels in blood can be maintained using a parenteral sustained release drug formulation (Bagchus et al., 2005; Kalicharan et al., 2016c; Minto et al., 1997; Wijnand et al., 1985). An example of such a parenteral drug formulation is an oil depot. In general, slow release from oil depots is a result of the high partition coefficient of lipophilic compounds; The release rate decreases when the compound is more lipophilic. Increased lipophilicity can be accomplished through esterification with a fatty acid. For nandrolone, the decanoate has been selected as the appropriate moiety. In contrast to nandrolone (the active parent compound), nandrolone decanoate (ND) is an inactive prodrug. Oil depots with ND have been applied in several clinical studies, in which they were administrated by intramuscular (i.m.) (Bagchus et al., 2005; Minto et al., 1997; Wijnand et al., 1985) or subcutaneous (s.c.) routes (Kalicharan et al., 2016c).
Although pharmacokinetic profiles of nandrolone depots have been published, the fundamental mechanisms of drug release and absorption into the central circulation have hardly been studied. In theory, ND is released from the oil depot into the interstitial (tissue) fluid. The rate at which this occurs is largely determined by the compound concentration in the oil formulation and its partition coefficient. Subsequently, ND is hydrolysed into nandrolone. Until now, it is generally assumed that ND is hydrolysed in serum and not in the tissue (fluid) at the site of injection (Wijnand et al., 1985)
Recently, we have demonstrated that there exists a delay (lag time) in the appearance of nandrolone in the central circulation (Kalicharan et al., 2016c). Since nandrolone has a log P of 3.0 (“ChemSpider,” 2015) and therefore will be absorbed relatively rapidly, this observed lag time indicates that immediate hydrolyses of ND does not occur. The lag time can be affected by several factors, such as diffusion in tissue fluid, cell membrane adsorption and cell absorption (Kalicharan et al., 2016c). On the one hand, at the site of injection, interstitial fluid transport is slower than blood flow. On the other hand, this transport is faster than diffusion. Another factor, cell membrane adsorption is relevant, because lipophilic prodrugs have high affinity with lipophilic cell structures such as cell membranes and membrane proteins. Due to the adherence to the cell membrane, cell absorption seems a logical consequence. Once absorbed, the lipophilic prodrug can be hydrolysed by esterases (if present) localized in cytosol and microsomes (Jewell et al., 2007; Li et al., 2005; Prusakiewicz et al., 2006). After hydrolysis, efflux of the active parent compound out of the cell must occur in order to reach the central circulation. All these factors may contribute to a prolonged residence time of the lipophilic prodrug in tissues and fluids around the injection site.
Hydrolysis can occur via chemical processes or by carboxylesterases (Imai and Ohura, 2010; Jewell et al., 2007; Prusakiewicz et al., 2006). These enzymes hydrolyse a different ester prodrug, haloperidol decanoate (Nambu et al., 1987; Oh-E et al., 1987). Because the ester bond in this prodrug is similar to the ester bond in ND, it is likely that ND hydrolysis also occurs by carboxylesterases. To our knowledge, this has however never been published yet and will be studied in this paper.
Interestingly, carboxylesterases are inhibited by benzil (Hatfield and Potter, 2011). This compound shows great similarity on molecular structure with a commonly added oil depot additive: benzyl alcohol (BOH). Although BOH is processed in a significant quantity in oil depots of 1–10% (m/v) (Bagchus et al., 2005; Kalicharan et al., 2016c; Minto et al., 1997; Van Weringh et al., 1994; Wijnand et al., 1985), any inhibitory effect of BOH on carboxylesterases is yet unknown but can be clinically relevant if it inhibits carboxylesterases.
The time period between ND release from the oil depot and metabolism in the liver may account for the complete lag time, but it is also well possible that hydrolysis occurs earlier in the absorption phase. Until now, this has never been unambiguously demonstrated.
4. Discussion
This research aimed to study whether locally injected ND as an oil depot is hydrolysed at the site of injection. Drug absorption from a locally injected oil depot into the central circulation exhibits a delayed absorption pattern, which is defined as lag time (Kalicharan et al., 2016c). About 8 h after a s.c. ND oil depot injection, N is observed in the central circulation (Kalicharan et al., 2016c). Remarkably, this lag time is much shorter when the oil depot is injected in the vastus lateralis muscle (2.7 h), about the same when injected in the gluteal muscle (11–12 h) and much longer when injected in the deltoid muscle (26.4 h) (Kalicharan et al., 2016c). This phenomenon of various lag times will be discusses in another article. This lag time is most likely caused by the postponed ND hydrolysis which would mean that hydrolysis does not take place in the tissue around the site of injection, but rather occurs in the central circulation or in the liver. This research elucidates whether the hydrolysis of N occurs at the injection site, or in the central circulation.
In literature, a half-life time hydrolysis of 4 min for 2.5 * 103mmol/mL (1mg/mL) nandrolone phenyl propionate in rat plasma has been reported (14). Based on these data, the duration time to hydrolyse 2.3 * 105mmol/mL (=0.01mg/mL) ND in HS was proposed by Wijnand et al. (Wijnand et al., 1985) to be below one hour. However, as reported by Li et al. and Rudakova et al. (Li et al., 2005; Rudakova et al., 2011), no carboxylesterases are present in HP. This implies that no enzymatical hydrolysis of ND to N can occur in HP and HS, which is confirmed in this current study: In human serum and plasma, no hydrolysis of ND occurred during 5 h of incubation. Also, no chemical hydrolysis of ND occurred in these media. Moreover, this entails that ND hydrolysis does not occur in interstitial fluid since interstitial fluid originates from blood plasma. It contains water and molecules less than approximately 40 kD molecular weight (Charman and Stella, 1992; Wiig et al., 2012). This means that even when carboxylesterases would be present in human blood plasma, they would not appear in the interstitial fluid as the molecular weight of human carboxylesterases is approximately 60 kD (Imai and Ohura, 2010).
*This paper demonstrates that ND hydrolysis does occur in human blood. Apparently human blood contains carboxylesterases. The appearance of N was observed after an average lag time of 34.9 2.5 min. Noticeable was the absence of Michaelis-Menten behaviour. Instead of nonlinear enzyme kinetics, a linear rate of hydrolysis was seen (Fig. 4). These observations suggest that the carboxylesterases, involved in ND hydrolysis, must be present in the blood cells since the distinction between whole blood and plasma is the presence of cells. Hydrolysis can occur either on the blood cell membrane, or occurs intracellularly. Because a lag time of N appearance was noticed, carboxylesterase activity on the cell membrane is apparently not very pronounced. Otherwise, the N recovery should be seen instantaneously. It can therefore be concluded that hydrolysis occurs after membrane diffusion and, subsequently intracellularly in erythrocytes cytosol (Quon and Stampfli, 1985) and probably in leukocytes. This is in line with literature, where it is reported that carboxylesterases are present in the endoplasmic reticulum and cytosol in human cells.
In porcine muscle and subcutaneous tissues, little hydrolysis of ND occurred in porcine muscle tissue during 5 h of incubation (Fig. 5). The tissues were chosen to be originated from a pig instead of other species (such as rats) to prevent false-positive results. Similar to human tissues, interstitial fluid between porcine tissue cells does not contain carboxylesterases (Li et al., 2005). Because the penetration depth of lipophilic molecules into tissue is very low (Lerner et al., 2006), it is relevant to study the carboxylesterase activity in cells that are in the direct proximity of the oil depot. The results with tissue conclude that hydrolysis in porcine muscle and subcutaneous cells occurs slowly, indicating that only little ND has reached an enzyme. In line with hydrolysis in HB, this also suggests that in vivo hydrolysis only occurs intracellularly, because immediate (within 30 min) N appearance was absent
Before intracellular hydrolysis can occur, ND must diffuse through the cell membrane. As can be seen in Fig. 4, the enzymatic conversion is proportional to the concentration of the substrate.
This suggests that there must be another process that plays a role. This process is the diffusion of ND through the cell membrane which is the rate-limiting step. Otherwise, Michaelis-Menten kinetics would have been observed and a lag time absent. As ND is more lipophilic (log P = 8.1 (“ChemSpider,” 2015)) than N (log P = 3.0 (“ChemSpider,” 2015)), we assume that the limiting step in mass transfer is the cell influx of ND rather than the cell efflux of N. Therefore, the N efflux will be neglected in the following estimation. With the value of lag time being roughly 2100 s and the literature value of the erythrocyte membrane thickness (h) (7*107 cm (Changjun Liu et al., 2003)), the diffusion coefficient (D) of ND through this membrane can be estimated to be 3.9*1017 cm2 /s according to the following equation:
This calculated value of the diffusion coefficient is much lower than other steroid diffusion coefficients. For example, the D of testosterone in percutaneous absorption has been reported to be 1.95*1011 cm2 /s (Scheuplein et al., 1969). Calculated lag time for this testosterone would be 4.2*103 s (h was kept constant), which gives a negligible corrected (for the N efflux) diffusion coefficient for ND in current study (D remains 3.9*1017 cm2 /s). There is a significant difference between the values of ND and testosterone and this may be a source of some discussion. However, it is at least a reflection of the large difference that exists between a prodrug having a log P = 8 and a parent drug having a log P = 3. The conclusion of this observation is that ND hardly does permeate through tissue. This indicates that ND must be absorbed into the central circulation via other routes, as direct absorption is excluded due to the high partition coefficient. It is assumed that ND subsequently adheres to small proteins (<40 kD) and migrates into lymph vessels to be absorbed into the central circulation.
Recently, it was shown that the in situ surface of a 0.5 mL injected oil depot is 755.4 mm2 (Kalicharan et al., 2016a). As can be seen in Table 2, the amount of cells per 1000 mm2 tissue is around 105 –108 cells, which is negligible with the amount of cells in blood (1015 cells/mL). Another advantage of blood, is that it is continuously refreshed (sink conditions).
Benzyl alcohol (BOH) is commonly used in oil depots at a concentration of 1–10% (m/v) (Bagchus et al., 2005; Kalicharan et al., 2016c; Minto et al., 1997; Van Weringh et al., 1994; Wijnand et al., 1985). It is used as viscosity reducer, local anaesthetic and as co-solvent in oil depots (Rowe et al., 2009). Recently, BOH was reported to have a very different absorption profile than N (Kalicharan et al., 2016c). In contrast to N, BOH was detected in the bloodstream within minutes after injection. Furthermore, whereas N was measured for 5 weeks after injection, BOH was cleared form the central circulation within 36 h (Kalicharan et al., 2016b).
The BOH molecular structure shows great similarity to the one of benzil (Table 3), which is an inhibitor of carboxylesterases (Hatfield and Potter, 2011). The inhibition is due to steric interaction of the benzene ring of benzil with the pocket of human carboxylesterase 1 (hCE1) (Harada et al., 2009). This was the reason to study the influence of BOH on ND hydrolysis. As observed, the addition of 1.0% BOH significantly inhibits the ND hydrolysis in human blood.
Although not investigated, the effect of BOH on the carboxylesterases in porcine muscle and subcutaneous tissues can be predicted. This can be estimated by calculating the amount of BOH locally using the log P of BOH. The log P of BOH is 1.03 (““ChemSpider,” 2015) which indicates a distribution in the water phase (interstitial fluid) of approximately 10% of the total amount injected BOH. The processed amount BOH in oil depots is 10% (m/ v), which would result in a concentration less than 1% in the water phase. In the current study, 1.0% BOH significantly inhibited carboxylesterases in whole blood. Therefore, it is possible that this inhibition will also occurs in the proximity of the oil depot. This is only temporary however, since nearly all BOH in the oil depot is released within 36 h. Indicating a large amount of BOH that enter the interstitial fluid and diffuses through the tissues near the injection site, and, consequently, it will temporary inhibit the carboxylesterases in these tissues
This study reveals that ND is stable in NBCS during incubation for 90 min at 37 C (Fig. 1). This implies the absence of chemical and enzymatic degradation of ND in NBCS (Fig. 1). It also has been shown that carboxylesterases from porcine liver are active in tissues from other species than pigs since porcine carboxylesterase also induced hydrolysis in new born calf serum (NBCS) and in human blood. This experimental setup was therefore suitable for use with human intravascular fluids.
5. Conclusion: contribution of nandrolone decanoate hydrolysis to nandrolone absorption
After injection of the oil depot, the released ND molecules appear in the interstitial tissue. Due to the high partition coefficient (log P = 8.1 (““ChemSpider,” 2015)) of ND, it is likely that ND adheres to small proteins ( < 40 kD) in the interstitial fluid. As argued above, the prodrug is neither hydrolysed in the interstitial fluid nor is able to diffuse in a significant way into the tissue. The interstitial fluid is drained via the lymph vessels, and therefore ND absorption into the central circulation via the lymphatic system is in fact the only likely route. Therefore, it can be reasoned that ND is hydrolysed when entering the central circulation. Alternatively, it is possible that the cellular components in the lymphatic system take care of (a part of) the hydrolysis.
Once entered in the central compartment, it will take about half an hour before ND hydrolysis will occur. All these phenomena, transport through the lymph and diffusion through cell membranes, contribute to the lag time observed after the injection of an oil depot.
Although this article focusses on the location and rate of ND hydrolysis, the outcome is also applicable for other esterified prodrugs processed in oil depots and i.m. or s.c. injected, such as haloperidol decanoate (Van Weringh et al., 1994), fluphenazine decanoate (Soni et al., 1988) or testosterone undecanoate (Morgentaler et al., 2008).
