Cytoplasmic p21(Cip1/WAF1) enhances axonal regeneration and functional recovery after spinal cord injury in rats.

p21(Cip1/WAF1), known as a cell-cycle inhibitory protein, facilitates neurite outgrowth from neurons when present in the cytoplasm. The molecular mechanism of this action is that p21(Cip1/WAF1) forms a complex with Rho-kinase and inhibits its activity. As myelin-derived inhibitors of axonal outgrowth act on neurons by activating Rho, that is responsible for the lack of spontaneous regeneration of the injured central nervous system (CNS), Rho-kinase may be a good molecular target against injuries in the CNS. In this study, we delivered TAT-fusion protein of cytoplasmic p21(Cip1/WAF1) locally after dorsal hemisection of the thoracic spinal cord in rats. The treatment significantly stimulated axonal regeneration and recovery of hindlimb function, and inhibited the cavity formation in the spinal cord after the injury. Cytoplasmic p21(Cip1/WAF1) may provide a potential therapeutic agent that produces functional regeneration following CNS injuries.

Hepatic immunopotentiation by galactose-entrapped liposomal IL-2 compound in the treatment of liver metastases.

To activate hepatic sinusoidal lymphocytes (HSL) and increase the local antitumor activity in the liver, we developed a liver-targeted interleukin-2 (IL-2) compound using a galactose residue-entrapped liposome. We prepared various kinds of IL-2-containing liposomes made by the hydration of powdered dimyristoyl-phosphatidylcholine with aqueous recombinant IL-2 followed by the combination with galactose residues to facilitate the selective uptake by liver parenchymal cells bearing galactose receptors. The IL-2 liposomes were given to C3H/He mice followed by the determination of: (1) organ distribution by 125I-labeled IL-2, (2) antitumor activity of hepatic sinusoidal lymphocytes by 51Cr-release assay, and (3) in vivo antitumor efficacy by the measurement of hepatic metastases. When galactose-entrapped IL-2 liposomes (Gallip-IL-2) were administered, a significantly greater hepatic accumulation of IL-2 was seen for up to 2 weeks compared to IL-2 liposomes or free IL-2. According to these results, the antitumor activity of HSL was significantly augmented. Moreover, when mice with hepatic micrometastases were treated with Gal-lip-IL-2, the area of hepatic metastases was significantly reduced. These findings thus indicate that Gal-lip-IL-2 may enhance the therapeutic efficacy of IL-2 against hepatic metastases and thereby facilitate a more practical daily dosing regimen.

The effect of adriamycin against a liver metastatic model by encapsulation in liposomes.

Antitumor activities of liposomes containing adriamycin (L-ADM) and their distribution process into tumour cells were analysed. The lipid composition of the liposomes was dimyristoylphosphatidylglycerol (DMPG)/egg phosphatidylcholine/cholesterol/adriamycin (ADM) in a molar ratio of 11.4:2:12:1.3. Liver-metastasizing murine tumour models, M5076 and L5178Y-ML, were used. In vivo antitumour effect against these tumour models was assessed from increase in life span (ILS). The survival prolongation effect of L-ADM in mice with liver failure caused by M5076 was significantly higher than that of F-ADM. In contrast, significant enhancement of the effects by encapsulation in liposomes was not observed in L5178Y-ML-bearing mice. In vitro cytostatic activities of L-ADM against M5076 cells as well as against other tumour cell lines were lower than those of F-ADM. The in vitro kinetic study on the distribution of L-ADM to the tumour cells revealed that ADM in L-ADM was taken up into the tumour cells mainly after it was released from the liposomes rather than taken up as the liposomal form. Among the cell lines tested, M5076 cells had the highest phagocytic activity and therefore the highest uptake activity of ADM during incubation with L-ADM. These findings suggest that the augmented antitumour activity of L-ADM in M5076-bearing mice was the result of phagocytosis of L-ADM by M5076 cells as well as the reduction of toxicity, prolonged retention of ADM in systemic circulation, and liver accumulation of ADM after administration of L-ADM.

Biopharmaceutical evaluation of the liposomes prepared by rehydration of freeze-dried empty liposomes (FDELs) with an aqueous solution of a drug.

We have evaluated a method for preparation of a dispersion of liposomes encapsulating a drug, namely rehydration of freeze-dried empty (not containing drug) liposomes with an aqueous drug solution (FDEL method). In the present study, we characterized and compared this method with the conventional method using a lipid composition of DPPC-DPPG-cholesterol in a molar ratio of 27:3:20. Two hydrophilic compounds, [3H]-inulin and [3H]-mannitol, were used as model drugs. Liposomal preparations by the FDEL method had an encapsulation efficiency of 2.9 and 6.7% for [3H]-inulin and [3H]-mannitol, respectively, when rehydrated and incubated at 70 degrees C. Since non-specific adsorption of these markers to liposomal membrane is negligible, this method produces liposomes which encapsulate a drug in the intravesicular space. One-tenth of the marker encapsulated in the liposomes prepared by the FDEL method (F-liposomes) was released very rapidly on incubation with rat plasma, followed by the slow release of the remaining fraction thereafter. No such rapid-release phase was observed for the liposomes prepared by the conventional method (C-liposomes). This suggests the existence of two types of encapsulation, loose encapsulation and tight encapsulation, in F-liposomes at least. Pharmacokinetic parameters of marker encapsulated tightly in F-liposomes were comparable to those in C-liposomes. It is likely that amphipathic drugs such as doxorubicin are incorporated into liposomes more easily than inulin and mannitol when formulated by the FDEL method. These results therefore suggest that the FDEL method is useful in the preparation of a liposomal formulation of a drug.

Pharmaceutical and biological properties of doxorubicin encapsulated in liposomes (L-ADM): the effect of repeated administration on the systemic phagocytic activity and pharmacokinetics.

We investigated the biodistribution and antitumour activity of doxorubicin (ADM) encapsulated in liposomes (L-ADM) after two administrations in tumour bearing mice. The effect of the first administration on phagocytic activity was also examined. The biodistribution of L-ADM after the second dosing at an interval of 4d was remarkably different from that after the first. The concentration of ADM in plasma and tumour after the second injection was higher, but that in the liver was lower than after the first administration. This decrease in distribution to the liver is thought to have contributed to the difference in the biodistribution characteristics of L-ADM. With regard to antitumour effect, the activity was similar between L-ADM and a solution of ADM (F-ADM). To investigate the effect of the first administration of L-ADM on biodistribution, systemic phagocytic activity was measured after the injection of F-ADM, L-ADM, or 'empty' liposomes not containing ADM. F-ADM and L-ADM (7.5 mg ADM/kg body weight) reduced phagocytic activity to approximately 50% and 30% of control, respectively. This finding suggests that entrapment of ADM in liposomes enhances both the distribution of the drug to the reticuloendothelial system (RES) and its suppressive effect on RES activity. These results indicate that the decrease in RES activity by L-ADM must be considered in estimation of the pharmacokinetics, antitumour activity, and toxicity of L-ADM in clinical use when given by repeat administration or used in combination with other antitumour agents.

Distribution of liposomes containing mannobiose esters of fatty acid in rats.

The biodistribution of liposomes modified by mannobiose residues was studied in rat. The purpose of the modification was to target the liposomes to macrophages. Mannobiose mono arachidic acid esters (MAEs) were synthesized and used to modify the surface of liposomes. It was shown by gel permeation chromatography that the MAE was preferentially incorporated into the membrane of the liposomes. After intravenous administration, mannobiose-modified liposomes were eliminated from the systemic circulation more rapidly than control liposomes without the modification. Whilst the modification did not affect the distribution of liposomes to kidney, lung, or thymus, it increased the distribution to liver and spleen. The uptake in the hepatic parenchymal cell fraction was not influenced by MAE incorporation. Taking into account the fact that endothelial cells do not take up particles whose size is > 100 nm, the increase in the distribution to liver were ascribed to an increase in uptake by Kupffer cells. These results suggest that mannobiose mono fatty acid esters are useful in the targeting of liposomes to Kupffer cells and other macrophages.

Activation of systemic and mucosal immune response following nasal administration of liposomes.

Adjuvant effects of liposomes on systemic and mucosal immune response were investigated following nasal administration to Balb/c mice. Bovine serum albumin (BSA)-specific serum IgG and salivary IgA levels were significantly elevated when BSA-associated liposomes were administered intranasally twice at 4-week intervals. Systemic immune response was activated only by negatively charged liposomes, while activation of mucosal immune response was independent of liposomal charge. Antigen localization in liposomes affected immune adjuvant effect; the mucosal immune response could be activated only by liposomes to whose surface BSA was attached, but the systemic immune response was activated by both liposomes to which antigens were attached and in which the encapsulating antigens occurred. The results suggest that the contribution of antigen-presenting cells in activation of systemic and mucosal immunity following intranasal administration is different.

Kinetic analysis of tissue distribution of doxorubicin incorporated in liposomes in rats (II).

The objective of this study is to perform kinetic modelling of the tissue distribution of doxorubicin encapsulated into liposomes (L-DXR), especially to the heart and liver. The release process of doxorubicin (DXR) from liposomes in blood was quantified by a release clearance. This parameter defines a release rate of DXR based on the concentration of L-DXR in blood and was estimated from kinetic modelling of DXR distribution to the heart after L-DXR administration. The distribution of free DXR to the heart was modelled separately. The experimental data for this modelling were reported previously (Harashima et al., Biopharm. Drug. Disposit., 13, 155-170 (1992)). This analysis provided a free DXR concentration profile as well as a release clearance of DXR after L-DXR administration. There was a remarkable difference in the free DXR concentration in blood between free and liposomal administration. The area under the DXR curve in the heart was reduced by approximately one third from that for the first two hours after DXR administration by liposomal encapsulation, which could be the reason for reduced cardiac toxicity. In our previous report, the distribution of L-DXR to the liver was shown to be explained by a sequentially linked two-compartment model with efflux process. The validity of this efflux model was examined in this study by a repeated dose study. The apparent uptake clearance decreased with time and showed a second peak after the repeated dose, which justified the efflux model. These kinetic analyses give quantitative understanding of the effect of liposomal encapsulation on the tissue distribution of DXR.

Stability of liposomes in vitro and their uptake by rat Peyer's patches following oral administration.

To evaluate the usefulness of liposomes as a carrier for the targeted delivery of antigens to gut-associated lymphoid tissue, liposomal stability and uptake by rat Peyer's patches were investigated. Liposomes composed of distearoylphosphatidylcholine, phosphatidylserine, and cholesterol (DSPC-liposome), or dipalmitoylphosphatidylcholine, phosphatidylserine, and cholesterol were stable in acidic solution (pH 2.0), diluted bile, and pancreatin solution. Following the oral administration of liposomes to rats, rhodamine B-PE incorporated in the lipid phase of DSPC-liposomes was preferentially taken up by Peyer's patches in the lower ileum. The uptake of rhodamine B-PE from DSPC-liposomes larger than 374 nm in mean diameter was high. Orally administered DSPC-liposomes of a large diameter thus appear to serve effectively as a vehicle for delivering antigens to Peyer's patches.

Enhancement of systemic and mucosal immune responses following oral administration of liposomes.

The effects of liposomes on the systemic and mucosal immune response were investigated using bovine serum albumin (BSA) in BALB/c mice. Following three oral administrations of varied formulations at 1-week intervals, serum BSA-specific IgG levels were increased significantly by BSA encapsulated in liposomes and moderately by a mixture of liposomes and BSA. Serum and salivary BSA-specific IgA levels were elevated by BSA-encapsulating liposomes only. Liposomes thus activate not only the systemic immune response but also the mucosal immune response following their oral administration. However, no increase in salivary IgA levels was observed by intraperitoneal or subcutaneous injection of BSA-encapsulating liposomes. The production of IgA is closely related to the oral administration of liposomes encapsulating antigens. Liposomes thus function as carriers of oral vaccines against various infections of the mucosal surface.

Uptake of phosphatidylserine liposomes by rat Peyer's patches following intraluminal administration.

Uptake of the nonabsorbable marker 6-carboxyfluorescein was investigated both free and encapsulated in liposomes as a function of their surface charge and hydrodynamic diameter in rat Peyer's patch and nonpatch tissue. Significant uptake of the marker occurred only when encapsulated in liposomes consisting of at least 25 mol% phosphatidylserine and was highest in Peyer's patches. 6-Carboxyfluorescein encapsulated in liposomes equal to or greater than 374 nm was preferentially taken up by Peyer's patches. There was a trend to higher uptake in lower intestinal segments. These findings were supported by fluorescence microscopic observations. Uptake by Peyer's patches was specific for negatively charged liposomes as judged from competition studies.

Effect of basic drugs on the hepatic uptake of ouabain by sinusoidal plasma membrane vesicles isolated from rat liver.

Although antiarrhythmic drugs are used to treat digitalis-induced cardiac disorders, some of these drugs have been reported to increase the serum digoxin concentration in patients, causing the severe side-effects. We have previously shown that many basic drugs including antiarrhythmic drugs inhibited the hepatic uptake of cardiac glycosides into isolated rat hepatocytes, which could be a cause for the increased serum digoxin concentration. The present study was designed to examine the mechanism of this inhibition using isolated rat sinusoidal plasma membrane vesicles. The effect of nine basic drugs (dipyridamole, nifedipine, verapamil, chlorpromazine, lidocaine, quinidine, ajmaline, disopyramide, and propranolol) on the uptake of ouabain was studied. Quinidine reduced the initial uptake rate of ouabain (30 s) while it did not change the uptake of ouabain in an equilibrium condition (30 min). Other basic drugs, such as verapamil, dipyridamole, and nifedipine also significantly reduced the initial uptake rate of ouabain. These basic drugs had no effect on the membrane fluidity. The inhibitory effects on the vesicular uptake were significantly correlated with the inhibitory effects on ouabain uptake by the isolated rat hepatocytes. These findings may suggest that the mechanism of the inhibition involves the inhibition of the transport process via the sinusoidal plasma membrane.

Kinetic analysis of tissue distribution of doxorubicin incorporated in liposomes in rats: I.

The purpose of this study was to perform a kinetic analysis of the tissue distribution of doxorubicin (DXR) and liposomes separately after intravenous administration of DXR entrapped in liposomes in rats. Liposomes were double labeled with 14C-DXR (L-DXR) and 3H-inulin (L-INU). Blood and tissues were sampled at specified times until 120 min. Blood clearance of L-DXR was similar to that of L-INU. Distribution of both L-DXR and L-INU into the liver was parallel and extensive, while in the heart, the pattern of distribution differed between L-DXR and L-INU after peak concentration. Time courses of tissue concentration were explained well by dividing tissue into a shallow compartment with efflux and a deep compartment without efflux. In the liver, pharmacokinetic parameters of L-DXR and L-INU were similar, and the two kinetically different compartments may correspond to different uptake processes in hepatic endocytosis. In the heart, the shallow compartment was considered to correspond to the cardiac vascular space, and the intercompartmental rate constant (k3) for L-DXR was much larger than that for L-INU. The estimated half-life for this process was 20 min. The half-life for the degradation of liposomes in blood circulation was also estimated at 20 min from data on the urinary excretion of released 3H-inulin. These results suggest that the release of DXR from liposomes may be the rate-limiting process in the tissue distribution of DXR to the heart.

Possibility of heat sterilization of liposomes.

Several kinds of liposomes were sterilized at 121 degrees C for 20 min. They tended to aggregate after heat sterilization (HS) in saline, while no aggregation was observed in an isotonized sugar or polyol solution. The dispersions containing egg phosphatidylcholine (EggPC) with a high peroxide value (POV) turned slightly yellowish after HS. This color change was prevented by using EggPC with a low POV, hydrogenated EggPC (H-EggPC) or dipalmitoylphosphatidylcholine (DPPC). Nitrogen gas bubbling at neutral pH also prevented the color change, but vitamin E did not. The particle size of the EggPC liposomes extruded through a 0.4 micron membrane filter did not change significantly after HS, whereas the H-EggPC or DPPC liposomes extruded through a 0.8 micron membrane filter tended to be reduced in size. On this change the type of medium had a considerable influence. The anionic 6-carboxyfluorescein leaked from the negatively charged liposomes (EggPC/cholesterol (Chol)/egg phosphatidylglycerol) during HS, while no leakage was observed from the positively charged liposomes (EggPC/Chol/stearylamine) not only during HS but also during a long period of storage. It was suggested that sterilization of liposomes by heating was practicable as well as that by filtration, if the liposomes were prepared as follows: the charged liposomes made of lipids with low POV's were dispersed in a sugar or polyol solution adjusted to nearly pH 6.5, where the amount of dissolved oxygen was minimized. An ionic water-soluble drug had to be encapsulated in the oppositely charged liposomes.

[Treatment of a refractory chronic osteomyelitis of the jaw].

Two cases of refractory chronic osteomyelitis of the jaw were presented, the first in a patient with pycnodysostosis, the second that had been receiving radiotherapy. Both cases were primary chronic osteomyelitis with a markedly protracted course of recovery, in spite of the antibiotic and local irrigation therapy. However the lesion was cured by the removal of sequesters and granulation tissues without radical treatment such as a decorticotomy. Radical surgery, decortication, is an effective procedure in the treatment of chronic osteomyelitis of the jaw, but treatment with the surgical procedures which remove only the necrotic bones and granulation tissues followed by antibiotic therapy is also possible.

Characterization of Rose Bengal binding to sinusoidal and bile canalicular plasma membrane from rat liver.

The binding of Rose bengal, a model organic anion, to sinusoidal and bile canalicular membrane fractions isolated from rat liver was compared. The fluorescence change of Rose bengal after being bound to liver plasma membranes was utilized for measuring the binding. The dissociation constants (Kd = 0.1-0.12 microM) and the binding capacities (n = 11-15 nmol/mg protein) for Rose bengal are comparable between the two membrane fractions, although the n value for sinusoidal membrane is somewhat larger than that for bile canalicular membrane. The Rose bengal binding to both membrane fractions was inhibited by various organic anions at relatively low concentrations, i.e., the half-inhibition concentrations (IC50) for Indocyanine green, sulfobromophthalein, Bromophenol blue and 1-anilino-8-naphthalene sulfonate were 0.1, 100, 1.5-2.5 and 100 microM, respectively, while taurocholate did not inhibit the Rose bengal binding to either membrane fraction at these low concentration ranges. The type of inhibition of sulfobromophthalein and Indocyanine green for Rose bengal binding is different between the two membrane domains. That is, in sinusoidal and bile canalicular membrane fractions, these organic anions exhibit mixed-type and competitive-type inhibition, respectively. It was suggested that the fluorescence method using Rose bengal may provide a simple method for detecting the specific organic anion binding protein(s) in the liver plasma membrane.

Comparison of specific binding of human epidermal growth factor (EGF) to sinusoidal and bile canalicular membranes isolated from rat liver.

The binding characteristics of human epidermal growth factor (EGF) were compared between highly purified canalicular (CMV) and sinusoidal (basolateral) rat liver plasma membrane (SMV) preparations. The dissociation constants (2-3 nM) for these membranes were comparable, while the binding capacity for CMV was approximately half that for SMV. The binding capacity for CMV was too high to be accounted for only by the contamination with sinusoidal membranes, since the measurements of specific activities of various enzymes (Na+,K+-ATPase, alkaline phosphatase, and leucine aminopeptidase) indicated that the extents of the cross contamination with other membrane fractions were at most 10%. Although the physiological function of specific binding of EGF to bile canalicular membrane domain remains to be determined, it may have a role in biliary excretion of EGF. The specific binding of EGF to bile canalicular membranes from rat liver was identified for the first time.

Comparison of bile acid binding to sinusoidal and bile canalicular membranes isolated from rat liver.

Simon et al. (J. Clin. Invest., 70 (1982) 401) studied cholate binding to crude liver plasma membrane vesicles and suggested that the binding may represent mainly the binding to the receptor (carrier) on the canalicular membrane. This hypothesis was supported by finding a good correlation between the number of cholate binding sites on liver plasma membrane and the maximal rate of biliary secretion (Tm) for taurocholate. We studied bile acid binding to sinusoidal and canalicular membrane vesicles isolated from rat liver by a rapid filtration technique. Scatchard analysis of the saturation kinetics showed both [3H]cholate and [3H]chenodeoxycholate bind to two classes of binding site on each membrane. However, little difference was observed between the binding to sinusoidal and canalicular membrane vesicles for each bile acid (cholate, Kd1 = 10.4 and 19.8 microM, n1 = 31.0 23.6 pmol/mg protein, Kd2 = 1.32 and 1.73 mM, n2 = 13.1 and 23.4 nmol/mg protein; and chenodeoxycholate, Kd1 = 0.207 and 0.328 microM, n1 = 36.7 and 27.4 pmol/mg protein, Kd2 = 1.16 and 2.26 mM, and n2 = 20.6 and 24.2 nmol/mg protein; numbers show the mean values sinusoidal and canalicular membrane vesicles, respectively). Chenodeoxycholate binding to sinusoidal membrane vesicles was markedly inhibited by cholate but not by Rose bengal, an organic anion dye. These studies indicate that both membranes (sinusoidal and canalicular membrane vesicles) have two kinds of binding site for bile acids, although no clear difference in the binding properties was observed between the two membranes. Consequently, the cholate binding Simon detected may represent the binding not only to canalicular membrane vesicles but also to sinusoidal membrane vesicles.