[(2R)-2,3-di(hexadecanoyloxy)propyl] 2-(trimethylazaniumyl)ethyl phosphate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: FIG. 24. Phase contrast photomicrographs of the ethanol-induced DPPC interdigitated sheets and DPPC IFVs. A. Photomicrographs (400×) of ethanol-induced DPPC interdigitated sheets formed at 20 mg/ml (3.0M ethanol, 150 mM NaCl, 10 mM Tris-HCl, pH 7.2, room temperature). The sheet suspension was then disrupted by a five-fold dilution with 3.0M ethanol NaCl/Tris buffer. B. Photomicrograph (400×) of 20 mg/ml DPPC IFVs in 3.0M ethanol NaCl/Tris buffer at room temperature. The IFVs were formed from ethanol/DPPC interdigitated sheets by raising the sample temperature above the Tm of the DPPC. The scale bars in both micrographs indicate 10 microns. at room temperature

Conditions: temp:45.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Liposomes were formed by the reversed-phase evaporation method, as described in Szoka, et al., Biochim. Biophys. Acta, 601 (1980) 559, and O'Connell, et al., Anal. Chem., 31 (1985) 142, the disclosures of which are hereby incorporated by reference, from a mixture of DPPC, cholesterol, DPPG, and Alachlor-DPPE conjugate in a molar ration of 5:5:0.5:0.01. Forty-three μmol of this mixture were dissolved in 4.2 ml of a solvent mixture containing chloroform-isopropyl ether-methanol (6:6:1,v/v). This solution was warmed to 45° C. and 0.7 ml of the dye solution was added with swirling. This mixture was sonicated for 5 minutes under a low flow of nitrogen. The organic phase was removed under vacuum on a rotary evaporator at 40° until all frothing had stopped. An additional 1.3 ml aliquot of the dye solution was added, and the liposomes were then sequentially extruded twice through each of two polycarbonate filters of decreasing pore sizes of 1.0 μm and 0.4 μm. The diameters of the liposome preparations were measured by laser scattering in a LA-900 particle size distribution analyzer (Horiba, Irvine, Calif.), using the manufacturers method, except that the usual sonication step was omitted to avoid lysis (rupture) of the liposomes. Finally, to remove any unencapsulated dye, the liposomes were gel filtered on a 1×14 cm Sephadex G-50 column and dialyzed overnight against TBS at 4° C. When stored at 4° C., there was no significant leakage of dye over a period of 9 months as described below. Liposomes were formed by the reversed-phase evaporation method This mixture was sonicated for 5 minutes under a low flow of nitrogen The organic phase was removed under vacuum on a rotary evaporator at 40° until all frothing An additional 1.3 ml aliquot of the dye solution was added except that the usual sonication step Finally, to remove any unencapsulated dye filtered on a 1×14 cm Sephadex G-50 column dialyzed overnight against TBS at 4° C stored at 4° C. of 9 months

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: FIG. 24. Phase contrast photomicrographs of the ethanol-induced DPPC interdigitated sheets and DPPC IFVs. A. Photomicrographs (400×) of ethanol-induced DPPC interdigitated sheets formed at 20 mg/ml (3.0M ethanol, 150 mM NaCl, 10 mM Tris-HCl, pH 7.2, room temperature). The sheet suspension was then disrupted by a five-fold dilution with 3.0M ethanol NaCl/Tris buffer. B. Photomicrograph (400×) of 20 mg/ml DPPC IFVs in 3.0M ethanol NaCl/Tris buffer at room temperature. The IFVs were formed from ethanol/DPPC interdigitated sheets by raising the sample temperature above the Tm of the DPPC. The scale bars in both micrographs indicate 10 microns. at room temperature

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Effect of all-trans Retinoic acid (atRA), 13-cis-Retinoic acid (13cRA) and N-(4-hydroxyphenyl)retinamide (4-HPR) on IMH: To 1 mL of buffered suspension of RPE membranes (100 mM Tris pH 8.0, 76.7 μg of protein) was added 60 μM or 6 μm of atRA, 13cRA or 4-HPR and incubated at room temp. for 15 min. A control reaction mixture without any inhibitor was also incubated at room temperature for 15 min. At the end of the 15 min incubation, all-trans-retinol [11-12-3H2] (0.2 μM) was added to the reaction mixtures (100 mM Tris pH 8.0, 76.7 μg of RPE protein, 0.2% BSA 100 μM of DPPC, 1 mM of DTT and 0.2 μM all-trans-retinol [11-12-3H2]) and incubated at room temperature for 30 min. At the end of this 30 minutes of incubation, an aliquot of the reactions were quenched to verify the equal addition of all-trans-retinol [11,12-3H2] and the effect of these inhibitors on LRAT. After this the control reaction mixture was incubated with atRA (60 & 6 μM), 13cRA (60 & 6 μM) or 4-HPR (60 & 6 μM) for 15 min. Now all the reaction mixtures were incubated with 30 μM of apo-rCRALBP (100 mM Tris pH 8.0, 7.7 μg of RPE protein, 0.2% BSA 100 μM of DPPC, 1 mM of DTT 30 μM apo-rCRALBP and 0.2 μM all-trans-retinol [11-12-3H2]) at 37° C. for 30 minutes. At the end of this incubation period the 200 μL reaction mixture was quenched by the addition of 750 μL ice cold methanol after which 100 μL of 1M sodium chloride solution was added, and 500 μl hexane (containing butylated hydroxy toluene at 1 mg/mL) was added to effect extraction of the retinoids. The retinoids were analyzed as previously described (27). The amount of 11-cis-retinol formed was used as a measurement of IMH activity. All experiments were performed in triplicate and the average values of these measurements were used for analysis. A control reaction mixture without any inhibitor was also incubated at room temperature for 15 min incubated at room temperature for 30 min At the end of this 30 minutes of incubation, an aliquot of the reactions were quenched the equal addition of all-trans-retinol [11,12-3H2] After this the control reaction mixture Now all the reaction mixtures At the end of this incubation period the 200 μL reaction mixture was quenched by the addition of 750 μL ice cold methanol after which 100 μL of 1M sodium chloride solution was added 500 μl hexane (containing butylated hydroxy toluene at 1 mg/mL) was added to effect extraction of the retinoids

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Isomeric preference of the mRPE65/tLRAT mediated esterification of retinols: The effect of mRPE65 on the processing of 11-cis-and all-trans-retinols was determined by monitoring the formation of tLRAT catalyzed retinyl esters from added all-trans-retinol [11,12-3H2], 11-cis-retinol [15-3H] and mRPE65. The reaction mixture (volume 0.1 mL) contains 100 mM Tris (pH 8.4), 5 μM of tLRAT 0.3% CHAPS, 1 mM dithiothreitol and 0.2 μm of all-trans-retinol [11,12-3H2] or 11-cis-retinol [15-3H] and mRPE65 (0.02 μM) or 200 μM/0.4% DPPC/BSA was incubated for 10 min at room temperature. After 10 min the reaction was quenched with 500 μL methanol, 100 μL of water and 500 μL of hexane. The amount of retinyl palmitate formed, as determined by normal phase-HPLC, was used as a measure of activity. Each experiment was done in triplicate, and the data points used are an average of these three points. The standard error was presented as error bars. After 10 min the reaction was quenched with 500 μL methanol, 100 μL of water and 500 μL of hexane

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Effect of all-trans Retinoic acid (atRA), 13-cis-Retinoic acid (13cRA) and N-(4-hydroxyphenyl)retinamide (4-HPR) on IMH: To 1 mL of buffered suspension of RPE membranes (100 mM Tris pH 8.0, 76.7 μg of protein) was added 60 μM or 6 μM of atRA, 13cRA or 4-HPR and incubated at room temp. for 15 min. A control reaction mixture without any inhibitor was also incubated at room temperature for 15 min. At the end of the 15 min incubation, all-trans-retinol [11-12-3H2] (0.2 μM) was added to the reaction mixtures (100 mM Tris pH 8.0, 76.7 μg of RPE protein, 0.2% BSA 100 μM of DPPC, 1 mM of DTT and 0.2 μM all-trans-retinol [11-12-3H2]) and incubated at room temperature for 30 min. At the end of this 30 minutes of incubation, an aliquot of the reactions were quenched to verify the equal addition of all-trans-retinol [11,12-3H2] and the effect of these inhibitors on LRAT. After this the control reaction mixture was incubated with atRA (60 & 6 μM), 13cRA (60 & 6 μM) or 4-HPR (60 & 6 μM) for 15 min. Now all the reaction mixtures were incubated with 30 μM of apo-rCRALBP (100 mM Tris pH 8.0, 7.7 μg of RPE protein, 0.2% BSA 100 μm of DPPC, 1 mM of DTT 30 μM apo-rCRALBP and 0.2 μM all-trans-retinol [11-12-3H2]) at 37° C. for 30 minutes. At the end of this incubation period the 200 μL reaction mixture was quenched by the addition of 750 μL ice cold methanol after which 100 μL of 1M sodium chloride solution was added, and 500 μl hexane (containing butylated hydroxy toluene at 1 mg/mL) was added to effect extraction of the retinoids. The retinoids were analyzed as previously described (27). The amount of 11-cis-retinol formed was used as a measurement of IMH activity. All experiments were performed in triplicate and the average values of these measurements were used for analysis. A control reaction mixture without any inhibitor was also incubated at room temperature for 15 min incubated at room temperature for 30 min At the end of this 30 minutes of incubation, an aliquot of the reactions were quenched the equal addition of all-trans-retinol [11,12-3H2] After this the control reaction mixture Now all the reaction mixtures At the end of this incubation period the 200 μL reaction mixture was quenched by the addition of 750 μL ice cold methanol after which 100 μL of 1M sodium chloride solution was added 500 μl hexane (containing butylated hydroxy toluene at 1 mg/mL) was added to effect extraction of the retinoids

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The lipids (DPPC:cholesterol=55:45 mol %) were dissolved in ethanol and the solution was injected into a sildenafil/ammonium sulphate solution (pH 3.5-4.5), whereupon, immediately after spontaneous vesicle formation, a 5% glucose solution (pH 7), which comprised further sildenafil citrate, was added for dilution and alkalinization of the reaction mixture, i.e. of the resulting liposome suspension. As a result of the production of this H+ gradient immediately after the vesicle formation, sildenafil is not only included in the liposomes in one step but is also retained there in a stable manner. The amount of sildenafil included liposomally in this manner was likewise in a range of about 160 to 230 nmol of sildenafil per μmol of total lipids (DPPC+cholesterol)—depending on the pH or H+ gradient. was added for dilution and alkalinization of the reaction mixture, i.e. of the resulting liposome suspension

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Effect of all-trans Retinoic acid (atRA), 13-cis-Retinoic acid (13cRA) and N-(4-hydroxyphenyl)retinamide (4-HPR) on IMH: To 1 mL of buffered suspension of RPE membranes (100 mM Tris pH 8.0, 76.7 μg of protein) was added 60 μM or 6 μm of atRA, 13cRA or 4-HPR and incubated at room temp. for 15 min. A control reaction mixture without any inhibitor was also incubated at room temperature for 15 min. At the end of the 15 min incubation, all-trans-retinol [11-12-3H2] (0.2 μM) was added to the reaction mixtures (100 mM Tris pH 8.0, 76.7 μg of RPE protein, 0.2% BSA 100 μM of DPPC, 1 mM of DTT and 0.2 μM all-trans-retinol [11-12-3H2]) and incubated at room temperature for 30 min. At the end of this 30 minutes of incubation, an aliquot of the reactions were quenched to verify the equal addition of all-trans-retinol [11,12-3H2] and the effect of these inhibitors on LRAT. After this the control reaction mixture was incubated with atRA (60 & 6 μM), 13cRA (60 & 6 μM) or 4-HPR (60 & 6 μM) for 15 min. Now all the reaction mixtures were incubated with 30 μM of apo-rCRALBP (100 mM Tris pH 8.0, 7.7 μg of RPE protein, 0.2% BSA 100 μM of DPPC, 1 mM of DTT 30 μM apo-rCRALBP and 0.2 μM all-trans-retinol [11-12-3H2]) at 37° C. for 30 minutes. At the end of this incubation period the 200 μL reaction mixture was quenched by the addition of 750 μL ice cold methanol after which 100 μL of 1M sodium chloride solution was added, and 500 μl hexane (containing butylated hydroxy toluene at 1 mg/mL) was added to effect extraction of the retinoids. The retinoids were analyzed as previously described (27). The amount of 11-cis-retinol formed was used as a measurement of IMH activity. All experiments were performed in triplicate and the average values of these measurements were used for analysis. A control reaction mixture without any inhibitor was also incubated at room temperature for 15 min incubated at room temperature for 30 min At the end of this 30 minutes of incubation, an aliquot of the reactions were quenched the equal addition of all-trans-retinol [11,12-3H2] After this the control reaction mixture Now all the reaction mixtures At the end of this incubation period the 200 μL reaction mixture was quenched by the addition of 750 μL ice cold methanol after which 100 μL of 1M sodium chloride solution was added 500 μl hexane (containing butylated hydroxy toluene at 1 mg/mL) was added to effect extraction of the retinoids

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Isomeric preference of the mRPE65/tLRAT mediated esterification of retinols: The effect of mRPE65 on the processing of 11-cis-and all-trans-retinols was determined by monitoring the formation of tLRAT catalyzed retinyl esters from added all-trans-retinol [11,12-3H2], 11-cis-retinol [15-3H] and mRPE65. The reaction mixture (volume 0.1 mL) contains 100 mM Tris (pH 8.4), 5 μM of tLRAT 0.3% CHAPS, 1 mM dithiothreitol and 0.2 μm of all-trans-retinol [11,12-3H2] or 11-cis-retinol [15-3H] and mRPE65 (0.02 μM) or 200 μM/0.4% DPPC/BSA was incubated for 10 min at room temperature. After 10 min the reaction was quenched with 500 μL methanol, 100 μL of water and 500 μL of hexane. The amount of retinyl palmitate formed, as determined by normal phase-HPLC, was used as a measure of activity. Each experiment was done in triplicate, and the data points used are an average of these three points. The standard error was presented as error bars. After 10 min the reaction was quenched with 500 μL methanol, 100 μL of water and 500 μL of hexane

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Effect of all-trans Retinoic acid (atRA), 13-cis-Retinoic acid (13cRA) and N-(4-hydroxyphenyl)retinamide (4-HPR) on IMH: To 1 mL of buffered suspension of RPE membranes (100 mM Tris pH 8.0, 76.7 μg of protein) was added 60 μM or 6 μM of atRA, 13cRA or 4-HPR and incubated at room temp. for 15 min. A control reaction mixture without any inhibitor was also incubated at room temperature for 15 min. At the end of the 15 min incubation, all-trans-retinol [11-12-3H2] (0.2 μM) was added to the reaction mixtures (100 mM Tris pH 8.0, 76.7 μg of RPE protein, 0.2% BSA 100 μM of DPPC, 1 mM of DTT and 0.2 μM all-trans-retinol [11-12-3H2]) and incubated at room temperature for 30 min. At the end of this 30 minutes of incubation, an aliquot of the reactions were quenched to verify the equal addition of all-trans-retinol [11,12-3H2] and the effect of these inhibitors on LRAT. After this the control reaction mixture was incubated with atRA (60 & 6 μM), 13cRA (60 & 6 μM) or 4-HPR (60 & 6 μM) for 15 min. Now all the reaction mixtures were incubated with 30 μM of apo-rCRALBP (100 mM Tris pH 8.0, 7.7 μg of RPE protein, 0.2% BSA 100 μm of DPPC, 1 mM of DTT 30 μM apo-rCRALBP and 0.2 μM all-trans-retinol [11-12-3H2]) at 37° C. for 30 minutes. At the end of this incubation period the 200 μL reaction mixture was quenched by the addition of 750 μL ice cold methanol after which 100 μL of 1M sodium chloride solution was added, and 500 μl hexane (containing butylated hydroxy toluene at 1 mg/mL) was added to effect extraction of the retinoids. The retinoids were analyzed as previously described (27). The amount of 11-cis-retinol formed was used as a measurement of IMH activity. All experiments were performed in triplicate and the average values of these measurements were used for analysis. A control reaction mixture without any inhibitor was also incubated at room temperature for 15 min incubated at room temperature for 30 min At the end of this 30 minutes of incubation, an aliquot of the reactions were quenched the equal addition of all-trans-retinol [11,12-3H2] After this the control reaction mixture Now all the reaction mixtures At the end of this incubation period the 200 μL reaction mixture was quenched by the addition of 750 μL ice cold methanol after which 100 μL of 1M sodium chloride solution was added 500 μl hexane (containing butylated hydroxy toluene at 1 mg/mL) was added to effect extraction of the retinoids

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The lipids (DPPC:cholesterol=55:45 mol %) were dissolved in ethanol and the solution was injected into a sildenafil/ammonium sulphate solution (pH 3.5-4.5), whereupon, immediately after spontaneous vesicle formation, a 5% glucose solution (pH 7), which comprised further sildenafil citrate, was added for dilution and alkalinization of the reaction mixture, i.e. of the resulting liposome suspension. As a result of the production of this H+ gradient immediately after the vesicle formation, sildenafil is not only included in the liposomes in one step but is also retained there in a stable manner. The amount of sildenafil included liposomally in this manner was likewise in a range of about 160 to 230 nmol of sildenafil per μmol of total lipids (DPPC+cholesterol)—depending on the pH or H+ gradient. was added for dilution and alkalinization of the reaction mixture, i.e. of the resulting liposome suspension

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Amikacin sulfate 12.0 g was dissolved in 200 mL water and pH was adjusted to 6.5 by adding necessary amounts of 25% NaOH solution. Lipids, 1.480 g DPPC and 0.520 g cholesterol, were dissolved in a mixture of 60 mL ethanol and 10 mL water. These amounts result in a 300 mL batch after infusion at a lipid/amikacin flow rate of 300/500 mL/min, respectively. Volumes can be proportionally adjusted for larger scale or if different flow rates are desired.