Reduction in antiviral activity of human interferon-gamma in acidic media with reference to structural change.

The fluorescence intensity of a unique tryptophan 36 in human interferon-gamma was drastically decreased below pH 4 with a concomitant decrease of antiviral activity. The region of residues 32-42 of human interferon-gamma was found by calculation to have a low hydrophobicity together with a high helical hydrophobic moment, and the net electric charge of this region having an amphiphilic helical structure changed significantly near pH 4. These results suggest that the region of residues 32-42 plays an important role in exhibiting antiviral activity.

Biodegradable microspheres in drug delivery.

General aspects of biodegradable microspheres prepared from natural and synthesized polymers used in drug delivery systems are reviewed first from various viewpoints: characteristics of biodegradable polymers (physicochemical properties, bioerosion mechanism, biocompatibility), preparation method for the microspheres, drug release from parenteral products and briefly nonparenteral products. The relationship between release pattern and pharmacological activity of therapeutic peptides and proteins and rational controlled release design are also discussed. In the latter half, successful sustained release depot formulations of peptides, leuprorelin acetate, and thyrotropin-releasing hormone (TRH), utilizing poly(lactic acid) (PLA) and poly(lactic/glycolic acid) (PLGA) microspheres are reviewed with respect to preparation, drug release, biocompatibility, pharmacological effects, and results of clinical studies. Thereafter, studies on antitumor therapy by chemoembolization using PLGA microspheres containing an angiogenesis inhibitor (TNP-470) are described as an example of targeted drug delivery with biodegradable microspheres.

New method for analysis of biodegradable polyesters by high-performance liquid chromatography after alkali hydrolysis.

A new method was developed for analysis of biodegradable polyesters, which involves alkali hydrolysis of polyesters to the corresponding hydroxyacids and determination of the hydroxyacids by high-performance liquid chromatography. It can be used to monitor weight change in polyesters and change in molecular ratio of the hydroxyacid constituents of polyesters during in vitro and in vivo degradation.

Formulation study of leuprorelin acetate to improve clinical performance.

Leuprorelin acetate, a highly potent gonadotrophin-releasing hormone agonist, was originally launched in the USA as a daily injection for the treatment of metastatic prostatic cancer. A once-monthly injectable depot form was subsequently developed. Biodegradable copoly(DL-lactic/glycolic) acid was chosen as the release-controlling polymer, and microspheres containing leuprorelin acetate were prepared by the in-water drying method. Results of studies in rats showed that a copolymer with a molecular weight of 14,000 and a lactic/glycolic acid ratio of 75/25 had the most satisfactory release-controlling properties. Microspheres given once monthly reduced serum testosterone levels in male rats. Microspheres also reduced serum oestradiol levels and caused a marked regression in experimental endometriosis in female rats. In clinical studies of prostatic cancer, use of the depot formulation has effectively reduced the dose required to as low as one-eighth of that needed for administration by daily injection. A sophisticated manufacturing system has now been developed and products now available have many advantages.

Sterility assurance of microspheres.

The International Conference on Harmonization (ICH) provides a forum for constructive dialogues between regulatory authorities and the pharmaceutical industry on the real and perceived differences in the technical requirements for product registration in the EU, US and Japan. Achievement obtained so far is beyond expectation, having strong impacts both favorable and unfavorable on Japanese regulatory authorities and the pharmaceutical industry. The ICH guidelines are very science-oriented and little consideration seems to have been paid to the cultural and legal differences among the three regions. An example of such a difference is the interpretation of the guidelines between the US and Japan. In the US, they are generally recognized as good examples, whereas in Japan, they are usually taken as minimum and rigid requirements. A more flexible approach to guidelines will be necessary in the development of new dosage forms in Japan. In this paper, how to assure sterility of a new dosage form, that is, once-monthly injectable and biodegradable microspheres of Leuprorelin, a super agonist of LHRH, whose pharmaceutical development the author was in charge of at Takeda Chemical Industries, will be introduced.

Membrane modification by negatively charged stearyl-polyoxyethylene derivatives for thermosensitive liposomes: reduced liposomal aggregation and avoidance of reticuloendothelial system uptake.

In order to avoid reticuloendothelial system (RES) uptake and prolong systemic circulation of cisplatin (CDDP)-encapsulating thermosensitive liposomes, stearylpolyoxyethylene (POE) derivatives [SnC, stearyl-O-(CH2CH2O)n-CH2COONa] were incorporated as membrane modifiers into lipid bilayers composed of dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC). The incorporation of S2C, S5C, S10C or S15C [lipid/SnC = 10/2 (w/w)] greatly reduced liposomal aggregation without impairing liposomal stability. After being intravenously administered to rats, the liposomes remained longer in the systemic circulation and showed lower RES levels than the control liposomes. When incorporated into liposomes [DPPC/DSPC = 7/3 (w/w)], S10C provided the greatest increase in systemic circulation time and the RES-avoiding activity among the modifiers tested. The systemic elimination rate (the ratio of the percent of the dose systemically eliminated to the AUC of the liposome level) for this type of liposome was 0.24/hr, about one fourth the rate for the control liposomes, and the RES uptake rate (the ratio of the percent of the dose taken up by the RES to the AUC) was 0.04/hr, one seventh the rate for the control liposomes. The RES uptake rate for S10C 7/3-liposomes was similar to the rate reported for GM1 liposomes, although the systemic elimination rate was double that for the GM1 liposomes. The obtained RES avoidance activity can be attributed to decreased liposomal aggregation and increased surface hydrophilicity. This type of thermosensitive liposome should be more useful in hyperthermia-mediated targeted tumor drug delivery systems than the thermosensitive liposomes without the modifiers to avoid RES uptake.

In vitro and in vivo evaluation of thyrotrophin releasing hormone release from copoly(dl-lactic/glycolic acid) microspheres.

In vitro and in vivo release of thyrotrophin releasing hormone (TRH) from copoly(dl-lactic/glycolic acid) (PLGA) microspheres were evaluated. Factors affecting the TRH release from the microspheres were examined to clarify the release mechanisms by changing the medium composition in the in vitro release test. The hydrolysis rate of PLGA, the matrix-forming substance in the microspheres, was faster in acidic medium than in neutral medium. The release rate of TRH from the PLGA microspheres increased with the increase in the degradation rate of PLGA. A decrease in an osmolarity of the medium also caused an increase in the TRH release rate even though no significant change in PLGA degradation was observed. The effect of osmolarity appears to be characteristic of water-soluble drug-containing microspheres composed of hydrophobic polymer. The release rate of TRH from PLGA microspheres was largely affected by the medium composition in the in vitro release test. A proper choice of medium was found to be important for the estimation of in vivo release. The in vivo release rate of TRH from the PLGA microspheres following administration to rats correlated with the in vitro release in pH 7, 1/30 M buffer.

In vitro cytotoxicities and in vivo distribution of transferrin-platinum(II) complex.

In vitro cytotoxic studies of protein-bound cis-diamminedichloroplatinum(II) (CDDP) against human epidermoid carcinoma A431 cells showed that transferrin (Tf)-bound CDDP (Tf-Pt, Pt/Tf 7:1 mol/mol), and human serum albumin (HSA)-bound CDDP (HSA-Pt, Pt/HSA 7:1 mol/mol) exerted antiproliferating activities with IC50 values of 7.2 and 85 microM, respectively. Tf-Pt inhibited the binding of 0.2 nM 125I-labeled human diferric transferrin (hTf(Fe)2) to A431 cells with a inhibition constant (Ki) of 42 nM, whereas HSA-Pt did not. In vivo distribution studies showed that hTf(Fe)2, the Ki of which was 5.3 nM to mouse melanoma B16 cells, was eliminated from plasma biexponentially in the B16-bearing and control mice after intravenous injection at a dose of 87 mg/kg, and AUCplasma values were 29 and 39 mg.h/mL, respectively. In the B16-bearing mice the AUCtumor was 5.6 mg.h/mL, while the AUCs of liver, kidney, and spleen were not distinguishable between the B16-bearing and control mice. Subsequently Tf-Pt (Pt/Tf 3:1 mol/mol) and free CDDP solution were administered intravenously to the B16-bearing mice. The systemic circulation of Pt was significantly prolonged by the administration of the complex. In conclusion, Tf could be a promising carrier protein for the transport of Pt to tumors.

Increased tumor cisplatin levels in heated tumors in mice after administration of thermosensitive, large unilamellar vesicles encapsulating cisplatin.

Hyperthermia (HT)-dependent cisplatin (CDDP) release and tumor CDDP level increase after the administration of thermosensitive, large unilamellar vesicles (LUVs: LUV-1 and LUV-2) and a thermosensitive, small unilamellar vesicle (SUV: SUV-1) were examined in comparison with those following administration of a non-thermosensitive LUV (LUV-3) and a CDDP solution (Sol) in tumor bearing mice. The LUV-1 and LUV-2 released CDDP at a faster rate than SUV-1 when incubated in saline at temperatures between 41 and 44 degrees C. The blood CDDP levels after liposome administration were higher than those after Sol administration. The systemic clearance of LUV-2 was slightly larger than those of the other liposomes. The tumor CDDP levels after thermosensitive liposome administration were increased in response to HT in comparison to LUV-3 or Sol. The increased ratio for LUV-1 was the largest. The ratio of the area under the tumor CDDP level versus time curve (AUC) for LUV-1 + HT to the AUC for Sol + HT was approximately 5. The results indicate that (1) the tumor-CDDP level increase after thermosensitive liposome administration is due to CDDP release from the liposome in the blood at or adjacent to the heated tumor, (2) the increase is highly dependent on the heat sensitivity and systemic stability of the liposome, and (3) LUV, such as LUV-1, exhibit higher heat sensitivity and larger, targeted drug delivery efficiency than SUV.

Controlled release of thyrotropin releasing hormone from microspheres: evaluation of release profiles and pharmacokinetics after subcutaneous administration.

The drug-release kinetics of thyrotropin releasing hormone (TRH) containing copoly(dl-lactic/glycolic acid) (PLGA) microspheres were evaluated both in vitro and in vivo. The drug was encapsulated in PLGA using an in-water drying method through a water in oil in water emulsion. The drug release from the PLGA microspheres in vitro correlated well with that in vivo, and pseudo-zero-order release kinetics were observed. The pharmacokinetics of TRH following administration of this controlled-release parenteral dosage form have been also examined in rats. Following a transient increase in the plasma level due to an initial burst, steady-state plasma levels were observed. The duration of drug release estimated from the plasma level was comparable with the results in the in vitro and in vivo release studies. The steady-state plasma levels correlated well with the levels predicted from the pharmacokinetic parameters following a single subcutaneous or intravenous injection of TRH solution. The results of this study confirm the previously reported in vivo sustained release of TRH achieved with this drug-delivery system.

pH-independent controlled-release microspheres using polyglycerol esters of fatty acids.

The release of a drug having low solubility in a certain pH range from controlled-release microspheres using tetraglycerol pentastearate and tetraglycerol monostearate in combination as the matrix base showed pH dependence. Trepibutone, an acidic drug having lower solubility in an acidic medium, was released pH-independently from the microspheres which incorporated magnesium oxide, a solid base. It might have resulted from the pH inside the matrix being kept in an optimum range for drug release due to the incorporation of a solid base. On the other hand, the addition of water soluble acidic or basic excipients was ineffective to achieve pH-independent release. For papaverine, a basic drug, pH-independent drug-release characteristics could be achieved by adding Eudragit L100-55, an enteric polymer. It is thought that the enteric polymer increased the pores for drug release by dissolving in a higher pH range, where the solubility of papaverine is low, and thereby made the release pH-independent. Further, selecting a polyglycerol ester of a fatty acid with an appropriate hydrophile-lipophile balance as the matrix could yield a drug with the desired release rate at any pH.

Anti-hypertensive effect of oral controlled-release microspheres containing an ACE inhibitor (delapril hydrochloride) in rats.

An oral controlled-release drug delivery system based on microspheres of polyglycerol esters of fatty acids (PGEFs), was applied to an anti-hypertensive, delapril hydrochloride. The in-vitro release profile was controlled by selecting a PGEF with an appropriate hydrophilic-lipophilic balance value for the matrix. The microspheres from which 80% of the drug was released in 6 h were orally administered to rats. The plasma concentration of the active metabolite was sustained after administration of the microspheres in comparison with administration of a solution. The in-vivo release profile was in good agreement with the in-vitro release profile. When the microspheres were administered, the pharmacological effect of delapril hydrochloride on the angiotensin I-induced pressor response was also sustained showing consistency with the plasma concentration-time curve.

Pharmaceutical manipulation of leuprorelin acetate to improve clinical performance.

Leuprorelin acetate, a highly potent luteinizing hormone releasing hormone agonist, was originally launched in the USA to be administered once daily by self-injection for the treatment of metastatic prostatic cancer. A once-monthly intramuscularly or subcutaneously injectable depot form of leuprorelin acetate has, subsequently, been developed. Biodegradable copoly(DL-lactic acid/glycolic acid) was chosen as the release-controlling polymer and the microcapsules containing leuprorelin acetate were prepared by an in-water drying method. Results of studies in rats showed that a copolymer with a molecular weight of 14,000 and a lactic acid/glycolic acid ratio of 75/25 had the most satisfactory releasing properties. Microcapsules given once monthly reduced serum testosterone levels in rats, dogs and man. In clinical studies, the depot preparation effectively reduced the dose of leuprorelin acetate required to up to one-eighth of that needed when injected daily. A sophisticated manufacturing system has now been developed and a very reliable controlled-release product is now available that has many advantages.

[Once-a-month injectable microcapsules of leuprorelin acetate].

Leuprorelin (leuprolide, D-Leu6-(des-Gly10-NH2)-LH-RH ethylamide) acetate is a super-active agonist of luteinizing hormone-releasing hormone (LH-RH). We developed once-a-month injectable microcapsules of this agonist by our novel in-water drying method. This depot formulation can release the drug at an apparent zero-order rate over one month with bioerosion of copoly (lactic/glycolic acid) utilized as a wall material of the polycore microcapsules. A dramatic prolonged depression of pituitary-gonadal axis, chemical castration, was achieved by the once-a-month injection in experimental animals; it expects a reliable efficacy for treating hormone-dependent prostatic, breast cancers and endometriosis. Studies on the dosage design of this new delivery system of leuprorelin are summarized.

Application of a spray drying technique in the production of TRH-containing injectable sustained-release microparticles of biodegradable polymers.

Copoly (dl-lactic/glycolic acid) microparticles for sustained release of a water-soluble drug (Thyrotropin releasing hormone: TRH) were prepared by a spray drying method. A higher entrapment ratio was achieved with the spray drying method with the in-water drying method. In order to avoid agglomeration of the microparticles, a double-nozzle spray drying method was designed using mannitol as an anti-adherent. The surface of the spray-dried microparticles was coated with mannitol, and the extent of agglomeration was decreased. Acetonitrile was the most suitable solvent for microencapsulation using the double-nozzle spray drying method because the initial burst of TRH from the microparticles during the first day was the smallest. When PLGA with a weight-average molecular weight of 14,000 was used, constant release of TRH continued for one month with a small initial burst. In conclusion, the production of biodegradable microparticles by the double-nozzle spray drying method appears to be an attractive alternative to conventional microencapsulation methods.

[Targeting therapy with a drug using temperature-sensitive liposomes entrapped antitumor drug together with localized hyperthermia].

The characteristics and feasibility of targeting therapy with combination of temperature-sensitive liposomes entrapped antitumor drug with localized hyperthermia were discussed on the theoretical as well as experimental basis. An optimum liposome form and a drug for this delivery were proposed as follows: the liposome is LUV which has a phase transition temperature of 41 to 42 degree and the osmotic pressure of the internal aqueous phase is 1.5 or higher than the physiological one, the drug has a large body clearance and has a synergistic effect with hyperthermia. The LUV preparation containing cisplatin was administered to mice together with tumor heating. The uptake of the drug in the tumor (as a criterion of targeting) were 3 to 5 times greater than those of an aqueous cisplatin solution. The tumor responses were well correlated with the drug-targeting.

Heat-induced drug release rate and maximal targeting index of thermosensitive liposome in tumor-bearing mice.

To evaluate the rate of drug release at the tumor and maximal drug targeting after administration of thermosensitive liposomes with hyperthermia, a theoretical and experimental method was derived assessing the fraction of drug released from liposomes in a single pass through the heated tumor, F, and the drug targeting index when drug release occurs completely in response to heat (F = 1), DTImax. The F and DTImax were evaluated for four types of liposomes (LUV-1 and LUV-2, thermosensitive large unilamellar liposomes; LUV-3, a nonthermosensitive large unilamellar liposome; and SUV-1, a thermosensitive small unilamellar liposome) using reported data on blood liposome levels and tumor drug levels after the liposomes were administered to tumor bearing mice. DTImax values for LUV-1 and SUV-1 were approximately 6, while the value for LUV-2 with a relatively large systemic clearance was only 2.3. The F values for LUV-1, LUV-2, and SUV-1 with hyperthermia were 0.71, 1.17, and 0.34, respectively, whereas the values for these liposomes without hyperthermia and for LUV-3 with or without hyperthermia were nearly zero. These results confirm earlier findings that LUV-1 and LUV-2 release CDDP almost completely at the heated tumor and that the large DTI value obtained in LUV-1 (DTI = 4.6) was due to its high heat sensitivity and its small systemic clearance.

Rates of systemic degradation and reticuloendothelial system (RES) uptake of thermosensitive liposome encapsulating cisplatin in rats.

The systemic degradation and reticuloendothelial system (RES) uptake of cisplatin (CDDP)-encapsulated thermosensitive liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC) (DPPC/DSPC = 9/1, 7/3, and 5/5, w/w) after intravenous administration to rats were examined by measuring the platinum (Pt) levels in the blood and RES (liver and spleen). The blood liposome level profile showed first-order rate elimination for each liposome administration. The elimination rate (Kel) was faster when the content of DSPC was lower (Kel: 1.3/hr for 9/1-liposomes, 0.7/hr for 7/3-liposomes, 0.5/hr for 5/5-liposomes). On the other hand, the RES liposome level profile showed distribution of liposomes followed by elimination therefrom. The RES level of the liposomes was lower when the content of DSPC was smaller (maximal level: 25% for 9/1-liposomes at 1 hr, 32% for 7/3-liposomes at 1 hr, 37% for 5/5-liposomes at 2 hr). The kinetic analysis demonstrated that the RES uptake rate (Kres) was almost the same among the liposomes (0.4/hr), while the systemic degradation rate (Kdeg; Kel-Kres) became larger as the content of DSPC decreased (0.9/hr for 9/1-liposomes, 0.3/hr for 7/3-liposomes, and 0.1/hr for 5/5-liposomes) and that the RES liposome distribution amount was dependent not only on the Kres but also on the Kdeg and the rate of RES liposome degradation. The Kdeg for each type of liposome corresponded with the systemic CDDP release rate.

Effects of the physicochemical properties of the emulsion formulation on the bioavailability of ethyl 2-chloro-3-[4-(2-methyl-2-phenylpropyloxy)phenyl]propionate in rats.

The effect of the physicochemical properties of the emulsion formulation on the absorption of ethyl 2-chloro-3-[4-(2-methyl-2-phenylpropyloxy)phenyl]propionate (AL-294) in rats and dogs was studied. When emulsions of different particle sizes were administered to rats, the higher the ratio of Tween-80 to the drug was, the smaller was the particle size and the higher was the absorption. When the emulsions of similar particle size (2 microns) with different Tween-80 ratios were administered to rats, no significant difference was observed in the extent of absorption. The absorption of AL-294 was correlated with the dissolution rate from the oil phase to the aqueous phase but not correlated with the amount of AL-294 solubilized by Tween-80. These results indicate that the absorption of AL-294 from emulsions depends mainly on the particle size in the gastro-intestinal fluid and that Tween-80 serves only to reduce the particle size in the emulsion.