Bile Salt Ratio Research Articles

Neuroprotective efficiency of celecoxib vesicular bilosomes for the management of lipopolysaccharide-induced Alzheimer in mice employing 23 full factorial design

The aim of this study was to develop and evaluate bilosomes loaded with Celecoxib (CXB) for the efficient treatment of Alzheimer. The thin-film hydration approach was utilized in the formulation of CXB bilosomes (CXB-BLs). The study used a 23-factorial design to investigate the impact of several formulation variables. Three separate parameters were investigated: bile salt type (X1), medication amount (X2), and lipid–bile salt ratio (X3). The dependent responses included entrapment efficiency (Y1: EE %), particle size (Y2: PS), and zeta potential (Y3: ZP). The formulation factors were statistically optimized using the Design-Expert® program. The vesicles demonstrated remarkable CXB encapsulation efficiency, ranging from 94.16 ± 1.91 to 98.38 ± 0.85%. The vesicle sizes ranged from 241.8 ± 6.74 to 352 ± 2.34 nm. The produced formulations have high negative zeta potential values, indicating strong stability. Transmission electron microscopy (TEM) revealed that the optimized vesicles had a spherical form. CXB release from BLs was biphasic, with the release pattern following Higuchi's model. In vivo studies confirmed the efficiency of CXB-BLs in management of lipopolysaccharide-induced Alzheimer as CXB-BLs ameliorated cognitive dysfunction, decreased acetylcholinesterase (AChE), and inhibited neuro-inflammation and neuro-degeneration through reducing Toll-like receptor (TLR4), and Interleukin-1β (IL-1β) levels. The findings suggested that the created CXB-BLs could be a potential drug delivery strategy for Alzheimer's treatment.

Pitavastatin-loaded bilosomes for oral treatment of hepatocellular carcinoma: a repurposing approach

Albeit its established efficacy as an anti-hyperlipidemic agent, pitavastatin (PIT) has been shown to have other various therapeutic effects. One of these effects is the anti-cancer activity against hepatocellular carcinoma (HCC). This effect has been evaluated in this study for the first time via its oral delivery loaded in bilosomes both in vitro in hepatocellular carcinoma (HCC) cell line; HepG2 and in vivo in an Ehrlich ascites carcinoma (EAC) model. Moreover, the impact of surface modification of bilosomes with lactoferrin (LF) as an active targeting ligand for HCC was investigated. Bilosomes were prepared by thin-film hydration and different molar phospholipid to bile salt ratios were used to optimize the bilosomal formulation. The molar phospholipid to bile salt ratio was adjusted to 4:1 at pH 7.4. LF-coated bilosomes possessed a particle size, PDI, entrapment efficiency, and zeta potential of 112.28 nm ± 6.35, 0.229 ± 0.06, 90.56% ± 3.22, and −7.86 mV ± 1.13, respectively. LF-coated bilosomes also increased permeation of PIT when tested on Caco-2 cells by 3.1-folds (compared to uncoated ones or free PIT solution). It also improved the cytotoxicity of HepG2 spheroids 44-folds more than PIT-free solution. RT-PCR analysis showed that LF-coated PIT-loaded bilosomes caused an improvement (2-fold increase) in the apoptotic potential of PIT mediated by caspase-3. In conclusion, the optimized LF-coated PIT-loaded bilosomes were cytotoxic to HCC with improved hepatocytes permeation and cellular uptake. Thus, the proposed formula could be a promising treatment for HCC.

Formulation and optimization of lornoxicam-loaded bilosomes using 23 full factorial design for the management of osteoarthritis in rats: Modulation of MAPK/Erk1 signaling pathway

The objective of this study was to formulate and evaluate Lornoxicam (LOX) loaded bilosomes for effective treatment of osteoarthrotis. Lornoxicam bilosomes were formulated using thin film hydration method. A 23 full factorial design was employed to evaluate the effect of different formulation variables. Three independent variables were studied; bile salt type (X1), drug amount (X2) and lipid: bile salt ratio (X3). The dependent responses were; entrapment efficiency (Y1: EE %), vesicles size (Y2: VS) and zeta potential (Y3: ZP). Design Expert® software was utilized to statistically optimize formulation variables. The vesicles revealed high LOX encapsulation efficiency ranging from 56.01 ± 3.26 to 82.70 ± 1.80% whereas vesicle size ranged from 94.04 ± 6.5 to 292.5 ± 5.3 nm. High negative zeta potential values indicated good stability of the prepared formulations. The optimized vesicles exhibited spherical morphology under transmission electron microscopy (TEM). LOX release from bilosomes was biphasic and the release pattern followed Higuchi's model. In-vivo studies confirmed the efficiency of LOX bilosomes in treatment of osteoarthritis by improving locomotor activity and coordination, inhibiting pro-inflammatory cytokines; IL-6, and NF-κβ as well as blocking mitogen-activated protein kinases (MAPKs)/extracellular signal-regulated kinase (ERK) pathway. Moreover, in-vivo histopathological studies of knee joint tissues treated with LOX bilosomal formulations showed a reduction in inflammatory cells infiltration with average synovial lining and average articular cartilage. The results revealed that the developed LOX bilosomes could be a promising drug delivery system to be used in the treatment of osteoarthritis.

Characterising the behaviour of poorly water soluble drugs in the intestine: application of biorelevant media for solubility, dissolution and transport studies

Based on the knowledge of human intestinal fluids, compositions of biorelevant media and their impact on solubility, dissolution and permeability studies of poorly soluble drug compounds are discussed. Human intestinal fluids show large variations with regard to composition and pH, which complicate the selection of biorelevant media. The influence of concentration and ratio of bile salts, phospholipids and hydrolysis products, such as monoglycerides and free fatty acids, in well characterised media, on the solubility, dissolution and permeability of a given drug provides valuable information on the behaviour of the drug in the intestine, thus enabling the prediction of the in-vivo absorption. This review discusses the implications of biorelevant media composition on the solubility, dissolution and permeability of poorly soluble drug compounds. Biorelevant media contain bile salts and phospholipids and when simulating the fed state also monoglycerides and free fatty acids. Solubility of some poorly soluble drugs increase independently of the type of surfactants included in the biorelevant media, while others have a higher solubility in monoglyceride- and fatty acid-containing media. This is independent of the log P (the octanol-water partition coefficient) of the drug. The use of biorelevant dissolution media improves the correlation to in-vivo data, compared with compendial media, and although the field of permeability studies is complex the use of biorelevant media in this setting shows promise with respect to a better prediction of absorption.

A phylogenetic survey of biliary lipids in vertebrates ,

Biliary lipids (bile salts, phospholipids, cholesterol, plant sterols) were determined in 89 vertebrate species (cartilaginous and bony fish, reptiles, birds, and mammals), and individual phospholipid classes were measured in 35 species. All samples contained conjugated bile salts (C(27) bile alcohol sulfates and/or N-acyl amidates of C(27) and/or C(24) bile acids). Phospholipids were generally absent in the bile of cartilaginous fish and reptiles and were present in low amounts relative to bile salts in bony fish and most birds. In mammals, the phospholipid-bile salt ratio varied widely. The bile from species with low biliary phospholipid-bile salt ratios often contained a high proportion of sphingomyelin, confirmed by HPLC-MS. In species with a high phospholipid-bile salt ratio, the predominant biliary phospholipid was phosphatidylcholine (PC). The phospholipid-bile salt ratio correlated weakly with the calculated weighted hydrophobic index value. Cholesterol was present in the bile of virtually all species, with plant sterols uniformly being present in only trace amounts. The cholesterol-bile salt ratio tended to be higher in mammals than in non-mammals, but bile of all species was unsaturated. Thus, most nonmammalian vertebrates have relatively low levels of biliary phospholipid and cholesterol, suggesting that cholesterol is eliminated predominantly as bile salts. Mammals have a higher phospholipid and cholesterol to bile salt ratio, with the dominant phospholipid being PC.

Studies on the uptake of fatty acids by brush border membranes of the rabbit intestine

Initial studies revealed that the uptake of palmitic acid and oleic acid into brush border membranes was similar when these were isolated from either whole small intestine, jejunum, or ileum. The uptake of these fatty acids was somewhat lower with membranes obtained from duodenum. Subsequent studies, all with membranes obtained from whole intestine, indicated an increase in binding with chain length of fatty acid of up to 16 carbons. Unsaturation decreased this uptake somewhat. Taurocholate and 1-palmitoyl lysolecithin had a moderate stimulatory effect on the binding of oleic acid and palmitic acid at concentrations of 10 and 0.5 mM, respectively, and inhibited at higher concentrations. Addition of 1.4 mM egg lecithin to the fatty acid - bile salt micelles, such that the lecithin - bile salt ratio was 0.2, decreased the uptake of fatty acids generally, but did not significantly affect the pattern of binding by membrane fractions isolated from different segments nor did it change the pattern of labelling when fatty acid chain length and unsaturation were varied. At lower concentrations, egg lecithin had little effect on the uptake of oleic acid, whereas dipalmitoyl phosphatidylcholine stimulated binding of both palmitic acid and oleic acid over the entire range of concentrations tested. Preincubation of the membranes with this saturated phospholipid stimulated the uptake of oleic acid, and addition of this choline lipid to the oleic acid - bile salt containing micelles did not substantially enhance fatty acid uptake in lipid-treated membranes. The binding of fatty acid was very rapid either in the presence or the absence of Ca2+, such that even in zero-time controls essentially equilibrium bindings were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile salt transport in intestinal lymph of the rat.

The concentrations of conjugated cholate and chenodeoxycholate have been measured in samples of rat thoracic duct lymph, portal and systemic blood. Both these bile salts were present in lymph. After administration of a corn oil meal the flux of both these bile salts in lymph increased significantly (P less than 0.001; Student's t test), the cholate flux rising from 0.9 +/- 0.6 nmol/h (mean +/- SD) to 3.5 +/- 1.1 nmol/h postprandially and the chenodeoxycholate flux rising from 2.5 +/- 0.8 to 4.6 +/- 1.6 nmol/h. This postprandial increase in bile salt flux was due to both an increase in bile salt concentration and an increased lymph flow which rose significantly (P=0.007) from 0.5 +/- 0.2 to 0.7 +/- 0.2 ml/h. Biliary drainage significantly (P less than 0.001) reduced the flux of cholate and chenodeoxycholate in lymph to 0.08 +/- 0.05 and 0.17 +/- 0.10 nmol/h respectively. These biliary drained animals produced no significant rise (P greater than 0.1) in bile salt flux when fed the corn oil meal. The ratio of the concentrations of conjugated cholate to chenodeoxycholate in systemic and portal blood was 1.9:1 and 3.3:1 respectively. In contrast, this bile salt ratio was only 0.44:1 in lymph. These studies show that bile salts, in particular chenodeoxycholate, can pass directly from the intestine into lymphatics thus establishing an enterolymphatic circulation of bile salts.

The bile salt activation of leucocyte sphingolipid hydrolase activity and the modifying effects of triton X-100

The activation of sphingomyelinase, galactosyl- and lactosylceramide β-galactsidases, GM 1 β-galactosidase, cerebroside sulphate sulphatase, trihexosylceramide α-galactosidase and globoside β-hexosaminidase by a number of bile salts was studied. Most of the salts examined, except glycolithocholate and deoxycholate, were effective activators of enzyme activity. Pure, but not crude, taurocholate elicited poor trihexosylceramide α-galactosidase and cerebroside sulphate sulphatase activities. The bile salt activation was often dependent on a number of parameters including the bile salt concentration, pH, protein to bile salt ratio, and the nature of the enzyme preparation. In the absence of bile salts, Triton X-100 induced little activity of any of the hydrolases except sphingomyelinase; in their presence however, Triton either promoted or inhibited the activation depending on the enzyme. Our data suggest that bile salt structure is an important factor in determining the degree of activation of individual hydrolases and that this may be due to intramicellar lipid substrate/bile salt interactions, which either facilitate or inhibit the corresponding enzyme reaction.

Pigment vs cholesterol cholelithiasis: bacteriology of gallbladder stone, bile, and tissue correlated with biliary lipid analysis.

Although pigment (calcium bilirubinate) gallstones in Japanese subjects are associated with bacterial infection, the role of infection in Americans with pigment gallstones has not been assessed. Anaerobic and aerobic cultures of gallbladder bile, stone, and wall were obtained at cholecystectomy from nine patients with pigment stones and 25 with cholesterol stones. Among pigment-stone subjects, only 1 of 9 grew organisms in greater than 105 colony-forming units (CFU)/ml or g in gallbladder bile or wall. Likewise, growth greater than 105 CFU/ml or g was present in only 1 of 26 biles and 2 of 26 walls from cholesterol-stone patients.Propionibacterium acnes was found in less than 105 CFU/g or ml in at least 1 specimen from 6 of 9 pigment- and 12 of 26 cholesterol-stone patients. This organism was considered a contaminant because propionic acid concentrations in bile, an index of active bacterial metabolism, were similar in specimens with or without low-titer growth. The concentrations of bile salts, phospholipids, cholesterol, and bilirubin in gall-bladder bile was unaffected by the type of bacteria in low-titer growth. But the lipid concentrations were markedly depressed in two biles with bacterial growth greater than 105 CFU/ml. The molar ratio of bile salts and phospholipids to cholesterol was significantly higher in biles surrounding pigment stones than those surrounding cholesterol stones (P 105 CFU/ml) is not associated with pigment or cholesterol stones in asymptomatic American subjects at cholecystectomy. These data suggest that pigment-stone formation in the United States is not primarily related to bacterial alteration of bile composition, as the experience with Japanese patients would suggest.

The effects of tiadenol, clofibrate and clofibride on bile composition in the rat

Biliary secretion was studied in normolipedemic rats after a 7 day treatment with the hypolipidemic drugs, tiadenol (bis-(hydroxyethylthio)-1,10-decane), clofibrate and clofibride (chloro-4-phenoxy-2-methyl-2-propionate of dimethylcarbamoyl-3-propyl). All three drugs decreased blood cholesterol and total lipids, increased liver weight and liver catalase content, and decreased biliary excretion of cholesterol. The biliary concentrations of bile salts, phoslipids and cholesterol decreased to a variable extent, in such a way that ratio of bile salts + phospholipids to chloresterol was increased by the drugs. The bile salt independent fraction was increased. The effects were qualitatively similar for all three drugs tested, but quantitative differences appeared for some of the parameters.

Solubilization of β-carotene and retinol into aqueous solutions of mixed micelles

1. 1. The solubility of β-carotene (non-polar solute) and retinol (polar solute) into aqueous solutions containing mixed micelles has been studied in vitro using experimental conditions simulating those present in the lumen of the human small intestine during fat digestion with respect to bile salts, lipids, Na +, pH and temperature. 2. 2. Altering the glycine to taurine conjugated bile salt ratio had no effect on the solubility of either β-carotene or retinol. Altering the ratio of tri- to dihydroxy bile salts changed the solubility of both solutes; the higher the ratio the lower the solubility. The solubility of retinol in the systems studied was approximately 7–9-fold greater than that of β-carotene. 3. 3. Increasing the concentration of Na + greatly increased the solubility of β-carotene but only slightly increased the solubility of retinol. 4. 4. In a mixture containing both solutes, no competition for solubilization occurred. 5. 5. pH values between 4.5 and 7 had no effect on the solubility of either solute but at pH values below 4.5, the solubility of both solutes markedly decreased.

Cholesterol and bile salt studies on the bile of patients with cholesterol gallstones.

Gallbladder bile from patients with cholesterol stones and control patients without cholesterol stones was analysed for the relative concentrations of bile salts, phospholipids, and cholesterol. It was not possible to distinguish stone-forming from control biles when these were plotted on triangular coordinates. There was a significant increase in the dihydroxy: trihydroxy bile salt ratio in the patients with cholesterol gallstones. The maximum cholesterol holding capacity for the bile salts was determined and no difference could be detected in the ability of the bile salts from either cholesterol stone-containing and control bile samples to dissolve cholesterol.

An in vivo comparison of two bile salt binding agents, cholestyramine and lignin.

In 2 normal subjects, studied after administration of radioactive taurocholate, cholestyramine markedly accelerated the loss of labelled bile salts from the bile and caused an elevation of the glycine/taurine conjugation ratio and of the tri-hydroxy/di-hydroxy bile salt ratio. These effects are consistent with its known bile salt binding activity. Lignin, a new bile salt binding agent effective in vitro, had no convincing effect in studies on 3 subjects. It is suggested that the hydrophobic bonding postulated for lignin is too weak to sequestrate circulating bile acids.

HYPEROXALURIA AND RENAL CALCULI IN ILEAL DISEASE

It has been suggested that, because of bile-salt malabsorption, patients with ileal dysfunction " spill " glycine-conjugated bile-salts into their large bowel. There, colonic bacteria deconjugate the bile-salts and convert the liberated glycine to glyoxalate, which in turn is absorbed, oxidised in the liver to oxalic acid, and excreted in the urine to produce hyperoxaluria and, occasionally, oxalate renal stones. To test this hypothesis, urinary oxalate excretion was measured in eleven patients with ileal dysfunction, and eight of these showed severe hyperoxaluria ranging from 91 to 202 mg. per 24 hours (normal <35 mg. per 24 hours). Three patients had had urinary calculi. In one patient the stone was analysed by X-ray diffraction studies and was shown to be calcium oxalate monohydrate. In a second patient, chemical analysis of calculi removed on two separate occasions again showed that these were calcium-oxalate stones. Since taurine feeding changes the glycine/ taurine bile-salt ratio in favour of taurine, this aminoacid was fed to one hyperoxaluric patient in an attempt to remove this substrate for oxalate formation. 9 g. of taurine per day by mouth promptly abolished this patient's hyperoxaluria.

GALLSTONE FORMATION

Cholesterol, the major component of Summary gallstones, is held in solution in the bile by formation of micelles in which phospholipids and bile-salts are involved. The ratio of cholesterol to bile-salts plus phospholipids determines cholesterol solubility. Although the bile produced by the liver in patients with gallstones is abnormal, some features of gallbladder function might favour stone formation. Of the hepatic factors contributing to gallstone formation a high cholesterol content of bile, a low bile-salt pool, and interruption of the enterohepatic circulation seem to be important. One hypothesis for gallstone formation relates to events at the plasma membrane of the bile canaliculus, but whether the critical factor here is an increase in the amount of cholesterol passing through the membrane or an abnormal cholesterol to phospholipid ratio remains controversial. It seems likely that an understanding of the mechanism for gallstone formation will come from an investigation of the problem at the cellular, subcellular, or molecular level. This review ends with a note on the prospects for treatment, which should be directed at affecting the cholesterol to phospholipid plus bile-salt ratio or at the cholesterol precipitate directly by using detergent agents.

Bile Salt Synthesis in Transplanted Human Liver

Bilirubin and bile salt excretion were determined in a patient following orthotopic liver transplantation. Bile salt synthesis was found to increase within 24 hr and the total amount excreted per day exceeded 400 mg. During the recovery phase a decrease in the bilirubin to bile salt ratio in bile provided evidence for a specific interference in the transport of conjugated bilirubin.

Hydrolysis of long-chain monoglycerides in micellar solution by pancreatic lipase

1. 1. Certain long-chain monoglycerides have a high solubility in bile-salt solutions forming mixed monoglyceride-bile-salt micelles. Monoglycerides in such an optically clear, micellar solution are readily hydrolyzed by pancreatic lipase (glycerol ester hydrolase, EC 3.1.1.3). Using a rat-pancreatic-juice preparation containing negligible esterase activity, the hydrolysis of a number of pure monoglycerides in micellar solution has been studied. 2. 2. The rate of hydrolysis was essentially linear with time and enzyme concentration up to a level of around 60–70% hydrolysis of the substrate. The extent of hydrolysis fell when the concentration of micelles of identical composition was increased. There was a broad pH optimum 5.5–7.5. Bile salts greatly enhanced the rate of hydrolysis at low concentrations, but this effect was possibly chiefly due to their dispersing effect; at higher concentrations, they caused a marked inhibition of hydrolysis. Typical anionic detergents influenced hydrolysis in the same manner as bile salts. With a cationic detergent the enzyme concentration had to be considerably increased to obtain the same rate of hydrolysis. 3. 3. 2-Monoolein was not attacked to any appreciable extent by pancreatic lipase; thus the specificity of lipase for the 1-ester position could be directly shown. Glycol monooleate was readily hydrolyzed; 1-α,α-dimethyl-monodecanoin was not split. 4. 4. Under experimental conditions where 1-monoolein was extensively hydrolyzed, middle-chain saturated 1-monoglycerides were hydrolized to a very limited extent. Extensive hydrolysis could be obtained by increasing the micellar amphiphile to micellar bile-salt ratio, by adding other amphiphiles such as fatty acid, or by using a two-phase heptane-buffer system where the monoglyceride was concentrated at the interface. The results of these and other experiments were interpreted as showing the tightness of packing of the micelle to be a critical factor influencing the extent of hydrolysis. 5. 5. Titration of the fatty acid of a mixed bile salt-monoglyceride-fatty acid micelle indicated a pK a of 6.9 with the experimental conditions present.

Hydrolysis of long-chain monoglycerides in micellar solution by pancreatic lipase

1. 1. Certain long-chain monoglycerides have a high solubility in bile-salt solutions forming mixed monoglyceride-bile-salt micelles. Monoglycerides in such an optically clear, micellar solution are readily hydrolyzed by pancreatic lipase (glycerol ester hydrolase, EC 3.1.1.3). Using a rat-pancreatic-juice preparation containing negligible esterase activity, the hydrolysis of a number of pure monoglycerides in micellar solution has been studied. 2. 2. The rate of hydrolysis was essentially linear with time and enzyme concentration up to a level of around 60–70% hydrolysis of the substrate. The extent of hydrolysis fell when the concentration of micelles of identical composition was increased. There was a broad pH optimum 5.5–7.5. Bile salts greatly enhanced the rate of hydrolysis at low concentrations, but this effect was possibly chiefly due to their dispersing effect; at higher concentrations, they caused a marked inhibition of hydrolysis. Typical anionic detergents influenced hydrolysis in the same manner as bile salts. With a cationic detergent the enzyme concentration had to be considerably increased to obtain the same rate of hydrolysis. 3. 3. 2-Monoolein was not attacked to any appreciable extent by pancreatic lipase; thus the specificity of lipase for the 1-ester position could be directly shown. Glycol monooleate was readily hydrolyzed; 1-α,α-dimethyl-monodecanoin was not split. 4. 4. Under experimental conditions where 1-monoolein was extensively hydrolyzed, middle-chain saturated 1-monoglycerides were hydrolized to a very limited extent. Extensive hydrolysis could be obtained by increasing the micellar amphiphile to micellar bile-salt ratio, by adding other amphiphiles such as fatty acid, or by using a two-phase heptane-buffer system where the monoglyceride was concentrated at the interface. The results of these and other experiments were interpreted as showing the tightness of packing of the micelle to be a critical factor influencing the extent of hydrolysis. 5. 5. Titration of the fatty acid of a mixed bile salt-monoglyceride-fatty acid micelle indicated a pK a of 6.9 with the experimental conditions present.