The bile acid binding and hypocholesterolemic action of two water-soluble polymers.

The in vitro bile acid binding properties of 2 water-soluble, linear, cationic resins, poly-[(dimethylimino)trimethylene chloride] or 3,3-ione C1, and poly-diallyldimethylammonium chloride) or CAT-FLOC were determined. Both polymers were substantially more active than cholestyramine. All were compared for hypocholesterolemic effect in normo-cholesterolemic dogs. CAT-FLOC and 3,3-ionene C1, administered at 1.8 and 1.2 g/day, respectively, exhibited cholesterol-lowering action equivalent to cholesteryramine given at 12 g/day. The results of this study suggest that effective reduction of plasma cholesterol may be achieved with significantly lower doses of bile acid sequestrants.

The effects of mevinolin on serum cholesterol levels of rabbits with endogenous hypercholesterolemia.

Mevinolin, a fungal metabolite isolated from cultures of Aspergillus terreus, is a potent competitive inhibitor of 3-hydroxy-3-methyl glutaryl coenzyme A reductase, the rate-controlling enzyme in cholesterol biosynthesis. In the current studies we demonstrate that mevinolin significantly lowers serum cholesterol in rabbits fed a cholesterol free, low-fat semi-synthetic diet. Rabbits maintained on this diet developed endogenous hypercholesterolemia with average cholesterol concentrations of 310 mg/dl over a 66-day period. Treatment with mevinolin for 39 days at a dose of 2 mg/kg per day lowered serum cholesterol levels by an average of 37% (P less than 0.05), while a dose of 6 mg/kg per day resulted in a 48% (P less than 0.05) decrease when compared with the control group. When the administration of mevinolin was discontinued, serum cholesterol levels of the 6 mg/kg per day group increased significantly to a maximum post-treatment value of 319 mg/dl (P less than 0.0001). The results of this study demonstrate that rabbits with endogenous hypercholesterolemia are a useful animal model for the study of cholesterol biosynthesis inhibitors like mevinolin.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 5. 6-(Fluoren-9-yl)- and 6-(fluoren-9-ylidenyl)-3,5-dihydroxyhexanoic acids and their lactone derivatives.

A limited study was conducted to determine the biological consequences of rendering the phenyl rings of the previously reported 7-(3,5-disubstituted [1,1'-biphenyl]-2-yl)-3,5-dihydroxy-6-heptenoic acids coplanar. Such constraint substantially diminished intrinsic HMG-CoA reductase inhibitory activity.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 6. Trans-6-[2-(substituted-1-naphthyl)ethyl(or ethenyl)]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-ones.

A variety of trans-6-[2-(substituted-1-naphthyl)ethyl(or ethenyl)]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-ones were prepared and, upon conversion to their 3,5-dihydroxy carboxylates, were found to have good inhibitory activity against the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-determining enzyme in cholesterogenesis. The most active compounds are 2,4,6- and 2,4,7-trichloro derivatives and would be expected to display about the same potency as the standard compactin upon resolution.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 2. Structural modification of 7-(substituted aryl)-3,5-dihydroxy-6-heptenoic acids and their lactone derivatives.

A series of 7-(substituted aryl)-3,5-dihydroxy-6-heptenoic (heptanoic) acids and their lactone derivatives have been prepared and tested for inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in vitro. A systematic exploration of the structure-activity relationships in this series led to the synthesis of (+)-trans-(E)-6-[2-[2,4-dichloro-6-[(4-fluorophenyl) methoxyl]phenyl]ethyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (66(+)), which has one-half of the inhibitory activity of compactin.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 3. 7-(3,5-Disubstituted [1,1'-biphenyl]-2-yl)-3,5-dihydroxy-6-heptenoic acids and their lactone derivatives.

The syntheses of a series of 7-(3,5-disubstituted [1,1'-bephenyl]-2-yl)-3,5-dihydroxy-6-heptenoic acids and their lactones are reported. Intrinsic 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitory activity is enhanced markedly when the biphenyl moiety is substituted by chloro or methyl groups at positions 3 and 5 and a fluoro group at position 4'. These substitutions, followed by resolution, provided compounds 100(+) and 110(+) with 2.8 times the intrinsic inhibitory activity of compactin. Compound 100(+) was shown to possess the same chirality in the lactone ring as compactin by single-crystal X-ray crystallography.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 1. Structural modification of 5-substituted 3,5-dihydroxypentanoic acids and their lactone derivatives.

A series of 5-substituted 3,5-dihydroxypentanoic acids and their derivatives have been prepared and tested for inhibition of HMG-CoA reductase in vitro. In general, unless a carboxylate anion can be formed and the hydroxy groups remain unsubstituted in an erythro relationship, inhibitory activity is greatly reduced. Furthermore, only one enantiomer of the ring-opened form of lactone 6a(+/-) possesses the activity displayed by the racemate. Insertion of a bridging unit other than ethyl or (E)-ethenyl between the 5-carbinol moiety and an appropriate lipophilic moiety (e.g., 2,4-dichlorophenyl) attenuates activity.

Contact lens-induced edema in vitro. Pharmacology and metabolic considerations.

The effects of physiologic and pharmacologic manipulations on contact lens-induced edema were studied. In isolated superfused rabbit corneas bathed in Ringer's solution and covered with large-diameter polymethylmethacrylate (PMMA) lenses, corneal swelling rates of 17-26 microns/hr (versus -5-5 microns/hr in paired controls) were observed. Neither the calcium antagonist diltiazem (10(-4) M), the glucocorticoid dexamethasone (10(-7) M), the glucose substitute fructose (20 mM), nor 0.5 mM adenosine and 0.3 mM reduced glutathione mitigated the edema. Lens-induced edema was 25 microns/hr in corneas bathed at pH 8.2 and decreased to 9 microns/hr at pH 7.0. In corneas without lenses, however, decreasing the pH from 7.4-7.0 caused significant swelling (P less than 0.05). The pyruvate dehydrogenase stimulant sodium dichloroacetate (3.2 mM) on the tears side ameliorated the edema, and its congener, 3.2 mM 2-chloropropionate, was less effective. These latter agents are known to relieve lactic acidosis systemically and had no significant effect on corneas without lenses. In tissues bathed with 20 mM lactate Ringer's, normal thickness was maintained in both control and PMMA-treated corneas throughout the 3-hr period. These findings suggest that the contact lens-induced edema does not involve the acute cytotoxic mechanisms seen in severe tissue ischemia or hypoxia. The edema appears to result in part from acidosis but mainly from stromal lactate accumulation.

Effects of sodium lactate on isolated rabbit corneas.

Corneal stromal lactate accumulation may result from epithelial hypoxia and contact lens wear, but the possible corneal toxicity of lactate has not been reported. Isolated superfused whole rabbit corneas were examined for thickness changes during exposure to neutral sodium lactate (NaL) or excess sodium chloride (NaCl) in Krebs-bicarbonate Ringer's solution for a 3-hr period. Placed in the tears side bath, 5 mM NaL significantly thinned corneas (swelling rates of 1 +/- 1 micron/hr in Ringer's controls vs -11 +/- 1 micron/hr in lactate-treated corneas; mean +/- SD). Excesses of 5 mM NaCl had essentially identical effects (0 +/- 1 micron/hr in controls vs -13 +/- 3 micron/hr in experimentals). When placed on the aqueous side of normal-thickness corneas, neither 20 mM NaL nor 20 mM excess NaCl affected corneal thickness, but both solutions stimulated endothelium-mediated deswelling in preswollen deepithelialized corneas. When "loaded" into the stroma of deepithelialized corneas, Ringer containing 20 mM lactate caused more swelling than Ringer's alone (491 +/- 18 microns in controls vs 558 +/- 20 microns in loaded corneas; mean +/- SEM). A similar swelling occurred when 20 mM excess NaCl was loaded into the stroma (483 +/- 15 vs 565 +/- 20 microns in controls and loaded corneas, respectively), due to fluid uptake into the hypertonic stroma across the endothelium from the aqueous side (Ringer's) bath. Corneas both loaded and superfused with either NaL or excess NaCl swelled and subsequently deswelled similar to controls swollen and superfused in Ringer's.(ABSTRACT TRUNCATED AT 250 WORDS)

Contact lens-induced edema in vitro. Ion transport and metabolic considerations.

The relationship of contact lens-induced edema to epithelial and endothelial function was determined in isolated superfused rabbit corneas. Placement of a polymethyl methacrylate (PMMA) contact lens on the cornea caused swelling rates of 15-28 microns/hr compared to 0-6 microns/hr in paired control corneas. The edema increased with temperature (P less than 0.01). PMMA-induced swelling was significant in: 1) bicarbonate-free Ringer's solution; 2) chloride-free Ringer's; 3) 0.3 mM furosemide-treated corneas; and 4) deepithelialized corneas. The swelling did not occur in corneas with silicone oil replacing the endothelium to block fluid uptake. The effluent aqueous bathing fluid from edematous corneas did not induce edema in normoxic corneas. These studies demonstrate that contact lens-induced edema depends on metabolism, involves a significant stromal contribution, and requires fluid absorption across the endothelial layer, but is not a direct result of epithelial and endothelial ion transport inhibition.

New cryoprotectant for cryorefractive surgery.

Cryorefractive surgeries, keratomileusis, keratophakia, and epikeratophakia cause destruction of keratocytes, which may result in postoperative corneal haze. We examined the effects of two cryoprotectants on keratocyte survival following freeze injury. We compared the ability of CPTES and the standard cryoprotectant KM-26 to prevent keratocyte death by altering the length of time corneal tissue was exposed to the cryoprotectant. When corneal stroma was immersed in CPTES for five minutes prior to freezing, 66.5% of the keratocytes survived; when tissue was immersed in KM-26 for the same length of time, 27.5% survived (P less than .01). Immersion for one to 30 minutes in CPTES prior to freezing produced keratocyte viabilities that were 40% to 80% of those of fresh, unfrozen tissue; immersion in KM-26 produced keratocyte viabilities of 20% to 60%. We compared the ability of these cryoprotectants to reduce corneal haze following freeze injury using our rabbit model of lamellar keratoplasty. The postoperative data were comparable to those in the cell culture experiments. Based on our findings in rabbit corneas, a cryoprotective medium such as CPTES may promote cell survival and thereby speed recovery from cryorefractive procedures in humans.

Long-term storage of frozen lenticules for cryorefractive surgery.

We studied freezing rates, cryoprotectants, and storage times on keratocyte viability, using rabbit corneal buttons incubated in either KM-26, CPTES, K-Sol, or TC 199 for 30 minutes at 4 degrees C. Using a controlled freezing rate (2 degrees/min to -40 degrees C), viabilities were 69 +/- 7% for KM-26, 113 +/- 21% for CPTES, 0.1 +/- 0.08% for K-Sol, and 0 +/- 0% for TC 199. The KM-26 and CPTES were further studied in corneas stored one to 30 days at -80 or -196 degrees C; CPTES had a better cryoprotective efficacy over one, three, and seven days of -80 degrees storage, and at liquid nitrogen storage temperature (-196 degrees) over one, 14, and 30 days storage. The findings demonstrate the superiority of CPTES. It provides better viability than KM-26 under similar conditions, and may enable long-term frozen storage of lenticules for later use in cryorefractive surgeries, with minimal loss of keratocyte viability.

Contact lens-induced edema in vitro--amelioration by lactate dehydrogenase inhibitors.

Isolated rabbit corneas bathed in Krebs-bicarbonate Ringer solution were observed for thickness changes after a 90 minute equilibration period. Control corneas swelled an average of 0.5 micron/hr, and placement of a polymethylmechacrylate (PMMA) contact lens on the epithelial surface caused the corneas to swell 24.5 microns/hr, an effect similar to 0.5 mM epithelial cyanide exposure. The pronounced swelling induced by PMMA lens placement was much less however, in the epithelial presence of 3.2 mM sodium oxalate (3.22 microns/hr) or 3.2 mM sodium oxamate (5.38 microns/hr). An equiosmotic excess of 4.8 mM NaCl was least active (15.89 microns/hr). On normal isolated corneas (without contact lenses), the Ringer containing an excess of 4.8 mM NaCl significantly deswelled the corneas (-13.44 microns/hr), which contrasted with oxalate and oxamate containing Ringer solutions (1.17 and 1.33 micron/hr respectively). The present study supports the notion that contact lens-induced edema results from stromal lactate accumulation, and suggests a potential alternative to osmotic therapy for its amelioration. These LDH inhibitors, in the concentrations used, have no acute osmotic or toxic effect on normal corneas in vitro.

Demonstration of active sodium transport across the isolated rabbit corneal endothelium.

The recent demonstration of sodium-sensitivity and chloride-insensitivity of the bicarbonate transport system of the rabbit corneal endothelium promoted a further examination of sodium fluxes across this membrane. Unidirectional fluxes were measured on paired endothelia at steady state. A net sodium flux from stroma to aqueous was found with a value of 3.5 muequiv/cm2/hr (almost double that of bicarbonate transport), which was independent of the ambient bicarbonate concentration.

Proton NMR relaxation in hydrogel contact lenses: correlation with in vivo lens dehydration data.

A study of hydrogel contact lenses was undertaken to determine whether NMR relaxation data can be used as a predictor for on-eye lens dehydration. Proton NMR relaxation times (T1 and T2), were determined for a series of contact lenses for which on-eye dehydration data were also available. NMR relaxation times were found to depend upon lens water content, but the dependence was not monotonic. T1 values varied between 100 and 800 msec, and T2 values varied between 6 and 85 msec for the lenses studied. In this study, the NMR signal and corresponding relaxation times are average values, derived both from lens water protons as well as from exchangeable polymer protons. A simple analysis of the data indicates that the mobility of these protons varies by more than a factor of 10 for the lenses studied. A test for linear correlation between NMR relaxation rate, 1/T1 and relative change in lens water mass, % delta mw gave r = -0.830 for all data, and r = 0.904 if one lens was excluded.

Pharmacological modification of rabbit corneal endothelial transport and permeability.

Either isolated normal or preswollen rabbit corneas were perfused across their endothelial surface with various drugs in the specular microscope. The deswelling rate of preswollen corneas was uninfluenced by calmodulin (1 and 10 micrograms/ml), phenylephrine (0.1 and 1 mM), DbcAMP (10(-4) M), theophylline (10(-3) and 10(-4) M), isoproterenol (2 x 10(-7) and 2 x 10(-6) M), or cyclic GMP (10(-4) and 10(-6) M). The swelling rate of normal thickness corneas was increased by furosemide (10(-5) and 10(-4) M) and thiocyanate (5 x 10(-2) and 5 x 10(-3) M) but not by SITS (10(-4) M), dipyridamole (5 x 10(-5) M) or NAP-taurine (10(-3) M). The results suggest that alteration of endothelial cyclic AMP or cyclic GMP levels has no influence on transendothelial fluid flow. Modulation of the metabolic processes underlying anion fluxes by furosemide and thiocyanate caused corneal swelling but none of the agents which affect passive anion exchange in other systems influenced transendothelial fluid movement. The furosemide and thiocyanate effects confirm that bicarbonate, or chloride, exchange may be important in part in the regulation of corneal dehydration by the endothelium.