Hypolipidemic effect of mannans from C. albicans serotypes a and B in acute hyperlipidemia in mice.

Mannans, which are biological macromolecules of polysaccharide origin and function as immunomodulators, have been shown to stimulate macrophages in vivo by interaction with the mannose receptor. Thus, they can be used to stimulate macrophages in order to effectively remove circulating atherogenic lipoproteins. Our primary aim was to evaluate the hypolipidemic potential of mannans from C. albicans serotype A (mannan A) and serotype B (mannan B) in a murine model of hyperlipidemia. Mannan A and mannan B were shown to significantly (p<0.05) stimulate both the proliferation (p <0.05) and nitric oxide production of murine peritoneal macrophages in vitro. Pre-treatment of CBA/Lac mice with mannan A prior to induction of hyperlipidemia significantly (p<0.001) reduced serum atherogenic LDL-cholesterol, total cholesterol, and triglycerides. Mannan B exhibited a similar, but more potent, hypolipidemic effect. Electron microscopic analysis of liver revealed a significant (p<0.001) decrease in the volume of lipid droplets when hyperlipidemic mice were pretreated by both mannans. In conclusion, our findings would suggest that both polysaccharide-based biological macromolecules evaluated in the present study, specifically, the natural immunomodulators (mannans A and B), appeared to function as effective lipid-lowering macromolecules, which could potentially serve as adjunct therapy to more conventional hypolipidemic medications such as a statin drug.

Preventing cardiovascular heart disease: Promising nutraceutical and non-nutraceutical treatments for cholesterol management.

Hypercholesterolemia is one of the major risk factors for the development of cardiovascular disease. Atherosclerosis resulting from hypercholesterolemia causes many serious cardiovascular diseases. Statins are generally accepted as a treatment of choice for lowering low-density lipoprotein (LDL) cholesterol, which reduces coronary heart disease morbidity and mortality. Since statin use can be associated with muscle problems and other adverse symptoms, non-adherence and discontinuation of statin therapy often leads to inadequate control of plasma cholesterol levels and increased cardiovascular risk. Moreover, there is compelling evidence on the presence of still considerable residual cardiovascular risk in statin-treated patients. Ezetimibe improves cholesterol-lowering efficacy and provides mild additional cardiovascular protection when combined with statin treatment. Despite a favorable safety profile compared to statins, ezetimibe-induced cholesterol-lowering is modest when used alone. Hence, there is a critical need to identity additional effective hypolipidemic agents that can be used either in combination with statins, or alone, if statins are not tolerated. Thus, hypolipidemic agents such as proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, apolipoprotein B-100 antisense oligonucleotides, cholesteryl ester transfer protein (CETP) inhibitors, and microsomal triglyceride transfer protein (MTTP) inhibitors, as well as yeast polysaccharides (beta-glucans and mannans) and compounds derived from natural sources (nutraceuticals) such as glucomannans, plant sterols, berberine, and red yeast rice are being used. In this review, we will discuss hypercholesterolemia, its impact on the development of cardiovascular disease (CVD), and the use of yeast polysaccharides, various nutraceuticals, and several therapeutic agents not derived from 'natural' sources, to treat hypercholesterolemia.

Serum cystatin C and chitotriosidase in acute P-407 induced dyslipidemia: Can they serve as potential early biomarkers for atherosclerosis?

In an attempt to better understand potential biomarkers for, and the role of macrophages in, the development of atherosclerosis, the toxicologic, and any therapeutic pharmacologic effects of carboxymethylated ß-glucan, gadolinium chloride, and poloxamer 407 were studied in mice for their capacity to perturb serum lipids, cystatin C, and chitotriosidase-1. Gadolinium and carboxymethylated ß-glucan dosed separately to control mice had no effect on serum lipids, whereas carboxymethylated ß-glucan, but not gadolinium, exerted a significant (p<0.01) and unexpected hypolipidemic effect in poloxamer 407-induced hyperlipidemic mice. An acute hyperlipidemic state (∼4 days), induced with poloxamer 407 administration alone, resulted in a significant (p<0.01) time-dependent decrease and increase in serum cystatin C and chitotriosidase, respectively. Carboxymethylated ß-glucan administration to hyperlipidemic mice significantly (p<0.05) increased the serum concentration of cystatin C, but significantly (p<0.01) decreased chitotriosidase activity, when each was compared to mice treated with poloxamer 407 only. Gadolinium administration caused a significant decrease in serum chitotriosidase activity in both controls (p<0.01) and poloxamer 407-induced hyperlipidemic (p<0.001) mice, but had no effect on the concentration of cystatin C in either controls or poloxamer 407-induced hyperlipidemic mice. Gadolinium administration resulted in both morphological and functional changes to liver macrophages, which included incorporation of excess lipids, especially when simultaneously administered with poloxamer 407. It is suggested that serum cystatin C and chitotriosidase may represent potential early biomarkers for eventual atherosclerosis in the poloxamer 407-induced mouse model of atherogenesis, and that two compounds known to either increase (carboxymethylated ß-glucan) or decrease (gadolinium chloride) the number of macrophages in vivo were able to modulate serum chitotriosidase activity, This, in turn, would appear to support the premise that serum chitotriosidase activity may be a more sensitive indicator of macrophage involvement than cystatin C in the context of future atherosclerosis.

A primer on the requirements for select agents and toxins used in biomedical and agricultural research applications.

A basic overview of the U.S. Department of Health and Human Services and U.S. Department of Agriculture List of Regulated Select Agents and Toxins used in biomedical and agricultural research, as controlled by the Centers for Disease Control and Prevention, is presented. The objective of the primer is to introduce to the health physics community a brief history, discuss basic terminology, review current legislation, and describe rules and regulations for the possession, use, and transfer of select agents and toxins. As with many institutions facing staff and budget restrictions, there is increased frequency of health physics professionals being designated the Responsible Official. This summary is necessary since radiolabeled select agents and toxins are being utilized in research applications and require oversight by the health physicist.

Intestinal absorption and biodistribution of cosalane and its amino acid conjugates: novel anti-HIV agents.

Cosalane and its amino acid conjugates are potent inhibitors of HIV replication. The purpose of this study was to investigate: (1) the pharmacokinetic disposition of the diglycine (GC) and the diaspartic acid (ASPC) conjugates of cosalane in male Sprague-Dawley rats; (2) intestinal absorption of cosalane and its amino acid conjugates using in vitro (small intestinal segments), in situ (closed loop); and (3) biodistribution of GC and its absolute oral bioavailability in rat. Cosalane and its conjugates exhibited biexponential disposition with very long half-lives upon intravenous dosing. However, these compounds failed to permeate the small intestine unless sodium desoxycholate (5-20 mM) was used as an intestinal permeation enhancer. A rank order correlation in terms of permeation enhancement in a descending order is as follows: GC>Cosalane>ASPC. In situ studies revealed that although the bile salt enhanced the permeation of cosalane across the enterocyte, its hepatic uptake was extensive. However, 66% of the absorbed dose of GC escaped uptake by the reticuloendothelial system (RES) and its biodistribution studies showed that the uptake by the RES was significantly lower compared to the parent compound. GC had an absolute oral bioavailability of 5.10+/-1.51%. Therefore, GC appears to be a favorable candidate for further development.

Fine-Particle ethylcellulose as a tablet binder in direct compression, immediate-release tablets.

Ethylcellullose has traditionally been used in tablets as a binder in an alcohol solution form. In the present study, fine-particle ethylcellulose (FPEC) was used as a binder to manufacture immediate-release tablets by the direct compression technique. The binding potential of FPEC is compared to that of commercially available coarse-particle ethylcellulose at the same viscosity grade and to that of hydrophilic binders. The compression force setting was kept constant for all batches. The concentration of the binder was varied from 5% to 25%. Acetaminophen was used as a model drug because capping is a problem frequently observed during high-speed compaction and further processing of acetaminophen tablets. In this study, there would be an increase in the contact area with FPEC and hence greater bond formation. This greater bond formation should be able to reduce the problem of capping in tablets containing highly elastic materials such as acetaminophen. Tablets were evaluated based on the following tests: weight variation, extent of capping, hardness, friability, disintegration, and dissolution. Based on the results of these tests, FPEC proved to be an effective binder for directly compressed acetaminophen tablets. The 10% and 15% formulations of FPEC passed all the tests and also produced the hardest tablets.

Potency of select statin drugs in a new mouse model of hyperlipidemia and atherosclerosis.

Poloxamer-407 (P-407) is a nonionic surfactant that induces atheroma formation in the aortas of C57BL/6 mice with long-term (14 weeks) administration. The objectives of the present study were to determine the mechanism(s) responsible for the induction of hypercholesterolemia as well as to determine whether this animal model may be of potential use in rank ordering the efficacy (lipid lowering) of various statin drugs. The effect of long-term (16 weeks) administration of P-407 on the catalytic activities of rate-limiting enzymes of cholesterol biosynthesis [HMG-CoA reductase (HMGR)] and catabolism [microsomal cholesterol 7alpha-hydroxylase (C7alphaH) and mitochondrial sterol 27 hydroxylase (S27H)] was assessed in C57BL/6 mice. Effects of P-407 on these enzymes were compared in mice fed an atheroma-inducing diet (high-cholesterol, supplemented with cholic acid) and animals maintained on a basal diet and injected with saline (controls) after 16 weeks. The mean value for the activities of C7alphaH in P-407-injected mice was 24.3+/-3.8 pmol min(-1) mg(-1) and was significantly (P<0.05) less than the mean value determined for sham-injected control animals (37.0+/-14.3 pmol min(-1) mg(-1)). In contrast, the mean values for the catalytic activities of S27H and HMGR did not change with P-407 administration. Neither C7alphaH nor S27H activity in mice fed the high-cholesterol diet differed from values for control animals, whereas the mean HMGR activity was drastically reduced (-94%, P<0.05). The hypercholesterolemic effect of P-407 is not due to altered cholesterol biosynthesis, but is mediated by reduced cholesterol catabolism due to decreased activity of the rate limiting enzyme (C7alphaH) in the classic bile acid synthetic pathway. Plasma triglyceride lowering resulting from the oral administration of equal doses of various statin drugs appeared, in general, to be positively correlated with their relative aqueous solubility and paralleled the efficacy of these agents to lower low-density-lipoprotein-associated cholesterol (LDL-C) in humans. The plasma triglyceride lowering effect of the five statin drugs tested produced the following rank order; pravastatin sodium (-44%)>atorvastatin calcium (-36%)>simvastatin (-33%)>lovastatin (-25%)>fluvastatin sodium (-19%). While reductions in plasma total cholesterol following administration of the statin drugs was not as profound as that observed with triglycerides, the relative rank order or trend was preserved. The percent reduction in plasma triglycerides in the present model appears to be a useful parameter with which to predict the relative reduction in plasma LDL-C expected for these agents in humans.

Endothelial function and myogenic reactivity in small mesenteric arteries of hyperlipidemic pregnant rats.

Supraphysiological increases in serum triglycerides and cholesterol often occur during pregnancy, but their effects on vascular function are poorly understood. Intraperitoneal injection of the nontoxic surfactant poloxamer 407 (P-407) results in sustained elevation of triglycerides and cholesterol. We asked if P-407-induced hyperlipidemia during late pregnancy adversely affects mesenteric resistance artery vasodilator function. On days 13-15 of pregnancy, rats were given a single intraperitoneal injection of P-407, sterile water vehicle, or non-lipid-altering pluronic F-88 (P-88). Four days postinjection, serum triglycerides, cholesterol, free fatty acids, and the lipid peroxidation product malondialdehyde were significantly increased in P-407-treated rats. Mesenteric arteries from P-407-treated rats displayed significant increases in myogenic reactivity (constrictor responses to step increases in intraluminal pressure). The nitric oxide (NO) blocker N(alpha)-methyl-L-arginine increased the myogenic response in control but not in P-407 arteries, normalizing group differences. Endothelial removal increased myogenic reactivity beyond that of prior NO synthase inhibition in controls and potentiated myogenic reactivity in P-407 arteries such that responses again converged. Relaxation responses to the endothelium-dependent vasodilator methacholine did not differ. We conclude that that P-407-induced hyperlipidemia during pregnancy increases myogenic reactivity due to selective attenuation of an NO-mediated vasodilator component of the myogenic response.

Binding of cosalane--a novel highly lipophilic anti-HIV agent--to albumin and glycoprotein.

Cosalane is a potent inhibitor of HIV replication with multiple sites of action. The purposes of this study were to (a) determine the extent and nature of cosalane binding to mucin, alpha(1)-acid glycoprotein (AAG), plasma, and human (HSA) and bovine serum (BSA) albumin, and (b) determine the primary site(s) of cosalane binding to HSA. Plasma protein binding of cosalane was studied by a gel filtration technique. Cosalane binding to HSA was also determined in the presence of salicylic acid. Competitive inhibition studies were conducted using warfarin, digitoxin, and diazepam to determine the primary HSA binding site(s) of cosalane. The drug was bound extensively to HSA and BSA and required 500-550 moles to saturate 1 mole of protein. Stoichiometries of cosalane binding to alpha(1)-acid glycoprotein (AAG) and mucin were between 30 and 50 mol/mol of either glycoprotein. The binding isotherm deviated from a rectangular hyperbola, suggesting self-association of the ligand. Salicylic acid decreased cosalane binding to HSA by one order of magnitude. Inhibition studies of cosalane to HSA revealed that the compound binds primarily to warfarin site with a K(i) of 1.24 +/- 0.24 nM. In summary, cosalane binds extensively to serum albumins and to a lesser extent to both AAG and mucin.

Inhibition of a model protease--pyroglutamate aminopeptidase by a natural oligosaccharide gum from Hakea gibbosa.

The purpose of this study was to investigate the effect of the oligosaccharide gum from Hakea gibbosa on the activity of a model protease enzyme pyroglutamate aminopeptidase (5-oxoprolyl peptidase; EC 3.4.19.3) and to elucidate the mechanism responsible for the decreased activity. Enzyme kinetic studies were conducted at 37 degrees C in 100 mM potassium phosphate buffer with 10 mM EDTA, 5% (v/v) glycerol, and 5 mM DTT (pH 8) for 15 min and were performed both in the presence and absence of the gum. Enzymatic activity was determined by a colorimetric assay using the specific substrate L-pyroglutamic acid beta-napthylamide. The enzyme kinetics was studied at various substrate and gum concentrations. The velocity of the reaction was determined by the amount of the product (beta-napthylamine) liberated at each substrate and gum concentration. The Ks and Vmax of the enzyme in the absence of the gum were 24.40+/-2.14 microM and 502.95+/-28.90 nmoles x min(-1) x mg protein(-1), respectively. As the concentration of the gum was gradually increased from 0.1 to 2%, the value of the Vmax decreased from 318.94+/-21.46 to 158.83+/-24.51 nmoles x min(-1) x mg protein(-1) while Ks increased from 17.42+/-4.6 to 63.03+/-1.89 microM. The mechanism for the inhibition of the enzyme by Hakea appeared to be a mixed-linear type (a type of non-competitive inhibition) as suggested from Hanes-Woolf, Dixon and Cornish-Bowden plots. The turnover number, kcat, calculated for the enzyme also decreased from 14.09+/-0.81 to 4.45+/-0.69 min(-1) as the concentration of the inhibitor was incrementally increased from 0 to 2% (w/v). The K(i) and alphaK(i) calculated from Dixon and Cornish-Bowden plots were found to be 0.31+/-0.11% (w/v) and 1.33+/-0.42% (w/v), respectively. The natural gum from Hakea gibbosa was effective in non-competitively inhibiting the enzyme pyroglutamate aminopeptidase. Thus, the natural gum may be a promising additive not only for its sustained-release and mucoadhesive properties as shown previously, but also for its ability to slow the enzymatic degradation of therapeutic polypeptides incorporated in dosage forms.

Regression of poloxamer 407-induced atherosclerotic lesions in C57BL/6 mice using atorvastatin.

HMG-CoA reductase inhibitor drugs or 'statins' have been shown to effectively reduce plasma total cholesterol (CHOL), CHOL associated with low-density-lipoprotein (LDL), and triglycerides (TG). In addition, slight elevations in HDL-CHOL are also typically observed. Poloxamer 407 (P-407), a nonionic surfactant, effectively elevates both plasma CHOL and especially TG in a dose-controlled fashion and results in formation of atherosclerotic lesions in the aortas of C57BL/6 mice without the requirement of dietary cholic acid [1,2]. The purpose of the present study was to assess whether a typical statin, namely atorvastatin (Lipitor(R)) would significantly reduce P-407-induced hypercholesterolemia and hypertriglyceridemia as well as cause regression of atherosclerotic lesions resulting from administration of P-407 to C57BL/6 mice. C57BL/6 mice in the present study were treated with either normal saline (C, controls), 0.5 g/kg of P-407 (P), or a high-fat, high-cholesterol, cholate-containing diet (HF) for 120 days. Mice in all groups were then equally and randomly divided and treated with either atorvastatin or saline for an additional 120 days. Beginning at Day 121 and using mice in groups P and HF as an example, one-fourth of the mice in each group received 20 mg/kg per day of atorvastatin with either concomitant HF feeding or P-407 administration ('progression' treatment groups), one-fourth received 20 mg/kg per day of atorvastatin following cessation of HF feeding or P-407 administration, one-fourth received saline (placebo) with either simultaneous HF feeding or P-407 administration ('progression' placebo groups), and one-fourth received saline (placebo) following cessation of HF feeding or P-407 administration. Total plasma CHOL was significantly (P<0.01) lower for mice in groups P and HF when administered atorvastatin relative to saline, but remained significantly (P<0.05) elevated compared to total plasma CHOL of C mice. With discontinuation of either P-407 administration or HF feeding, total plasma CHOL declined rapidly in both P and HF mice with atorvastatin-treated mice generally demonstrating lower plasma CHOL concentrations relative to saline-treated mice. Total plasma TG was significantly (P<0.01) lower for mice in group P administered atorvastatin relative to saline, but remained significantly (P<0.05) elevated compared to plasma TG of C mice. With discontinuation of P-407 administration, total plasma TG declined rapidly in P mice with atorvastatin-treated mice typically demonstrating lower plasma TG concentrations relative to saline-treated P mice. Aortas of mice treated with 20 mg/kg per day of atorvastatin in both groups P and HF, whether maintained on the HF-diet or treated with P-407 from Day 120 to 240 or whether each treatment was terminated at Day 120, revealed no presence of atherosclerotic lesions relative to saline-treated mice and were indistinguishable from aortas retrieved from C mice. Atorvastatin at a dose of 20 mg/kg per day not only significantly reduced the plasma CHOL and TG concentrations, but also resulted in regression of atherosclerotic lesions induced in C57BL/6 mice by administration of P-407 or ingestion of a HF-diet containing cholic acid.

Pharmacokinetics, biliary excretion, and tissue distribution of novel anti-HIV agents, cosalane and dihydrocosalane, in Sprague-Dawley rats.

Cosalane and dihydrocosalane are potent inhibitors of HIV replication with a broad range of activity. The purpose of this study was to investigate: 1) the pharmacokinetic disposition of both cosalane and dihydrocosalane in male Sprague-Dawley rats, and 2) biliary excretion, enterohepatic circulation, and tissue distribution of cosalane after i.v. and/or oral administration. Animals were administered i.v. (10 mg/kg) cosalane or dihydrocosalane through a jugular vein to obtain plasma profiles. Dose dependence of cosalane was studied over a dose range of 1.0 to 10 mg/kg. The extent of enterohepatic recycling, biliary excretion, and tissue distribution were studied after i.v. administration. Both cosalane and dihydrocosalane exhibited a biexponential disposition with very long half-lives of 749 +/- 216 and 1016 +/- 407 min, along with very large volumes of distribution 23.1 +/- 4.4 and 24.4 +/- 2. 5 liter/kg, respectively. Both cosalane (nondetectable) and dihydrocosalane (<1%) showed very poor oral bioavailability. The biliary and renal excretions of cosalane were found to be negligible with no detectable metabolites either in urine or bile. After oral administration, more than 87% of the cosalane dose was excreted in the feces as the parent compound. Also, cosalane was sequestered significantly in liver with quantifiable levels in all tissues tested, even 48 h after the dose was administered. Therefore it was concluded that the poor oral bioavailability of cosalane may be due to its poor enterocytic transport coupled with sequestration in liver parenchymal cell membrane layers.

Potential downregulation of HMG-CoA reductase after prolonged administration of P-407 in C57BL/6 mice.

This study investigated the potential alteration in the amount of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase messenger RNA (mRNA) and lipoprotein lipase (LPL) mRNA in the livers of C57BL/6 mice after long-term (200 days) treatment with the nonionic surfactant called poloxamer 407 (P-407). Previously, P-407 has been used to produce a dose-controlled hyperlipidemic state in C57BL/6 mice with subsequent formation of atherosclerotic lesions. Five groups of mice were studied; controls (C); mice fed a standard chow diet enriched with only cholic acid (CH); mice fed the high-cholesterol, high-fat Paigen diet (HF); mice treated with 0.5 g/kg P-407 every third day (P); and mice administered 0.5 g/kg P-407 every third day while consuming a diet identical to that of mice in group CH (PC). Neither a significant (p < 0.05) weight loss nor alteration in liver enzymes (AST and ALT) were observed for any group throughout the study when compared with the control mice. Total plasma cholesterol (CHOL) was significantly elevated compared with controls for mice in groups HF, P, and PC, whereas total plasma triglycerides (TG) were significantly increased for mice in only groups P and PC. Long-term ingestion of a high-fat diet or a diet enriched in cholic acid resulted in a significant (p < 0.05) reduction in HDL-CHOL when compared with controls. Plasma samples assayed at 200 days for mice in groups HF and P showed a shift in the lipoprotein fraction distribution primarily to VLDL-CHOL as compared with mice in group C in which, as expected, most of the CHOL was contained in the HDL fraction. The biologic activity of HMG-CoA reductase assayed in hepatic microsomal homogenates was significantly reduced for mice in groups CH (p < 0.01), HF (p < 0.01), and PC (p < 0.05), but not for mice in group P, when compared with control. A statistical analysis of the data demonstrated significant (p < 0.05) reductions in the HMG-CoA reductase mRNA levels in hepatic tissue for all treatment groups relative to mRNA levels determined for mice in group C. In contrast, no treatment group demonstrated a significant difference in hepatic LPL mRNA levels when compared with mRNA levels determined for control animals. These data demonstrate that P-407 administration to C57BL/6 mice significantly decreased the amount of HMG-CoA reductase mRNA detected in liver.

Evaluation of a novel, natural oligosaccharide gum as a sustained-release and mucoadhesive component of calcitonin buccal tablets.

The objective of this study was to evaluate the gum from Hakea gibbosa (hakea) as a sustained-release and mucoadhesive component in buccal tablets for a model peptide, namely, salmon calcitonin. Flat-faced core tablets containing either 12 or 32 mg of hakea and 40 microg (200 IU) of salmon calcitonin (sCT) per tablet were formulated using a direct compression technique and were coated with Cutina on all but one face. The in vitro release profiles were sigmoidal in nature and according to a mathematical model indicated super Case II transport as the primary mechanism of release. The resulting plasma sCT and calcium concentrations were determined following both intravenous administration and buccal application of mucoadhesive tablets in rabbits. Following intravenous administration, the mean values determined for t(1/2) (alpha), t(1/2) (beta), V(d), and CL for sCT were 0.76 +/- 0.06 min, 67 +/- 18 min, 1484 +/- 454 mL/kg, and 19 +/- 2 mL/min.kg, respectively. Following the application of the mucoadhesive buccal tablets which contained 40 microg of sCT and either 12 or 32 mg of hakea, the calculated apparent bioavailability (F) and clearance (CL) were 37 +/- 6% and 19 +/- 3.3 mL/min.kg and 16 +/- 8% and 18 +/- 0.4 mL/min. kg, respectively. Serum calcium concentrations indicated that biologically active sCT was delivered across the rabbit buccal mucosa. The strength of mucoadhesion of the tablets was also quantitated in terms of the force of detachment as a function of time. The force of detachment for the mucoadhesive buccal tablets containing either 12 or 32 mg of hakea and 40 microg of sCT increased from 4.47 +/- 0.68 to 8.41 +/- 1.0 N and 8.23 +/- 1.62 to 14.98 +/- 1.63 N, respectively, from 5 to 90 min following application to excised rabbit intestinal mucosa. These results demonstrate that the novel, natural gum from Hakea gibbosa may be used to sustain the release of sCT from a unidirectional-release buccal tablet. The mechanism of in vitro release is likely to involve peptide diffusion/polymer dissolution. The mucoadhesive strength, as measured by the force of detachment, can be modulated by altering the amount of hakea in the tablet. The mucoadhesive buccal tablets described in this paper represent an improved transbuccal delivery system for therapeutic polypeptides.

Transmucosal sustained-delivery of chlorpheniramine maleate in rabbits using a novel, natural mucoadhesive gum as an excipient in buccal tablets.

The objective of this study was to evaluate the gum from Hakea gibbosa (Hakea) as a sustained-release and mucoadhesive component in buccal tablets following their application to the buccal mucosa of rabbits. Flat-faced core tablets containing either 22 or 32 mg of Hakea and 40 or 25 mg of chlorpheniramine maleate (CPM) per tablet with either sodium bicarbonate or tartaric acid in a 1:1.5 molar ratio were formulated using a direct compression technique and were coated with Cutina(R) on all but one face. The resulting plasma CPM concentration versus time profiles were determined following buccal application of the tablets in rabbits. The strength of mucoadhesion of the tablets was also quantitated in terms of the force of detachment as a function of time. Following the application of the mucoadhesive buccal tablets, the following values for several pharmacokinetic parameters were obtained. The force of detachment for the mucoadhesive buccal tablets containing 22 mg of Hakea and either 25 and 40 mg CPM, and 32 mg Hakea and 40 mg CPM increased from 1.64+/-0.47 to 7.32+/-0.34 N, 1.67+/-0.30 to 7.21+/-0.36 N, and 2.93+/-0.73 to 7.92+/-0.60 N, respectively from 5 to 90 min following application to excised intestinal mucosa. Addition of either sodium bicarbonate or tartaric acid, as well as higher amounts of CPM, did not affect the mucoadhesive bond strength. These results demonstrate that the novel, natural gum, H. gibbosa, may not only be used to sustain the release of CPM from a unidirectional-release buccal tablet, but also demonstrate that the tablets are sufficiently mucoadhesive for clinical application. The mucoadhesive strength as measured by the force of detachment, can be modulated by altering the amount of Hakea in the tablet. The mucoadhesive buccal tablets evaluated represent an improved transbuccal delivery system for conventional drug substances.

Evaluation of the gum from Hakea gibbosa as a sustained-release and mucoadhesive component in buccal tablets.

The objective of this paper was to evaluate the mucoadhesive and sustained-release properties of the water-soluble gum obtained from Hakea gibbosa (hakea), for the formulation of buccal tablets. Flatfaced tablets containing hakea were formulated using chlorpheniramine maleate (CPM) as a model drug. Two types of tablets were prepared: uncoated tablets (type I) and tablets in which all but one face of the type I tablet was coated with hydrogenated castor oil (Cutina) using a compression coating technique (type II). In an attempt to explain the observed sustained-release effect, the potential for a chemical interaction between hakea and CPM was evaluated by FTIR, differential scanning calorimetry (DSC), UV spectroscopy, and acid-base titrations. Mathematical modeling of the CPM release data was developed to elucidate the mechanism of drug release. The mucoadhesive strength was evaluated by quantitating the force of detachment. Finally, the rates of water uptake and erosion were determined for the buccal tablets. The time required for 90% of the CPM to be released in vitro (t90%) was used as a basis for comparison. For formulations that did not contain hakea, the t90% was 14 min for both directly compressed and wet granulated tablets, whereas the t90% for wet granulated tablets containing 2 or 32 mg hakea/tablet was 36 and 165 min, respectively. Directly compressed tablets containing 2, 12, 22, and 32 mg hakea/tablet displayed t90% values of 48, 120, 330, and 405 min, respectively. DSC, FTIR, UV spectroscopy and acid-base titration experiments suggested the absence of chemical interactions. The force of detachment for directly compressed and wet granulated tablets increased from 0.70 +/- 0.3 to 4.08 +/- 0.52 N and from 0.65 +/- 0.28 to 3.94 +/- 0.31 N as the amount of hakea per tablet was increased from 0 to 32 mg, respectively, at a 5 N attachment compression force. The novel natural gum, hakea, may not only be utilized to sustain the release of CPM from a unidirectional-release buccal tablet, but it also exhibited excellent mucoadhesive properties. The mechanism by which CPM release was sustained was more likely due to slow relaxation of the hydrated hakea. The mucoadhesive strength can be modulated by altering the amount of hakea in the tablet.

The effects of various excipients on the unfolding of basic fibroblast growth factor.

The purpose of this study was to determine the effects of various pharmaceutical additives on temperature and chaotrope-induced (guanidine HCl = GnHCl) denaturation of basic fibroblast growth factor (bFGF). The effect of various pharmaceutical additives on the stability (tertiary structure) of bFGF was assessed using fluoresence spectroscopy. An increase in the hydrophobicity of anionic surfactants (alkyl sulfonates) incubated with bFGF were shown to retard thermal-induced denaturation of bFGF. Increasing the concentration of decane sulfonate in an aqueous bFGF solution decreased the rate and extent of GnHCl-induced denaturation and demonstrated no inherent capacity to significantly alter the conformation of bFGF by itself. Structural differences in surfactants (e.g., aromatic, heterocyclics and straight-chain alkyl compounds) resulted in differences in the rate and extent of thermal-induced denaturation of bFGF. An increase in the concentration of citrate buffer increased the rate and extent of thermal-induced denaturation of bFGF. Results suggest that various excipients may affect the rate and extent of bFGF unfolding induced by chemical or thermal stress. Ionic charge and the degree of lipophilicity of an additive may significantly affect the rate and extent of the unfolding process.

Permeation of unfolded basic fibroblast growth factor (bFGF) across rabbit buccal mucosa--does unfolding of bFGF enhance transport?

PURPOSE: To investigate whether recombinant human basic fibroblast growth factor (rhbFGF) would permeate freshly-excised rabbit buccal mucosa. In addition, the effect of a permeation enhancer (Na+ glycocholate) and the possibility of reversibly unfolding the globular protein to a more linear conformation to increase the permeability of the test protein was evaluated. METHODS: The in vitro flux of bFGF through freshly-excised rabbit buccal mucosa was determined using side-by-side diffusion systems. Detection of bFGF was performed using gradient elution, reversed-phase high-pressure liquid chromatography (RP-HPLC). Fluorescence spectroscopy and heparin affinity chromatography were used to assess the tertiary structure of bFGF. RESULTS: Preliminary in vitro results have demonstrated that the bFGF flux increased from 1.4 +/- 0.13 ng min-1 cm-2 to 3.2 +/- 0.38 ng min-1 cm-2 with the addition of 15 mM Na+ glycocholate (NaG) to the donor solution. Subsequent addition of guanidine HCl (GnHCl) to the donor solution (3 M) was not followed by a further increase in the flux of bFGF (2.9 +/- 0.26 ng min-1 cm-2). However, when the order of addition of the additives was reversed (GnHCl first followed by NaG), the flux of bFGF across rabbit buccal mucosa was increased. Upon addition of GnHCl, there was a significant (p < .05) increase in bFGF flux from 1.2 +/- 0.15 ng min-1 cm-2 to 5.0 +/- 0.58 ng min-1 cm-2. Addition of NaG further increased the flux to 8.5 +/- 1.1 ng min-1 cm-2 which was approximately 3- to 3.5-fold greater than that determined with the protein alone in the absence of any donor phase additives. The percent of parent bFGF remaining following a 3-hr exposure of a bFGF solution to either the mucosal, serosal, or both sides of rabbit buccal mucosa were 54.3 +/- 5.7%, 71.8 +/- 6.3%, and 36.2 +/- 5.4%, respectively with the majority of parent bFGF lost during the first 15 minutes. A model endopeptidase (endoproteinase Arg-C from mouse submaxillary gland) was shown in vitro to contribute to the loss in parent bFGF. CONCLUSIONS: The permeation of bFGF across rabbit buccal mucosa may be significantly increased by initially unfolding the protein with GnHCl and then treating the tissue with the permeation enhancer, NaG. Refolding and possible reactivation of bFGF's bioactivity may occur following membrane transport and subsequent dilution into an infinite sink.

Poloxamer 407-induced atherogenesis in the C57BL/6 mouse.

Poloxamer 407 (P-407) induces hyperlipidemia in the rat. It was the purpose of this investigation to determine if chronic P-407 administration would produce atherogenic arterial lesions in the C57BL/6 mouse, a strain reported to be susceptible to hyperlipidemia-induced atherosclerotic plaque formation. One injection (i.p.) of P-407 (0.5g/kg) produced hypercholesterolemia in the mouse that peaked at 24 h and returned to control levels by 96 h following treatment. Four groups of mice were maintained: (1) saline injected (C); (2) P-407-injected (0.5g/kg every 3rd day) (P); (3) P-407 injected plus cholic acid in the diet (PC); and (4) mice fed a high cholesterol (CHOL) diet containing cholic acid (HF). Mice from each group were sacrificed following 90, 145, 200, or 300 days of treatment. Plasma lipid concentrations, hepatic CHOL concentrations (145 and 300 day), and aortic atherogenic lesion areas were measured. Plasma CHOL and triglyceride remained at control levels throughout the 300 days in the C group. CHOL of the HF animals plateaued at approximately 225 mg/dl. P-407 produced CHOL concentrations of 600 mg/dl in P mice and 1000-1500 mg/dl in PC animals. There was no lesion formation in C mice. However, by 90 days lesions were present in the three other groups. Size of the lesions progressed through day 300 with the largest lesions (184.33 + 27.99 mu2 x 10(-3)) being present in the PC mice. HF and P animals had lesions of 70.50 + 11.35 and 43.33 + 7.88 mu2 x 10(-3), respectively. This study provides an animal model where atherogenesis has been produced with hyperlipidemia induced using a chemical agent.

Evaluation of a mucoadhesive buccal patch for delivery of peptides: in vitro screening of bioadhesion.

We have assessed the bioadhesive properties of several different mucoadhesive buccal patches. The patches consisted of custom coformulations of silicone polymers and Carbopol 974P. The contact angle of water was measured for each of the test formulations, using an ophthalmic shadow scope. The corresponding work of adhesion between the water and the patches (W1), and between the patches and freshly-excised rabbit buccal mucosa (W2) was then calculated, using a modification of Dupre's equation. The bioadhesive strength between the patches and excised rabbit buccal mucosa was also assessed. The results of the contact-angle measurements indicated that the contact angle decreased with an increase in the amount of Carbopol in the formulation. Additionally, the calculated values of both W1 and W2 increased with an increase in the amount of Carbopol in the buccal-patch formulations. A correlation (r not equal to 0.9808) was found between the measured contact angle and the calculated values for W2. The direct measurement of the force required to separate a buccal patch from excised rabbit buccal mucosa with the INSTRON demonstrated that the adhesive strength increased with an increase in the amount of Carbopol. This preliminary study has shown that the measurement of contact angles alone may provide a useful technique for estimating the work of adhesion, and may serve as a convenient and rapid screening procedure to identify potential mucoadhesive buccal-patch formulations.