Chemistry: analytical and immunological testing – Lipids – triglycerides – cholesterol – or lipoproteins
Reexamination Certificate
2001-03-01
2004-12-07
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Lipids, triglycerides, cholesterol, or lipoproteins
Reexamination Certificate
active
06828155
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for forecasting and evaluating the pharmacokinetic parameter of an injection containing lipid A analog, a quality evaluating method of the injection for ensuring the injection to have constant pharmacokinetic parameter, and a preparation process of the injection.
PRIOR ART
Lipid A is the main moiety causing activities of lipopolysaccharide (hereinafter referred to as LPS), has various biological activities such as macrophage stimulation, antitumor effect and pyrogenicity (for example, Haruhiko Takada and Shozo Kotani,
Protein, Nucleic Acid and Enzyme,
31(4), 361 (1986)).
Various lipid A analogs have recently been synthesized and examined for their biological activities (Yuji Ogawa et al.,
Metabolism
26(5), 415 (1989)). Most of the Lipid A analogs having the glycolipid structure are sparingly soluble in water, so that it is difficult to prepare an injection with lipid A analogs.
In order to prepare an injection and to obtain a highly transparent aqueous solution, the addition of triethylamine, bovine serum albumin, lipids, or the like (Y. B. Kim, et al,
Eur. J. Biochem.
31, 230 (1972) and R. B. Ramsey, et al,
Blood,
56, 307 (1980), J. Dijkstra, et al,
J. Immunol.,
138, 2663 (1987)) and use of basic amino acids or polyamines (JP-A 4-198192) as a solubilizing agent have been reported.
On the other hand, as a method of dispersing a lipid such as lecithin or the like in water to form aggregates of liposomes or the like, known is a method to add a lipid to a buffer having a pH around neutrality, followed by heating and sonication.
The inventors have prepared an injection preparation having a high transparency, which contains aggregates having a diameter not greater than 30 nm, prepared by dissolving the lipid A analog or a pharmacologically acceptable salt thereof produced according to the methods disclosed in JP-A 5-194470 and WO96/39411 in an alkaline aqueous solution and then adding a buffer thereto.
Administration of this injection preparation to the living body of a rat or a beagle is however accompanied with the problem that the blood level of a lipid A analog shows a large variation, depending on the difference in the raw material medicament or preparation lot. This occurs because the state of the aggregates of the medicament (lipid) in the solution of the injection preparation is not uniform.
It is the common practice to evaluate the state of the aggregates of a lipid in a solution from the appearance observed through an electron microscope, particle size distribution measured by a laser diffraction particle size distribution measuring apparatus or consideration based on the physical properties such as critical micelle concentration or surface tension.
Not so many reports have however been proposed on circular dichroism spectroscopy and/or membrane fluidity evaluation method employed as an evaluation method of the invention.
In circular dichroism spectroscopy (which will hereinafter be abbreviated as “CD spectroscopy”), measurement is carried out reflecting the difference in the refractive index and absorbance of an optically active substance between those relative to left hand circular polarized light and those relative to right hand circular polarized light. This method is frequently employed for the conformation analysis of peptides or proteins or optical activity analysis of low-molecular compounds, but it has hardly been employed for the analysis of lipid-related substances. Reported are only the case wherein a dynamic change of a lipid in a liposome membrane depending on temperature was investigated by the analysis of variations in a CD spectrum (JP-A 62-252795); the case wherein CD spectroscopy is useful for evaluation of the characteristics of lipid particles in water, is an evaluation method markedly simple in operation and permits measurement of a dynamic state change in the membrane at a diluted concentration (N. Nakashima et al, CHEM. LET., 1503, 10 (1985)); and a case wherein CD spectroscopy is employed as one of the evaluation methods of the correlation between the characteristics of aggregates in various formulations of Prostaglandin E1 in liposome and elution characteristics (Sharon M. K. et al, Biochim. Biophys. Acta, 1327, 97 (1997)).
As a method for evaluating membrane fluidity (softness of membrane), known are fluorescence probe method, electron spin resonance (ESR) method and nuclear magnetic resonance (NMR) method (Robert B Gennith; Biomembrane, p146 (1992), published by Springer Verlag, Tokyo). Among them, the fluorescence probe method is a method for evaluating membrane fluidity having a bimolecular membrane structure of phospholipid. In this method, the state of the membrane in the vicinity of a fluorescent substance is observed by mixing a fluorescence probe such as diphenylhexatriene (which will hereinafter be abbreviated as “DPH”) in the membrane of lipid and then, measuring the polarity of fluorescence emitted upon exposure to polarized incident light. There is a report of the application of this method to a lipid A analog (Braudenburg K, et al., Biochim. Biophys. Acta, 225, 775 (1984)). The fluorescence probe method makes it possible to calculate fluorescence polarity (P: ranging from 0 to 0.5) and/or fluorescence anisotropy (r: ranging from 0 to 0.4) and/or order parameter (S: ranging from 0 to 1.0) by measuring the vertical polarizing component and horizontal polarizing component of fluorescence emitted from a lipid sample (Hiroshi Terada, Tetsuro Yoshimura; Liposome in Life Science (1992), published by Springer Verlag, Tokyo). Herein, as the order parameter (S) approaches 0, the membrane fluidity becomes larger. As it approaches 1, on the other hand, the membrane fluidity becomes smaller.
No report has however been published yet on the definite forecasting or evaluation of the pharmacokinetic parameter of a lipid A analog based on the correlation between the measuring and evaluating results of the aggregate condition of the medicament in a solution and the pharmacokinetic parameter. In addition, a report has been made neither on a process for producing an injection preparation wherein the pharmacokinetic parameter of a lipid A analog has been controlled nor a quality assurance method for ensuring the injection preparation to have predetermined pharmacokinetic parameter, each from the viewpoints of the state of the aggregates of the medicament (lipid) in a solution.
It is possible to prepare an injection preparation containing aggregates having a diameter not greater than 30 nm by dissolving a lipid A analog or pharmacologically acceptable salt thereof in an aqueous alkaline solution and then adding a buffer thereto. In the resulting injection preparation, the lipid A analog or pharmacologically acceptable salt thereof constitutes endoplasmic reticulum with lipid biomolecular membrane or micelle structure. In other words, an injection of a lipid A analog which has high transparency in the form of an aqueous solution, a preferred pH range as an injection and good stability can be prepared by the above-described process.
The injection preparation thus prepared is however accompanied with the problem that when it is administered to a rat or beagle, the blood level varies largely from one lot to another of a raw material medicament or the preparation. This owes to that the existing state of a lipid A analog in a solution, that is, the aggregate structure in the form of endoplasmic reticulum of lipid biomolecular membrane or micelle having a diameter not greater than 30 nm differs with the lot of the raw material medicament or injection preparation. There is accordingly a strong demand for the development of a practically usable injection of a lipid A analog, that is, an injection having uniform pharmacokinetic parameter, which is typified by the blood level, without being influenced by the difference among the lots of a raw material medicament or injection preparation; and a forecasting and evaluating method of the pharmacokinetic parameter of the injection.
DISCLOSURE OF
Asai Yasuyuki
Ashizawa Kazuhide
Kaneko Kazuhiro
Kikuchi Kiyomi
Kushida Ikuo
Cole Monique T.
Eisai Co. Ltd.
Warden Jill
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