Drug – bio-affecting and body treating compositions – Nonspecific immunoeffector – per se ; or nonspecific...
Reexamination Certificate
1999-07-13
2001-10-23
Bui, Phuong T. (Department: 1638)
Drug, bio-affecting and body treating compositions
Nonspecific immunoeffector, per se ; or nonspecific...
C424S184100, C424S192100, C424S193100, C424S194100, C424S197110, C424S282100, C424S283100, C424S234100, C424S257100, C424S258100, C435S071200, C435S243000
Reexamination Certificate
active
06306404
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to improved adjuvant and vaccine compositions, methods for preparing said improved adjuvant and vaccine compositions, and methods of using the improved compositions.
2. Description of the Prior Art
Conventional vaccines have been used for many years to protect humans and animals from a wide variety of infectious diseases. Typically, these conventional vaccines contain one or more antigens which may include an attenuated pathogen, killed pathogen, or an immunogenic component of a pathogen. In some vaccines, the antigen or antigens may be employed alone to elicit protective immune responses. In other vaccines, the antigen or antigens may be employed together with one or more adjuvants to enhance the immunogenicity of an antigen. One such adjuvant known to the art is monophosphoryl lipid A, which is derived from the lipopolysaccharide of
Salmonella minnesota
R595. It is also known to the art that monophosphoryl lipid A is a lipidic material which spontaneously aggregates with itself in an aqueous environment. Moreover, it is known that the degree of aggregation has an effect on the activity of monophosphoryl lipid A as an immunostimulant in that the aggregated monophosphoryl lipid A is less stimulatory.
Monophosphoryl lipid A is typically obtained as the triethylamine salt in the form of a lyophilized white powder. Being very hydrophobic, the lyophilized monophosphoryl lipid A does not readily form a clear solution when reconstituted with water but instead yields a turbid mixture with visible white particulates of heterogeneous size that settle out and further aggregate upon standing. To make an acceptable aqueous preparation of monophosphoryl lipid A, it is known to suspend the lyophilized monophosphoryl lipid A triethylamine salt at 1 to 2 mg/mL (w/v) in water containing 0.2% triethylamine, to heat the suspension at 65-70° C., and then to sonicate the mixture. The resulting aqueous preparation, slightly opalescent or clear, is an aqueous colloidal suspension. The triethylamine aids in the solubilization of the monophosphoryl lipid A and may be substituted with similar amounts of triethanolamine.
When aqueous preparations of monophosphoryl lipid A prepared as described hereinabove are frozen and then thawed, however, the monophosphoryl lipid A aggregates resulting in a turbid mixture quite similar in appearance to the turbid mixture of monophosphoryl lipid A prior to sonication. Similarly, when an aqueous preparation of monophosphoryl lipid A as described hereinabove is lyophilized and then rehydrated, the result is also a turbid mixture of aggregated monophosphoryl lipid A.
SUMMARY OF THE INVENTION
The present invention provides to the art a lyophilized composition containing monophosphoryl lipid A, which composition exhibits an enhanced reconstitution feature and which avoids the settling out and aggregation problems of the prior art. In particular, the present invention provides a lyophilized composition comprising monophosphoryl lipid A, sugar and, optionally, an added amine based surfactant, and is capable of being reconstituted or rehydrated with an aqueous diluent to form, without further sonication, an aqueous colloidal suspension of monophosphoryl lipid A having a light transmission of at least 88%, as measured spectrophotometrically. The lyophilized composition according to the present invention comprises up to about 5 wt % monophosphoryl lipid A, greater than about 70 wt % sugar and from about 0 to about 30 wt % optionally added amine based surfactant, said wt % based on the total of the weights of monophosphoryl lipid A, sugar and, if present, amine based surfactant. Preferably, the lyophilized composition according to the present invention comprises up to about 5 wt % monophosphoryl lipid A, from about 70 to about 99.99 wt % sugar and from about 0 to about 28 wt % optionally added amine based surfactant. More preferably, the lyophilized composition according to the present invention comprises up to about 4 wt % monophosphoryl lipid A, from about 75 to about 99.99 wt % sugar and from about 0 to about 22 wt % optionally added amine based surfactant. The lyophilized composition may further comprise an immunologically effective amount of an antigen or antigens. The lyophilized composition of the present invention may be reconstituted or rehydrated with an aqueous diluent at concentrations up to about 210 mg of lyophilized composition per ml of aqueous diluent, preferably from about 10 mg of lyophilized composition per ml of aqueous diluent to about 210 mg of lyophilized composition per ml of aqueous diluent, to form, without further sonication, an aqueous colloidal suspension.
Another aspect of the present invention is a method of preparing an aqueous colloidal suspension of monophosphoryl lipid A in which the aqueous colloidal suspension is frozen for storage and then thawed for use without the problems of settling out and aggregation known in the prior art. By this method, monophosphoryl lipid A is mixed in an aqueous diluent and optionally with an amine based surfactant and also optionally an antigen or antigens. An aqueous colloidal suspension is formed by sonicating, optionally with heating, or other known methods, as described in greater detail hereinafter. Sugar, in an amount from about 10 mg/ml to about 200 mg/ml, is added to the mixture either before or after the formation of an aqueous colloidal suspension. The sugar may be in the form of a solid or in the form of an aqueous solution. The resulting aqueous colloidal suspension may then be frozen. Thawing the frozen aqueous colloidal suspension affords without further sonication an aqueous colloidal suspension containing monophosphoryl lipid A having a light transmission of greater than or equal to 88%, as measured spectrophotometrically. An antigen or antigens, as defined hereinafter, may be added to the thawed aqueous colloidal suspension to form a vaccine composition which may be administered to a vertebrate. Alternatively, if the aqueous colloidal suspension contains an antigen before freezing, the vaccine composition may be thawed and administered to a vertebrate.
The aqueous colloidal suspensions of the present invention are a special type of liquid suspension in which the particles of suspended monophosphoryl lipid A are present in very finely divided but not in dissolved form. The aqueous colloidal suspensions containing monophosphoryl lipid A, sugar and, optionally, an amine based surfactant according to the present invention are true suspensions not solutions, and do not have the property, unlike ordinary suspensions of monophosphoryl lipid A, of settling out and aggregation. The presence of the aqueous colloidal suspensions of the present invention can be determined by light transmission. Thus, an aqueous colloidal suspension containing monophosphoryl lipid A, sugar and optionally an amine based surfactant according to the present invention is one which exhibits a light transmission of greater than or equal to 88%, as measured spectrophotometrically.
The present invention solves the settling out and aggregation problems of the prior art, by providing the addition of sugar to an aqueous colloidal suspension of monophosphoryl lipid A prior to freezing or lyophilization. The sugar may be added either before or after formation of the aqueous colloidal suspension but must be added before freezing or lyophilization of the suspension. The addition of sugar to an aqueous colloidal suspension of monophosphoryl lipid A prior to freezing or lyophilization provides a composition which, after freezing can be thawed to afford an aqueous colloidal suspension without further sonication or, alternatively, after lyophilization, can be reconstituted with a suitable aqueous diluent and afford without further sonication an aqueous colloidal suspension as described hereinabove. Suitable sugars include the monosaccharides, dextrose, mannose, galactose and fructose as well as the disaccharides sucrose, lactose, Isomaltose, maltose and trehalose.
Eldridge John Hayward
LaPosta Vincent James
American Cyanamid Company
Bui Phuong T.
Moram Daniel B.
Szatkowski Thomas S.
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