The contribution of the in-vivo fate of an oil depot to drug absorption.

Sustained release of lipophilic compounds can be achieved with oil depots. These parenteral formulations are generally injected in the vastus lateralis and deltoid muscle. It is known that the absorption rate differs between these two muscles. The reason for this is not fully understood. The aim of the current study was to investigate the fate of an oil depot in different tissues to elucidate whether the disappearance rate of oil is the cause of observed differences in absorption rate. A study with healthy volunteers was conducted to determine 1.0mL oil depots in the vastus lateralis and deltoid muscle for two weeks. The spatial distribution of the oil depots was determined using MRI. Additionally, a study in rats was conducted to microscopically examine the oil immediately and after 31days of injection. All rats were injected with a 0.1mL oil depot with and without benzyl alcohol (BOH), a commonly used excipient in oil depots. In humans, it was shown that all oil depots were equal in volume and surface area directly after injection. Moreover, the disappearance rate for all oil depots was similar; within one week there was no depot visible anymore by MRI. This in contrast to the depots in rats, which were still microscopically visible after 31days. It is concluded from these observations that the oil is dispersed to small droplets in the course of time. The resulting increase in surface area does not lead to an increase in absorption rate however. The results of this paper show that the variation in drug absorption as found for the two muscles is not caused by a distinction in surface areas or disappearance rates of the oil depots. Therefore, it is argued that the local tissue drainage (e.g. lymph flow) plays a considerable role in drug absorption from oil depots, whereby the lymph flow differs between the muscles.

Where does hydrolysis of nandrolone decanoate occur in the human body after release from an oil depot?

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 23.3-233.3µM. 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±2.5min. 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.

Spatial distribution of oil depots monitored in human muscle using MRI.

Oil depots are parenteral drug formulations meant for sustained release of lipophilic compounds. According to mass transport models, the drug-release rate from these injections is determined by the surface area of the oil depot. Until now, the size of the surface area of injected depots has not been assessed, however. MRI provides an excellent possibility to distinguish between water and adipose tissue. The aim of this study was to investigate whether MRI can be used to determine the shape and hence the surface area of oil depots in muscle tissue. The developed MRI-scan protocol is demonstrated to be suitable for visualising oil depots. It was applied to determine the surface area of 0.5mL oil, i.m. injected in healthy volunteers. The mean (±RSD) surface area and volume of the depots recovered after injection was 755.4mm(2) (±26.5) and 520.1mm(3) (±24.6). It is shown that the depot disappearance from the injection site is very variable between volunteers. It is suggested that the oil is first solubilized and subsequently distributed. In all cases, the oil was not detectable after 14days. These factors are relevant for the understanding of the mechanism by which compounds are released out of oil depots.

Bone targeting potential of bisphosphonate-targeted liposomes. Preparation, characterization and hydroxyapatite binding in vitro.

The main constituent of bone is hydroxyapatite (HAP). Since HAP is only present in 'hard' tissues like bone and teeth, it represents a promising target for the selective drug delivery to bone. Due to the exceptional affinity of bisphosphonates (BP) for HAP, cholesteryl-trisoxyethylene-bisphosphonic acid (CHOL-TOE-BP), a new tailor-made BP derivative, was used as bone targeting moiety for liposomes. CHOL-TOE-BP-targeted liposomes were designed for the treatment of bone-related diseases to achieve prolonged local exposure to high concentrations of the bioactive compounds, thereby enhancing therapeutic efficacy and minimizing systemic side effects. The CHOL-TOE-BP-targeted liposomes were characterized regarding particle size and zeta potential. To study the bone targeting potential of these conjugates, an in vitro HAP binding assay was established. The obtained binding data indicate that CHOL-TOE-BP is useful as targeting device for liposomal drug delivery to bone.

Steric stabilization of poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes mediates prolonged circulation and tumor targeting in mice.

BACKGROUND: Efficient tumor targeting of polymeric gene transfer systems (polyplexes) represents a major challenge. To establish tumor targeting after intravenous (IV) administration, the circulation lifetime of these systems should be sufficiently long. Since naked polyplexes are rapidly eliminated from the circulation after IV adminstration, strategies have to be developed to improve their pharmacokinetics. METHODS: Complexes of plasmid DNA and poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA)-graft-PEG or AB di-block copolymers of pDMAEMA and PEG, as well as PEGylated complexes prepared via PEGylation of preformed complexes (postPEGylation), were evaluated for their physicochemical properties (size and charge) their interactions with blood constituents and transfection activity in vitro. The pharmacokinetics and biodistribution of PEG-polyplexes were studied in mice after IV administration. The degree of accumulation in two subcutaneous (SC) mouse tumors after IV administration was evaluated for the system with the longest circulation time. RESULTS: It is shown that the surface charge of the pDMAEMA-polyplexes was effectively shielded by two PEGylation methods (i.e. the use of pDMAEMA-graft-PEG polymers and postPEGylation). The shielding effect was the highest for the postPEGylation method with PEG(20000), yielding polyplexes that hardly show interactions with blood components (i.e. albumin and erythrocytes) and show substantially prolonged circulation time in mice after IV administration. The superior colloidal stability and circulation kinetics of the postPEGylated polyplexes translated into tumor accumulation which amounted to about 3.5% of the injected dose per gram tumor tissue in a SC Neuro2A tumor model and to about 4.2% of the injected dose per gram tumor tissue in a SC C26 tumor model. CONCLUSIONS: This study shows that postPEGylation of pDMAEMA-based polyplexes is the most attractive method to prepare polyplexes with long circulating properties. Tumor targeting capacity after intravenous administration was demonstrated in two subcutaneous tumor models.

Liposomes to target the lymphatics by subcutaneous administration.

Liposomes have been proposed as carriers for the delivery of therapeutic and diagnostic agents to the lymphatic system. Subcutaneous (s.c.) injection is the route of administration most extensively studied for this purpose. Decisive factors influencing lymphatic absorption and lymph node uptake of s.c. administered liposomes are liposome size and the anatomical site of injection. Generally, other factors such as lipid composition, charge and the presence of a hydrophilic PEG-coating on the liposome surface do not substantially affect lymphatic absorption and lymph node uptake of s.c. administered liposomes. Studies on the intranodal fate of liposomes demonstrate that phagocytosis by macrophages is the most important mechanism for lymph node uptake of liposomes. The observation of relatively high uptake of liposomes in regional lymph nodes after s.c. administration has stimulated research on lymphatic targeting of liposomes for diagnostic and therapeutic applications.

The fate of poly(2-dimethyl amino ethyl)methacrylate-based polyplexes after intravenous administration.

Poly(2-dimethyl amino ethyl) methacrylate (pDMAEMA) cationic polymers have been shown to be efficient vectors for gene delivery in vitro. This contribution deals with the in vivo properties of polyplexes based on this polymer. In mice, pDMAEMA/[32P]-pLuc complexes distributed primarily to the lungs. The gene expression profile matched the biodistribution profile. In vitro turbidity experiments in serum showed severe aggregation upon addition of cationic polyplexes, pointing out the involvement of aggregates in the dominant lung uptake of the positively charged polyplexes. Incubations of polyplexes with albumin yielded a decline of the zeta potential of the complexes to negative values, making an electrostatic mechanism in the dominant lung uptake less likely. Hemagglutination experiments showed that the polyplexes induce the formation of extremely large structures when incubated with washed erythrocytes. Altogether, the present data indicate that aggregate formation and trapping of the formed aggregates in the lung capillary bed is probably responsible for the dominant lung uptake and transfection. Poly(ethylene)glycol (PEG) of the polymeric structures prevented the increase in the observed turbidity in serum seen with polyplexes and was also able to reduce interactions with erythrocytes. Currently, the in vivo fate of the PEGylated polyplexes is under investigation.

Role of macrophages in the localisation of liposomes in lymph nodes after subcutaneous administration.

The macrophage 'suicide' technique, based on the ability of clodronate-containing liposomes to deplete lymph nodes of macrophages, was used to study the role of macrophages in lymph node localisation of subcutaneous (s.c.) administered liposomes. Reduced liposome localisation in macrophage depleted lymph nodes confirmed that phagocytosis by macrophages is an important mechanism for lymph node localisation of large (non-sized) liposomes. Depletion of macrophages had less effect on the lymph node localisation of small (about 0.1 microm) liposomes; small liposomes may reach macrophages in regions of lymph nodes not reached by large liposomes. Small liposomes may also be taken up by cells other than macrophages, such as endothelial cells lining the lymph node sinuses. Remarkably, inclusion of poly(ethyleneglycol)-distearoylethanolamine (PEG-PE) into liposomes did not reduce the degree of lymph node localisation in control lymph nodes. As macrophage depletion had a strong negative effect on the lymph node localisation of PEG-liposomes, it is concluded that, despite the presence of a PEG-coating, PEG-liposomes retained by lymph nodes are to a large extent taken up by lymph node macrophages.

Liposomes as carriers of the antiretroviral agent dideoxycytidine-5'-triphosphate.

The presence and replication of the human immunodeficiency virus (HIV) in cells of the mononuclear phagocyte system (MPS) together with the preferential uptake of liposomes in macrophages suggest that liposomes can become a valuable carrier of anti-HIV agents. Moreover, liposomes reduce toxicity of encapsulated drugs and protect encapsulated drugs against rapid degradation in the blood circulation. To overcome problems associated with the administration of free nucleosides and to improve targeting to the MPS, dideoxycytidine-5'-triphosphate (ddCTP) was encapsulated in liposomes. Liposomes were stable with regard to retention of the entrapped drug, particle size and chemical stability of ddCTP. Results obtained with liposome encapsulated ddCTP in the murine acquired immunodeficiency syndrome (MAIDS) model indicate that ddCTP encapsulated in liposomes can reduce proviral DNA in cells of the mononuclear phagocyte system (MPS) in both spleen and bone marrow.

Lymphatic uptake and biodistribution of liposomes after subcutaneous injection . IV. Fate of liposomes in regional lymph nodes.

The ability of clodronate-containing liposomes to deplete lymph nodes of macrophages was used as a tool to investigate the fate of liposomes in regional lymph nodes after subcutaneous (s.c.) administration. Reduced lymph node localization of liposomes in macrophage-depleted lymph nodes confirmed that phagocytosis by macrophages plays an important role in lymph node retention of liposomes. Depletion of macrophages had less effect on lymph node localization of small liposomes than on the lymph node localization of large liposomes. Inclusion of distearoylphosphatidylethanolamine (DSPE)-poly(ethyleneglycol) (PEG-PE) into the liposomes, which is known to oppose macrophage uptake, did not affect lymph node localization in macrophage-depleted or control lymph nodes. We conclude that PEG-liposomes retained by lymph nodes are also taken up by lymph node macrophages. Morphological observations visualizing the uptake of PEG-liposomes by lymph node macrophages support this conclusion.

The influence of the route of administration and liposome composition on the potential of liposomes to protect tissue against local toxicity of two antitumor drugs.

The present paper reports on the influence of the route of administration and liposome stability on the protective effect of liposome encapsulation of two model antitumor agents, mitoxantrone and doxorubicin. The results demonstrate that liposome encapsulation can protect surrounding tissue from the cytotoxic effects of the drugs after subcutaneous (s.c.) and intramuscular (i.m.) administration. The route of administration is an important factor influencing tissue damage. Liposomal mitoxantrone caused much less tissue irritation after im injection than after s.c. injection. Liposome stability is also an important factor. Liposomes composed of 'fluid-state' phospholipids only delayed the damaging effects of doxorubicin when injected sc. Liposomes with a more rigid nature were much more effective in preventing local tissue damage over a longer period of time when administered sc. Results suggest that slow release of liposome-associated drugs may eventually cause severe local tissue damage. The incorporation of the hydrophilic lipid derivative distearoylphosphatidylethanolamine-poly(ethyleneglycol) (PEG-PE) had no apparent effect on the protective effect of liposomes after sc administration.

Lymphatic uptake and biodistribution of liposomes after subcutaneous injection: III. Influence of surface modification with poly(ethyleneglycol).

PURPOSE: The aim of the present paper was to assess the effect of inclusion of distearoylphosphatidylethanolamine-poly(ethyleneglycol) (DSPE-PEG) into liposomal bilayers on the lymphatic uptake and lymph node localization of liposomes after subcutaneous administration. METHODS: [3H]-Cholesteryloleylether labeled liposomes of various composition and sizes were injected s.c. into the dorsal side of the foot of rats. At several time-points after injection, blood levels of liposomes were determined. Lymphatic uptake from the s.c. site of injection and lymph node localization in regional lymph nodes were determined at the end of the 52 h observation period. RESULTS: The results demonstrate that inclusion of DSPE-PEG into several types of liposomes has only a modest effect on lymphatic uptake. Also lymph node localization is only slightly affected by PEG-mediated steric stabilization. CONCLUSIONS: Factors other than the presence of a steric barrier are more important in determining lymphatic uptake from the s.c. injection site. The observation that lymph node localization was only slightly affected by PEG-coating strongly suggests that macrophage uptake is not the only important mechanism of lymph node localization of s.c. administered liposomes.

Lymphatic uptake and biodistribution of liposomes after subcutaneous injection. II. Influence of liposomal size, lipid compostion and lipid dose.

The present paper reports on the results of a systematic study on liposome variables potentially affecting lymphatic disposition and biodistribution of liposomes after sc injection. Liposomal size was found to be the most important factor influencing lymphatic uptake and lymph node localization of sc administered liposomes. Lymphatic uptake from the s.c. injection site of small liposomes (about 0.04 microm) was relatively high (76% of the injected dose (%ID)) as compared to large, non-sized liposomes, which remained almost completely at the site of injection. Small liposomes were less efficiently retained by regional lymph nodes than larger liposomes. Liposomal lipid composition did not influence lymphatic uptake with one exception: Lymphatic uptake was decreased in case of neutral liposomes composed of (DPPC). Lymph node localization was substantially enhanced by inclusion of phosphatidylserine (PS) into the liposomal bilayers. Saturation of lymphatic uptake and lymph node localization did not occur over a large liposomal lipid dose range, illustrating the efficient performance of lymph nodes in capturing s.c. administered particles.

Immunospecific targeting of immunoliposomes, F(ab')2 and IgG to red blood cells in vivo.

In this report a model to study the fate of target cells in the blood circulation after injection of appropriate immunoliposomes is discussed. The effect of intravenous administration of antimouse RBC immunoliposomes, F(ab')2 or IgG on the fate of intravenously injected 51Cr-labelled mouse RBC (Cr-mRBC) in the mouse and, particularly, in the rat was studied. The immunoliposome was of the Fab'-MPBPE-REV type (Fab'-fragments covalently linked to reverse phase evaporation vesicles by maleimido-4-(p-phenylbutyrate)phosphatidylethanolamine). In the rat model a high blood level (80%) of the injected dose of target cells, Cr-mRBC, was maintained for several hours. The elimination by Fab'-liposomes, F(ab')2 or IgG of Cr-mRBC, and subsequent uptake into liver and spleen was dose dependent. Administration of Fab'-liposomes or F(ab')2 resulted in a preferential uptake into the spleen (above a certain dose also, but much lower, uptake into the liver was observed), while after IgG administration 51Cr-label was mainly recovered in the liver. At equal protein doses (+/- 130 micrograms) Fab'-liposomes induced a faster elimination of the Cr-mRBC and a higher uptake into the spleen than F(ab')2. The potential advantage of the use of drug-loaded immunoliposomes to eliminate target cells from the blood stream and to induce a certain pharmacological effect in the target cells, in comparison with the free antibody administration of F(ab')2 or IgG is discussed.