Hepatitis B surface protein docked vesicular carrier for site specific delivery to liver

The intrinsic liver tropism of liposomes can be augmented by the addition of targeting features such as the incorporation of hepatotropic elements of the hepatitis viruses. Hepatitis B virus is known to infect hepatocytes after viremia by asialoglycoprotein receptor mediated uptake. However, the specificity of hepatitis B virus surface protein (HBsAg) towards hepatocytes has confronting reports. In the present study, we evaluated the functional ability of HBsAg to be employed as a ligand for targeting hepatocytes. We prepared ¹⁴C labeled small unilamellar vesicles (SUVs) composed of egg PC/Cholesterol/N-glutarylphosphatidylethanolamine (NGPE) in a 60:30:10 molar ratio. HBsAg was covalently linked to SUVs using a water-soluble carbodiimide (EDC) mediated conjugation with NGPE. In vitro cell binding and uptake studies revealed that bioprotein docked carrier system was efficiently taken up by HepG2 cells by the receptor mediated endocytosis. The biodistribution behaviour of plain and HBsAg coated liposomes was also examined followed by intravenous injection. The study revealed that almost 75% of the radioactivity was recovered in the liver after 4 h of injection that was nearly three-fold greater in magnitude than the plain liposomes. Further, fractionation of liver into liver parenchymal cells (PC) and non-parenchymal cells confirmed the preferential localization of the HBsAg coated liposomal carrier in the parenchymal cells.     

Hepatitis B surface protein docked vesicular carrier for site specific delivery to liver

The intrinsic liver tropism of liposomes can be augmented by the addition of targeting features such as the incorporation of hepatotropic elements of the hepatitis viruses. Hepatitis B virus is known to infect hepatocytes after viremia by asialoglycoprotein receptor mediated uptake. However, the specificity of hepatitis B virus surface protein (HBsAg) towards hepatocytes has confronting reports. In the present study, we evaluated the functional ability of HBsAg to be employed as a ligand for targeting hepatocytes. We prepared (14)C labeled small unilamellar vesicles (SUVs) composed of egg PC/Cholesterol/N-glutarylphosphatidylethanolamine (NGPE) in a 60:30:10 molar ratio. HBsAg was covalently linked to SUVs using a water-soluble carbodiimide (EDC) mediated conjugation with NGPE. In vitro cell binding and uptake studies revealed that bioprotein docked carrier system was efficiently taken up by HepG2 cells by the receptor mediated endocytosis. The biodistribution behaviour of plain and HBsAg coated liposomes was also examined followed by intravenous injection. The study revealed that almost 75% of the radioactivity was recovered in the liver after 4 h of injection that was nearly three-fold greater in magnitude than the plain liposomes. Further, fractionation of liver into liver parenchymal cells (PC) and non-parenchymal cells confirmed the preferential localization of the HBsAg coated liposomal carrier in the parenchymal cells.     

Disposition of intact liposomes of different compositions and of liposomal degradation products

Small unilamellar liposomes containing bovine serum albumin were prepared by a new double-emulsion technique and administered to mice and rats in intravenous injections. The elimination of intact liposomes, the association of phospholipid marker with lipoproteins, and the appearance of released internal marker and its degradation products were followed by column chromatography of plasma samples. In vitro labelled lipoproteins were administered to the animals in intravenous injections together with free bovine serum albumin and the elimination of the two substances was studied by closely related techniques. The clearance of intact PC:PS (4:1) liposomes from plasma was biphasic and much faster than that of labelled lipoproteins and bovine serum albumin either originating from liposomes or injected as such. The second elimination phase for these liposomes was barely detectable by our analytical methods. In contrast, DSPC:CHOL (2:1) liposomes showed a very significant second-phase elimination, with a half-life of 12 hr for the intact liposomes. In tissue distribution studies in mice, the major accumulation of liposomal markers was found in the liver and spleen, and less in the kidneys and intestinal wall. Uptake into liver and spleen appeared to be due to the uptake of intact liposomes, whereas the uptake into kidneys and gut wall was caused by the uptake of liposomal degradation products. The uptake of PC:PS (4:1) liposomes into the liver was higher than that of DSPC:CHOL (2:1) liposomes; the opposite was the case with their uptake into the spleen. In rats, too, liposomes of different compositions showed significant variations in stability and in plasma half-lives of intact liposomes. Generally, there was a considerable increase in the liposomal stability in the presence of cholesterol and when use was made of a phospholipid with a high transition temperature.     

Drug-induced lipid peroxidation in mice—III: Glutathione content of liver,kidney and spleen after intravenous administration of free and liposomally entrapped glutathione

The half-life of extracellular glutathione was found to be 1.9 min in fed mice with a hepatic glutathione content of 44 ± 10 nmol glutathione per mg protein. It was 4.9 min in animals that had been fed for 48 hr a liquid sucrose diet resulting in a decreased hepatic glutathione of 25 ± 7 nmol/mg. A single intravenous injection of 16.2 μmol liposomally entrapped glutathione led to an increase in hepatic glutathione to 45 nmol/mg in the sucrose-fed mice after 2 hr and had no effect in the fed group. The spleen glutathione content reached a maximum at 30 min after injection in both groups. The maximum uptake into liver was 21% of the applied dose, into the spleen 7% and into the kidneys 2.4%. Injection of glutathione in solution led to a similar increase of hepatic glutathione as observed with GSH- containing liposomes, while liposomes filled with the constituent amino acids had only a marginal effect. The spleen took up only liposomal GSH. In contrast, the kidney glutathione content increased within 10 min up to 150% upon injection of free glutathione. The findings are consistent with a rapid hydrolysis of extracellular free glutathione followed by an interorgan turnover utilizing the constituent amino acids for resynthesis in the liver. Pretreatment of the animals with the glutathione synthesis inhibitor buthionine sulfoximine essentially abolished the hepatic glutathione increase upon treatment with GSH-liposomes or with the free compound. The finding that only liposomally entrapped glutathione protects mice against liver necrosis induced by highly dosed paracetamol is discussed with respect to differential uptake and distribution of GSH-liposomes in the liver.     

Cell culture and animal studies for intracerebral delivery of borocaptate in liposomal formulation

The efficacy of a liposomal formulation for intracerebral delivery of borocaptate (BSH) to brain tumor cells has been investigated using cell culture to study BSH uptake and persistence and using tumor-bearing rats to determine BSH distribution in the brain. During a 16-hr incubation, cellular uptake of BSH solution or BSH liposomal formulation was similar. However, the cellular persistence of BSH greatly increased when BSH was present in liposome. The differences in cellular persistence for BSH solution and BSH-loaded liposomes were significant both in 12-hr and 24-hr incubation experiments (p < 0.05 and p < 0.01, respectively). For the studies involving tumor-bearing rats, BSH level in tumor tissue was significantly higher than that in normal brain tissue at 2 hr and 6 hr after intracerebral injection of BSH-loaded liposomes (p < 0.01). Our study indicated that the liposomal formulation enhanced cellular persistence of BSH in tumor cells and therefore favored the boron accumulation in the cells. With the prolonged physical retention of liposomes at the local injection site and the cellular retention of BSH enhanced by the liposomes, the intracerebral delivery of BSH using liposomal formulation may provide an effective boron delivery approach for boron neutron capture therapy of brain tumors.     

Biochemical mechanisms and morphological selectivity in hepatotoxicity: studies in cultures of hepatic-parenchymal and non-parenchymal cells

1. Primary cultures of rat-liver parenchymal and non-parenchymal cells have been used to study some of the factors influencing the selective injury that can be caused in vivo by the direct-acting hepatotoxins beryllium, cadmium, ricin and modeccin to either liver-parenchymal or non-parenchymal cells.

2. The studies on beryllium and cadmium compounds show that it is necessary to consider the chemical species generated in the culture medium, since particulate or colloidal forms are taken up predominantly by non-parenchymal cells whereas soluble forms more readily enter parenchymal cells.

3. The studies with the glycoproteins ricin and modeccin illustrate the importance in their selective cell toxicity of specific membrane-recognition processes present in liver cells, particularly uptake in non-parenchymal cells through interactioiis with terminal mannose oligosaccharides in the toxins.     

Cell-specific delivery of genes with glycosylated carriers.

Cationic liposomes and polymers have been accepted as effective non-viral vectors for gene delivery with low immunogenicity unlike viral vectors. However, the lack of organ or cell specificity sometimes hampers their application and the development of a cell-specific targeting technology for them attracts great interest in gene therapy. In this review, the potential of cell-specific delivery of genes with glycosylated liposomes or polymers is discussed. Galactosylated liposomes and poly(amino acids) are selectively taken up by the asialoglycoprotein receptor-positive liver parenchymal cells in vitro and in vivo after intravenous injection. DNA-galactosylated cationic liposome complexes show higher DNA uptake and gene expression in the liver parenchymal cells in vitro than DNA complexes with bare cationic liposomes. In the in vitro gene transfer experiment, galactosylated liposome complexes are more efficient than DNA-galactosylated poly(amino acids) complexes but they have some difficulties in their biodistribution control. On the other hand, introduction of mannose residues to carriers resulted in specific delivery of genes to non-parenchymal liver cells. These results suggest advantages of these glycosylated carriers in cell-specific targeted delivery of genes.     

Cell Culture and Animal Studies for Intracerebral Delivery of Borocaptate in Liposomal Formulation

The efficacy of a liposomal formulation for intracerebral delivery of borocaptate (BSH) to brain tumor cells has been investigated using cell culture to study BSH uptake and persistence and using tumor-bearing rats to determine BSH distribution in the brain. During a 16-hr incubation, cellular uptake of BSH solution or BSH liposomal formulation was similar. However, the cellular persistence of BSH greatly increased when BSH was present in liposome. The differences in cellular persistence for BSH solution and BSH-loaded liposomes were significant both in 12-hr and 24-hr incubation experiments (p &lt; 0.05 and p &lt; 0.01, respectively). For the studies involving tumor-bearing rats, BSH level in tumor tissue was significantly higher than that in normal brain tissue at 2 hr and 6 hr after intracerebral injection of BSH-loaded liposomes (p &lt; 0.01). Our study indicated that the liposomal formulation enhanced cellular persistence of BSH in tumor cells and therefore favored the boron accumulation in the cells. With the prolonged physical retention of liposomes at the local injection site and the cellular retention of BSH enhanced by the liposomes, the intracerebral delivery of BSH using liposomal formulation may provide an effective boron delivery approach for boron neutron capture therapy of brain tumors.     

Nasal Delivery. The Use of Animal Models to Predict Performance in Man

Abstract

Galactose and Mannose residues were tagged on the surface of n-glutaryl-phosphatidyl-ethanolamine (NGPE) containing liposomes with and without polyethylene glycol of molecular weight 2000 Da conjugated to distearoyl phosphatidylethanolamine (PEG-2000-DSPE). Biodistribution studies showed that sugar bearing liposomes were cleared more rapidly from circulation than those not bearing the sugar moieties. However, the rate of clearance of glycosylated conventional liposomes was much faster than the sugar bearing sterically stabilized liposomes. Intrahepatic distribution studies showed that a substantial amount of conventional liposomes without sugar residues were taken up by both parenchymal (P) (40%) and non-parenchymal (NP) cells (60%). However, incorporation of PEG-2000-DSPE shifted this uptake slightly in favour of parenchymal cells (47%). While ratio of distribution of galactosylated conventional liposomes to P and NP cells was found to be 74: 26, galactosylation of sterically stabilized liposomes further enhanced the affinity of these vesicles towards P cells (P: NP ratio being 93: 7). Thus, reduced uptake by Kupffer cells was observed with galactosylated sterically stabilized liposomes as compared to conventional liposomes. Whereas, mannosylation of both the liposomes shifted the distribution towards Kupffer cells in an analogous manner. These findings indicate that sterically stabilized liposomes tagged with galactose residues on their surface are more effective in targeting the entrapped material to hepatocytes as compared to conventional liposomes. This approach can therefore be employed for delivering therapeutic agents like drugs, enzymes, genetic materials, anti-sense oligonucleotides selectively to liver P cells for treatment of hepatic disorders.     

Repeated injection of pegylated liposomal antitumour drugs induces the disappearance of the rapid distribution phase

Upon repeated administration, empty pegylated liposomes lose their long‐circulating characteristics, referred to as the accelerated blood clearance (ABC) phenomenon. To investigate whether cytotoxic drug‐containing pegylated liposomes could also elicit a similar phenomenon, two pegylated liposomal antitumour drugs (doxorubicin and mitoxantrone) were prepared, and they were administrated twice in the same animals with a 10‐day interval at a dose level of 8 mg kg^?1 (pegylated liposomal doxorubicin) and 4 mg kg^?1 (pegylated liposomal mitoxantrone). By comparing the overall pharmacokinetics after a single‐dose injection with that in animals treated with two doses, it was surprising to find that repeated administration of pegylated liposomal antitumour drugs caused the disappearance of rapid distribution phase instead of the ABC phenomenon, resulting in the conversion of a two‐compartment model to a one‐compartment model. Further investigation revealed that repeated injection induced the decreased uptake of liposomal antitumour drugs by the spleen at the early time point of 0.5–8 h after injection. In contrast, the deposition of liposomal antitumour drugs into liver was not affected. Therefore, the disappearance of the rapid distribution phase might be related to the reduced spleen uptake at the early time point.     

Preparation,Characterisation and Biodistribution of 99mTc-labeled Liposome Encapsulated Cyclosporine

The present study investigated the effect of charge (neutral, negative and positive) on liposomal membrane on the distribution of cyclosporine encapsulated in it to various organs. Liposomes were prepared by using different phospholipids by thin film hydration followed by sequential extrusion through polycarbonate membranes to achieve a desired particle size, with high entrapment efficiency and then lyophilised using sucrose as cryoprotectant. The possible in vivo distribution of cyclosporine and its liposomes after direct labeling with reduced technetium-99m has been studied in mice. The blood kinetics and biodistribution study of these labeled complexes shows prolonged circulation of positive and neutral charged liposomes in blood compared to free drug and negative charged liposomal formulation. The biodistribution of the tagged liposomes confirms that increased radioactivity was seen in liver and spleen, with minimal involvement of the kidney. At 4h post injection the biodistribution data in kidney reveals approximately 1-2% of the injected dose was present for cyclosporine loaded liposomes, which elicits the possibility of reducing the nephrotoxicity, generally seen in free cyclosporine. Interestingly, the biodistribution and -y imaging studies of the charged cyclosporine liposomes indicated that an appreciable amount of these labeled complexes goes to bone marrow when compared to the free cyclosporine. The findings demonstrate the distribution of these liposomes within various organs and proved that the positively charged liposomes experience increased bone uptake and prolonged circulation half-life. Hence this finding implies the possibility of using these formulations for liver and bone marrow transplantation.     

Pulmonary toxicity,hepatic, and extrahepatic metabolism of 2-methylnaphthalene in mice

Male mice ($\text{C57BL}\text{6J}$) were injected once ip with 0.1, 1, 100, 200, 400, 600, 800, or 1000 mg/kg of 2-methylnaphthalene dissolved in corn oil. After 24 hr, the animals were killed and the lungs, livers, and kidneys were prepared for light microscopy. In addition, some lungs were subjected to scanning and transmission electron microscopy. A dose of 200 mg/kg produced a bronchiolar necrosis which affected the nonciliated bronchiolar (Clara) cell; the parenchymal cells remained unaffected. At higher doses of 2-methylnaphthalene (800 mg/kg), in addition to the damaged Clara cell, severe damage to the upper respiratory tract was noted. No liver or kidney pathology was detected by light microscopy in animals treated with the highest dose. No cellular damage was noted in any organ at doses less than 200 mg/kg. Forty-eight hours after a dose of 200–1000 mg/kg of 2-methylnaphthalene, less pulmonary damage was detected by light microscopy. The metabolism of 2-methylnaphthalene was investigated in hepatic, pulmonary, and renal microsomes from $\text{C57BL}\text{6J}$ mice. Lung and liver microsomes produced three isomeric dihydrodiols of 2-methylnaphthalene as well as other monohydroxylated metabolites. Only trace amounts of these metabolites were produced by the kidney.     

Sulfonamide Acetylation in Isolated Rabbit and Rat Liver Cells

Abstract: Suspensions of isolated liver cells were prepared from rabbit livers perfused with Ca⁺⁺ -free buffer and 0.05% collagenase. Primary cell suspensions (containing both parenchymal and non-parenchymal liver cells) metabolized sulfadimidine and sulfanilamide at first-order kinetics for at least 2–3 hrs. Suspensions of purified rabbit liver parenchymal cells had an equal metabolic capacity, and it could be demonstrated that the metabolic rate of both sufadimidine and sulfanilamide was correlated to the amount of viable parenchymal cells in suspension. Suspensions of non-parenchymal cells were lacking the ability to metabolize both drugs. By means of homogenates of purified rabbit and rat liver cells, it could be demonstrated that the enzyme N-acetyltransferase was located in the cytosolic fraction of the parenchymal cells. It was concluded that the cytosolic fraction of the liver parenchymal cells is the main site of sulfonamide acetylation in both rabbit and rat.     

Assessment of targeting potential of galactosylated and mannosylated sterically stabilized liposomes to different cell types of mouse liver

Galactose and Mannose residues were tagged on the surface of n-glutaryl-phosphatidylethanolamine (NGPE) containing liposomes with and without polyethylene glycol of molecular weight 2000 Da conjugated to distearoyl phosphatidylethanolamine (PEG-2000-DSPE). Biodistribution studies showed that sugar bearing liposomes were cleared more rapidly from circulation than those not bearing the sugar moieties. However, the rate of clearance of glycosylated conventional liposomes was much faster than the sugar bearing sterically stabilized liposomes. Intrahepatic distribution studies showed that a substantial amount of conventional liposomes without sugar residues were taken up by both parenchymal (P) (40%) and non-parenchymal (NP) cells (60%). However, incorporation of PEG-2000-DSPE shifted this uptake slightly in favour of parenchymal cells (47%). While ratio of distribution of galactosylated conventional liposomes to P and NP cells was found to be 74:26, galactosylation of sterically stabilized liposomes further enhanced the affinity of these vesicles towards P cells (P:NP ratio being 93:7). Thus, reduced uptake by Kupffer cells was observed with galactosylated sterically stabilized liposomes as compared to conventional liposomes. Whereas, mannosylation of both the liposomes shifted the distribution towards Kupffer cells in an analogous manner. These findings indicate that sterically stabilized liposomes tagged with galactose residues on their surface are more effective in targeting the entrapped material to hepatocytes as compared to conventional liposomes. This approach can therefore be employed for delivering therapeutic agents like drugs, enzymes, genetic materials, anti-sense oligonucleotides selectively to liver P cells for treatment of hepatic disorders.     

Reduced hepatic toxicity, enhanced cellular uptake and altered pharmacokinetics of stavudine loaded galactosylated liposomes.

The aim of the present investigation was to reduce the hepatic toxicity, enhance the cellular uptake and alter the pharmacokinetics of stavudine using galactosylated liposomes. beta-D-1-Thiogalactopyranoside residues were covalently coupled with dimyristoyl phosphatidylethanolamine, which was then used to form liposomes. The galactosylated liposomal system was assessed for in vitro ligand-specific activity. The drug release from liposomes was studied by dialysis method. Ex vivo cellular uptake study was performed using liver parenchymal cells harvested from male albino rats. Changes in hematological parameters, hepatic enzymes, hepatomegaly, plasma and tissue distribution of the formulations (free stavudine solution, uncoated liposomal and galactosylated liposomes) were determined using albino rats. Percent cumulative drug release in 24h was low (34.8+/-2.6%). Enhanced hepatic cellular d4T uptake (27.96+/-2.41pg d4T/million cells) was seen in case of galactosylated liposomal d4T. Galactosylated liposomes maintained a significant level of d4T in tissues rich in galactose specific receptors and had a prolonged residence (11.44+/-1.25h) in the body resulting in enhanced half-life of d4T (23.07+/-1.25h). This formulation did not show either hematological or hepatic toxicity. Galactosylation of liposomes alter the biodistribution of encapsulated drug thereby delivering the drug to cells bearing galactose specific receptors.     

The intracellular distribution of liver cell calcium in normal rats and one hour after administration of carbon tetrachloride

In order to recognize the significance of elevated liver calcium level in the early phase of carbon tetrachloride intoxication, the subcellular distribution of calcium in liver was investigated one hour after administration of CCl₄ intraperitoneally. During preparation of subcellular fractions attempts were made to prevent redistribution of calcium by adding Ruthenium Red and EGTA or LaCl₃ to homogenization medium, and by shortening of differential eentrifugation to a minimum. The latter caused a loss of purity in sub cellular fractions which was overcome by correction of calcium values from atom absorption spectrometry by means of marker enzyme activity. The calcium levels in normal rat liver were found to be 70 $\text{nmoles}\text{g}$ liver wet wt in cytosol, 310 $\text{nmoles}\text{g}$ liver in microsomes and about 500 $\text{nmoles}\text{g}$ liver in mitochondria. A minor part of the latter fraction may belong to nuclei and plasma membranes. One hour after CCl₄ administration calcium levels in cytoplasma were not altered, in microsomes were decreased to 200 $\text{nmoles}\text{g}$ liver and in mitochondria were elevated to 2.5 $\text{μmoles}\text{g}$ liver. In rats pretreated with vitamin D the whole additional calcium, after carbon tetrachloride application in the range of 9 $\text{μmoles}\text{g}$ liver, was sequestrated in mitochondria. In the early phase of carbon tetrachloride intoxication all border membranes of liver cells have to participate in bringing about the reversible increase of liver cell calcium.     

Enhancement of Tissue Delivery and Receptor Occupancy of Methylprednisolone in Rats by a Liposomal Formulation

A liposomal formulation of methylprednisolone (L-MPL) was developed to improve localization of this immunosuppressant in lymphatic tissues. Liposomes containing MPL were formulated from a mixture of phosphatydylcholine and phosphatydylglycerol (molar ratio, 9:1) and sized by extrusion through a 0.1-µm membrane. Male Sprague–Dawley rats received a bolus dose of 2 mg/kg of L-MPL or free MPL in solution (control). Samples of blood, spleen, liver, thymus, and bone marrow were collected at intervals over a 66-hr period. Concentrations of MPL in plasma and organs and free cytosolic glucocorticoid receptors (GCR) in spleen and liver were determined. The plasma MPL profiles for free and L-MPL were bi- and triexponential. Although the alpha phase kinetics of both dosage forms were similar, L-MPL showed a substantially slower elimination phase than did free drug. Incorporation of MPL into liposomes caused the following increases: terminal half-life, from 0.48 (MPL) to 30.13 hr (L-MPL); MRT, from 0.42 to 11.95 hr, V _ss, from 2.10 to 21.87 L/kg; and AUC, from 339 to 1093 ng · hr/mL. Uptake of liposomes enhanced significantly the delivery of drug to lymphatic tissues and liver; AUC tissue:plasma ratios for spleen increased 77-fold; for liver, 9-fold; and for thymus, 27-fold. The duration of GCR occupancy was extended 10-fold in spleen and 13-fold in liver by the liposomal formulation. Lymphatic tissue selectivity and extended receptor binding of liposome-delivered MPL offer promise for enhanced immunosuppression.     

Specific incorporation of asialofetuin-labeled liposomes into hepatocytes through the action of galactose-binding protein

Asialofetuin-labelled liposomes (AF-liposomes) having mean diameters of 0.13 micron ([S]) and 0.35 micron ([L]) were obtained by the subsequent extrusion method in combination with dialysis. Intravenously administered AF-liposomes [S] were rapidly cleared from the systemic circulation. By pre- and post-treatment of the rats with free asialofetuin (AF), the rate of elimination decreased to that of non-labelled liposomes (N-liposomes) [S]. The liver uptake of AF-liposomes [S] (60 per cent of dose in 30 min) was about twice that of N-liposomes [S]. Forty-seven per cent of the AF-liposomes [S] incorporated into the liver were found in the parenchymal cell fraction as against 20 per cent in the non-parenchymal cell fraction. In contrast, N-liposomes [S] were taken up primarily by non-parenchymal cells as was also the case for AF- or N-liposomes [L]. Both the lipid and aqueous markers of AF-liposomes [S] were detected in subcellular fractions of the liver, but their distribution was noted to change differently with the lapse of time. Intravenously administered AF-liposomes [S] were specifically recognized by galactose-binding protein and underwent disruption in the cell after being taken up by hepatocytes. AF-liposomes [S] may possibly be utilized to deliver drugs into hepatocytes.     

Uptake,distribution, and effects of carbon tetrachloride in rainbow trout (Salmo gairdneri)

Uptake, distribution, and effects of CCl₄ were studied in rainbow trout. Carbon tetrachloride (1 ml/kg, ip) produced 5- to 10-fold increases in serum GOT, GPT, and ICD activities, whereas exposure of trout to CCl₄ in the tank water (1–80 mg/liter) produced neither mortality nor significant changes in enzyme activities. CCl₄ residue(s) appeared highest in concentration in the adipose tissue, followed by liver, brain, and spleen, and was lowest in gill regardless of the administration route. The elimination rates of ¹⁴C residue(s) from the tissue samples were most rapid in muscle ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{β = 1.7}\text{hr}$) and relatively prolonged for liver ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{β = 38.9}\text{hr}$). Maximum liver concentrations of ¹⁴C residue(s) were reached at 2 hr by either ip (1 ml/kg) or water exposure (80 mg/liter) and were 4.8 μmol/g and 0.75 μmol/g, respectively. No increase in liver triglyceride (TG) concentrations were noted at liver CCl₄ concentrations that have been associated with increased TG levels in the rat. Histological examination of tissues revealed varying degrees of liver and splenic necrosis 6 hr after administration of CCl₄.     

Synthesis of cholinesterase following poisoning with irreversible anticholinesterases: effects of theophylline and N6,O2-dibutyryl adenosine 3',5'-monophosphate on synthesis and survival.

The half-time ($\text{t}\text{1}\text{2}$) for return of whole blood cholinesterase (ChE) activity in mice following poisoning with Soman and DFP is on the order of 30 hr; in rats, the $\text{t}\text{1}\text{2}$ for return of whole blood ChE is 32 hr for Soman and 106 hr for DFP. In fact, the recovery of ChE activity in other rat tissues (liver and brain) is also significantly (p < 0.001) slower after DFP poisoning than after Soman poisoning. It appears that DFP adversely affects the normal rate for synthesis of ChE in rats; DFP does not affect overall protein synthesis in the rat as measured by ¹⁴C-amino acid incorporation into protein. Pretreatment of mice with a combination of theophylline-DBcAMP before poisoning with Soman causes animals to be more sensitive to the poison. Treatment of mice and rats with theophylline-DBcAMP at 24, 29, and 45 hr after Soman poisoning produces a dramatic increase in whole blood ChE but not in brain ChE at 48 hr; this increase in enzyme activity is due to pseudo-ChE and not acetyl-ChE. Ethionine, injected 1 hr before the initial dose of theophylline-DBcAMP, completely abolished the enhanced ChE activity observed in whole blood. These results suggest that the enhanced ChE activity observed in blood is due to a stimulating effect of the drugs on de novo synthesis and/or a release of ChE by the liver.