This invention provides compounds containing a 2-deoxy-2-amino-xcex2-D-glucopyranose (glucosamine) glycosidically linked to an cyclic aminoalkyl (aglycon) group. The invention further provides methods for inducing an immune response using the compounds of the present invention in the presence or absence of an antigen. In addition, methods for treating disease with the compounds of the present invention with or without an antigen are also provided in this invention.

This invention relates generally to immunoeffector compounds, their use in pharmaceutical compositions, and methods for their production and their use in prophylactic and/or therapeutic vaccination. More particularly, the present invention relates to novel compounds comprising 2-deoxy-2-amino-xcex2-D-glucopyranose (glucosamine) glycosidically linked to a cyclic aminoalkyl (aglycon) group, and their use in pharmaceutical adjuvant systems.
Humoral immunity and cell-mediated immunity are the two major branches of the mammalian immune response. Humoral immunity involves the generation of antibodies to foreign antigens. Antibodies are produced by B-lymphocytes Cell-mediated immunity involves the activation of T-lymphocytes which either act upon infected cells bearing foreign antigens or stimulate other cells to act upon infected cells. Both branches of the mammalian immune system are important in fighting disease Humoral immunity is the major line of defense against bacterial pathogens. In the case of viral disease, the induction of cytotoxic T lymphocytes (CTLs) appears to be crucial for protective immunity. Thus, an effective vaccine preferably stimulates both branches of the immune system to protect against disease.
Vaccines present foreign antigens from disease causing agents to a host so that the host can mount a protective immune response. Often vaccine antigens are killed or attenuated forms of the microbes which cause the disease. The presence of non-essential components and antigens in these killed or attenuated vaccines has encouraged considerable efforts to refine vaccine components including developing well-defined synthetic antigens using chemical and recombinant techniques. The refinement and simplification of microbial vaccines, however, has led to a concomitant loss in potency. Low-molecular weight synthetic antigens, though devoid of potentially harmful contaminants, are often not sufficiently immunogenic by themselves. These observations have led investigators to add immune system stimulators known as adjuvants to vaccine compositions to potentiate the activity of the vaccine components.
Immune adjuvants are compounds which, when administered to an individual or tested in vitro, increase the immune response to an antigen in a subject to which the antigen is administered, or enhance certain activities of cells from the immune system. A number of compounds exhibiting varying degrees of adjuvant activity have been prepared and tested (see, for example, Shimizu et al. 1985, Bulusu et al. 1992, Ikeda et al. 1993, Shimizu et al. 1994, Shimizu et al. 1995, Miyajima et al. 1996). However, these and other prior adjuvant systems often display toxic properties, are unstable and/or have unacceptably low immunostimulatory effects.
Presently, the only adjuvant licensed for human use in the United States is alum, a group of aluminum salts (e.g., aluminum hydroxide, aluminum phosphate) in which vaccine antigens are formulated. Particulate carriers like alum reportedly promote the uptake, processing and presentation of soluble antigens by macrophages. Alum, however, is not without side-effects and is unfortunately limited to humoral (antibody) immunity only.
The discovery and development of effective adjuvant systems is essential for improving the efficacy and safety of existing and future vaccines. Thus, there is a continual need for new and improved adjuvant systems, particularly those that drive both effector arms of the immune system, to better facilitate the development of a next generation of synthetic vaccines. The present invention fulfills these and other needs.
The compounds of the present invention are immunoeffector molecules which enhance humoral and cell-mediated immune responses to vaccine antigens. The compounds can generally be descrbed as belonging to the class of cyclic AGP compounds, where AGP stands for aminoalkyl glucosaminide phosphates. The term xe2x80x9ccyclic AGPxe2x80x9d means an azacycloalkyl or (azacycloalkyl)alkyl glucosaminide phosphate, wherein a 2-deoxy-2-amino-b-D-glucopyranose (glucosamine) is glycosidically linked to an azacycloalkyl or (azacycloalkyl)alkyl (aglycon) group.
The compounds of this invention comprise a 2-deoxy-2-amino-xcex2-D-glucopyranose (glucosamine) glycosidically linked to an cyclic aminoalkyl (aglycon) group. The compounds are phosphorylated at the 4 or 6-position of the glucosarine ring and acylated with alkanoyloxytetradecanoyl residues on the aglycon nitrogen and the 2 and 3-positions of the glucosamine ring, The compounds of the subject invention are described generally by formula (I):
and pharmaceutically acceptable salts thereof,
wherein X is xe2x80x94Oxe2x80x94 or xe2x80x94NHxe2x80x94 and Y is xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94; R1, R2, and R3 are each independently a ( C9-C14)acyl group, including saturated, unsaturated and branched acyl groups; R4 is xe2x80x94H or xe2x80x94PO3R7R8, wherein R7 and R8 are each independently H or (C1-C4) aliphatic groups; R5 is xe2x80x94H, xe2x80x94CH3 or xe2x80x94PO3R9R10, where R9 and R10 are each independently selected from xe2x80x94H and (C1-C4)aliphatic groups; R6 is independently selected from H, OH, (C1-C4) oxyaliphatic groups, xe2x80x94PO3R11R12, xe2x80x94OPO3R11R12, xe2x80x94SO3R11, xe2x80x94OSO3R11, xe2x80x94NR11R12, xe2x80x94SR11, xe2x80x94CN, xe2x80x94NO2, xe2x80x94CHO, xe2x80x94CO2R11, and xe2x80x94CONR11R12, wherein R11 and R12 are each independently selected from H and (C1-C4)aliphatic groups; with the provisos that one of R4 and R5 is a phosphorus-containing group and that When R4 is xe2x80x94PO3R7R8, R5 is other than xe2x80x94PO3R9R10, wherein xe2x80x9c*1-3xe2x80x9d and xe2x80x9c**xe2x80x9d represent chiral centers;
wherein n, m, p and q are each independently an integer from 0 to 6, with the proviso that the sum of p and m is from 0 to 6.
In some embodiments of compounds of the present invention X and Y are each oxygen, R4 is PO3R7R8, R5 and R6 are H, and n, m, p, and q are integers from to 0 to 3. In a more preferred embodiment, R7 and R8 are xe2x80x94H. In an even more preferred embodiment, n is 1, m is 2, and subscripts p and q are 0. In yet an even more preferred embodiment, R1, R2, and R3 are C9-C13 acyl groups, most preferably C10-C12 acyl groups. In a still more preferred embodiment, *1-3 are in the R configuration, Y is in the equatorial position, and ** is in the S configuration. Particularly preferred are (N-[(R)-3-tetradecanoyloxytetradecanoyl]-(S)-2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-xcex2-D-glucopyranoside, Formula (II),
(N-[(R)-3-dodecanoyloxytetradecanoyl]-(S)-2-pyrrolidinyhnethyl 2-deoxy-4-O-phosphono-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O-[(R)-3-dodecanoyloxytetradecanoyl]-xcex2-D-glucopyranoside, Formula (III),
and (N-[(R)-3-decanoyloxytetradecanoyl]-(S)-2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-xcex2-D-glucopoyranoside, Formula (IV),
and their pharmaceutically acceptable salts.
The present invention also provides pharmaceutical compositions containing compounds of the general and specific formulas above. The pharmaceutical compositions can be combined with a variety of antigens and in a variety of formulations known to those of skill in the art.
The compounds of the present invention are also useful in methods of inducing an immune response in a subject. The method entails administering to the subject a therapeutically effective amount of one or more compounds of the present invention, preferably in a pharmaceutical composition that also contains a pharmaceutically acceptable carrier.
The present invention also encompasses methods of treating a mammal suffering from or susceptible to a pathogenic infection, cancer or an autoimmune disorder. The method entails administering to the mammal a therapeutically effective amount of one or more compounds of the present invention, preferably in a pharmaceutical composition that also contains a pharmaceutically acceptable carrier.
Still further, the present invention involves a method for treating diseases or conditions ameliorated by nitric oxide production in a subject. The method entails contacting the subject with an effective amount of a compound or compounds of the present invention, or with an effective amount of a composition containing one or more compounds of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the compounds of the present invention can be administered 48 hours prior to, up to, and during ischemia.
Definitions
The term xe2x80x9cacylxe2x80x9d refers to those groups derived from an organic acid by removal of the hydroxy portion of the acid. Accordingly, acyl is meant to include, for example, acetyl, propionyl, butyryl, decanoyl, and pivaloyl. xe2x80x9c(C9-C14)acylxe2x80x9d, for instance, refers to an acyl group having from 9 to 14 carbons.
The term xe2x80x9caliphaticxe2x80x9d by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic, hydrocarbon moiety, including a moiety that contains both cyclical and chain elements, which may be fully saturated or mono- or polyunsaturated, having the number of carbon atoms designated (i.e. C1-C4 means one to four carbons). Examples of saturated hydrocarbon radicals include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclopropyl, cyclopropylmethyl, methylene, ethylene, and n-butylene. An unsaturated alkyl group is one having one or more double bonds and/or triple bonds. Examples of unsaturated aliphatic groups include vinyl, 2-propenyl, crotyl, -2-(butadienyl), 1-propynyl and 3-propynyl.
The term xe2x80x9coxyaliphatic refers to those groups having an aliphatic group attached to the remainder of the molecule through an oxygen atom.
Each of the above terms (eg., xe2x80x9calkyl,xe2x80x9d xe2x80x9cacylxe2x80x9d) are meant to include both substituted and unsubstituted forms of the indicatedmoiety.
Substituents for the aliphatic groups can be a variety of groups selected from: xe2x80x94ORxe2x80x2, xe2x95x90O, xe2x95x90S, xe2x95x90NRxe2x80x2, xe2x95x90Nxe2x80x94ORxe2x80x2, xe2x80x94NRxe2x80x2Rxe2x80x3, xe2x80x94SRxe2x80x2, -halogen, xe2x80x94SiRxe2x80x2Rxe2x80x3Rxe2x80x2xe2x80x3, xe2x80x94OC(O)Rxe2x80x2, xe2x80x94C(O)Rxe2x80x2, xe2x80x94CO2Rxe2x80x2, xe2x80x94CONRxe2x80x2Rxe2x80x3, xe2x80x94OC(O)NRxe2x80x2Rxe2x80x3, xe2x80x94NRxe2x80x3C(O)Rxe2x80x2, xe2x80x94NRxe2x80x2xe2x80x94C(O)NRxe2x80x3Rxe2x80x2xe2x80x3, xe2x80x94NRxe2x80x3C(O)2Rxe2x80x2, xe2x80x94NHxe2x80x94C(NH2)xe2x95x90NH, xe2x80x94NRxe2x80x2C(NH2)xe2x95x90NH, xe2x80x94NHxe2x80x94C(NH2)xe2x95x90NRxe2x80x2, xe2x80x94S(O)Rxe2x80x2, xe2x80x94S(O)2Rxe2x80x2, xe2x80x94S(O)2NRxe2x80x2Rxe2x80x3, xe2x80x94CN and xe2x80x94NO2 in a number ranging from zero to (2mxe2x80x2=1), where mxe2x80x2 is the total number of carbon atoms in such radical, Rxe2x80x2, Rxe2x80x3 and Rxe2x80x2xe2x80x3 each independently refer to hydrogen and unsubstituted (C1-C4)aliphatic groups. From the above discussion of substituents, one of skill in the art will understand that the term xe2x80x9calkyrxe2x80x9d is meant to include groups such as haloalkyl (e.g., xe2x80x94CF3 and xe2x80x94CH2CF3) and the like.
The terms xe2x80x9chaloxe2x80x9d or xe2x80x9chalogen,xe2x80x9d by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. In compounds having multiple halogen substituents, the halogens may be the same or different.
The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d is meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substiatents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by addition of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by addition of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrozenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge, S. M., et al., xe2x80x9cPharmaceutical Saltsxe2x80x9d, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.
Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention.
The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.
Introduction
In an effort to improve the safety of vaccines, manufacturers are avoiding whole cell killed vaccines, and producing recombinant or subunit vaccines. In the preparation of these safer vaccines extraneous bacterial or viral components are eliminated, while the minimal structures or epitopes deemed necessary for protective immunity remain. The safety of these vaccines is improved due to the elimination of extraneous bacterial or viral components which often times prove to be toxic and pyrogenic. However, the same components that result in toxicity provide nonspecific immunostimulation that make whole cell vaccines so effective. Without the additional immunostimulation the minimal structures and epitopes comprising recombinant and subunit vaccines are often poorly immunogenic.
A disaccharide molecule derived from the LPS of Salmonella minnesota R595, MPL(copyright) immunostimulant (Corixa Corp.), has immunostimulant properties. MPL(copyright) immunostimulant, Monophosphoryl lipid A, is a structural derivative of lipid A (or LPS) and has an improved therapeutic index relative to lipid A (see U.S. Pat. No. 4,987,237 for the structure of Monophosphoryl lipid A; U.S. Pat. Nos. 4,436,727 and 4,436,728 for description of preparation of Monophosphoryl lipid A). Other useful immunostimulants include 3-de-O-acylated monophosphoryl lipid A (3D-MPL), which is described in U.S. Pat. No. 4,912,094. The compound can be safely administered to humans as doses up to at least 20 xcexcg/kg, although increases in temperature, flu-like symptoms, increasing heart rate and modest decreases in blood pressure can occur in some patients at dose levels of xe2x89xa710 xcexcg/kg. Cell culture and animal evaluations confirm MPL(copyright) immunostimulant still retains some of the immunostimulatory activity of the parent LPS in that pyrogenicity and the ability to induce pro-inflammatory cytokines such as TNF and IL-8 remain, albeit at higher dose levels. Thus, the need for effective vaccine adjuvants is well recognized. Ideally, these adjuvants will boost the protective immune response without inducing unwanted toxicity and pyrogenicity.
In an effort to obtain an immunostimulant that has low pyrogenicity, synthetic molecules have been prepared which share structural similarities with the MPLO immunostimulant. These novel molecules which are collectively called aminoalkyl glucosaminide phosphates (AGPs), consist of an acylated glucose moiety linked to an acylated aminoalkyl group (Johnson et al. (1999) Bioorg. Med. Chem. Lett. 9: 2273-2278; PCTAWO98/50399 and references therein). Each molecule possesses 6 fatty acid tails which is thought to be the optimal number for peak adjuvant activity. The substitution of different chemical moieties within the aminoalkyl structures was designed into the AGPs in anticipation of optimizing stability and solubility properties. Thus the AGPs can be broadly separated into several families based on the structure of their aminoalkyl groups. After initial biological evaluation, it became apparent that the aminoalkyl motifs could dramatically affect the pyrogenic properties of the AGPs (see U.S. patent application Ser. No. 09/074,720 filed May 7, 1998, and U.S. Pat. Nos. 6,113,918 and 6,303,347). As part of the initial screening process of the synthetic adjuvant compounds, rabbit pyrogenicity data was determined. It was noted that several of the compounds did not elicit a fever response when administered i.v. at doses of 10 xcexcg/kg. In general, these same compounds failed to induce detectable levels of inflammatory cytokines TNF-xcex1 or IL-1xcex2 in an ex vivo cytokine induction assay on human peripheral blood mononuclear cells. Here we report on studies of the adjuvant properties of a class of AGPs which induce minimal activity in both the rabbit pyrogen test and the ex vivo cytokine assay.
Compounds and Compositions
The present invention provides compounds described generally by formula (I):
and pharmaceutically acceptable salts thereof,
wherein X is xe2x80x94Oxe2x80x94 or xe2x80x94NHxe2x80x94 and Y is xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94; R1, R2, and R3 are each independently a (C9-C14) acyl group, including saturated, unsaturated and branched acyl groups; R4 is xe2x80x94H or xe2x80x94PO3R7R8, wherein R7 and R8 are each independently H or (C1-C4)aliphatic groups; R5 is xe2x80x94H, xe2x80x94CH3 or xe2x80x94PO3R9R10, wherein R9 and R10 are each independently selected from xe2x80x94H and (C1-C4)aliphatic groups; R6 is independently selected from H, OH, (C1-C4)oxyaliphatic groups, xe2x80x94PO3R11R12, xe2x80x94OPO3R11R12, xe2x80x94SO3R11, xe2x80x94OSO3R11, xe2x80x94NR11R12, xe2x80x94SR11, xe2x80x94CN, xe2x80x94NO2, xe2x80x94CHO, xe2x80x94CO2R11, and xe2x80x94CONR11R12, wherein R11 and R12 are each independently selected from H and (C1-C4)aliphatic groups; with the provisos that one of R4 and R5 is a phosphorus-containing group and that when R4 is xe2x80x94PO3R7R8, R5 is other than xe2x80x94PO3R9R10, wherein xe2x80x9c*1-3xe2x80x9d and xe2x80x9c**xe2x80x9d represent chiral centers;
wherein n, m, p and q are each independently an integer from 0 to 6, with the proviso that the sum of p and m is from 0 to 6.
Although the hexopyranoside in Formula I is shown in the gluco configuration, other glycosides are within the scope of the invention. For example glycopyranosides, including other hexopyranosides (e.g., allo, altro, manno, gulo, ido, galacto, talo), are within the scope of the invention.
In the general formula above, the configuration of the 3xe2x80x2-stereogenic centers to which the normal fatty acyl residues are attached, denoted xe2x80x9c*1xe2x80x9d, xe2x80x9c*2xe2x80x9d and xe2x80x9c*3xe2x80x9d, is R or S, but preferably R. The absolute stereochemistry of the carbon atoms of the cyclic aglycon unit to which R6 and the glucosamine unit are attached, directly or indirectly (denoted xe2x80x9c**xe2x80x9d) can be R or S. In the general formula above, Y can be in the equatorial or axial position, but is preferably equatorial. All stereoisomers, enantiomers, diastereomers and mixtures thereof are considered to be within the scope of the present invention.
In preferred embodiments, of the present invention, X and Y are xe2x80x94Oxe2x80x94, R4 is phosphono, R5 and R6 are H, and n, m, p, and q are integers of from 0 to 3, and more preferably 0 to 2. Most preferably the integer n is 1, the integer m is 2, and integers p and q are 0. In this preferred embodiment, the compounds of this invention are 2-pyrrolidinylmethyl xcex2-D-glucosaminide 4-phosphates having the general formula (V):
In a preferred embodiment of the present invention, the configuration of the 3xe2x80x2-stereogenic centers (xe2x80x9c*1-3xe2x80x9d) to which they are attached is R, Y is in the equatorial position, and the absolute stereochemistry of the pyrrolidine stereogenic center (xe2x80x9c**xe2x80x9d) is S.
Particularly preferred embodiments are N-[(R)-3-tetradecanoyloxytetradecanoyl]-(S)-2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylanino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-xcex2-D-glucopyranoside, and its pharmaceutically acceptable salts, depicted in formula (II):
(N-[(R)-3-dodecanoyloxytetradecanoyl]-(S)-2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O-[(R)3-dodecanoyloxytetradecanoyl]-xcex2D-glucopyrano side and pharmaceutically acceptable salts thereof; Formula (III),
and (N-[(R)-3-decanoyloxytetradecanoyl]-(S)-2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-xcex2-D-glucopyranoside and pharmaceutically acceptable salts thereof; Formula (IV),
Preparation of Compounds
The compounds of the present invention can be prepared using methods outlined in Johnson et al., Bioorg. Med. Chem. Lett. 9:2273-2278 (1999) and PCT/WO98/50399 and references therein. In general, the synthetic methods described in the above-noted references are broadly applicable to the preparation of compounds"" having different acyl groups and substitutions. One of skill in the art will appreciate that the convergent methods described therein can be modified to use alternate acylating agents, or can be initiated with commercially available materials having appropriate acyl groups attached.
Evaluation of Compounds
The compounds provided herein can be evaluated in a variety of assay formats to select a compound having a suitable pharmacophoric profile. For example, U.S. Pat. No. 6,013,640 describes animal models suitable for evaluating cardioprotective effects of compounds described herein. The examples below also provide assays for evaluating pyrogenicity of the subject compounds, and further assays for evaluating the proinflammatory effects of the compounds.
The present invention further provides pharmaceutical compositions comprising the compounds provided herein in admixture with one or more pharmaceutically acceptable carriers. Suitable carriers will depend on the condition being treated along with the route of administration. Accordingly, a discussion of the carriers is provided below in conjunction with the methods of use.
Pharmaceutical Compositions and Their Uses
In one embodiment, the present invention provides pharmaceutical compositions containing a compound of the present invention and a pharmaceutically acceptable carrier. The compound is present in a therapeutically effective amount, which the amount of compound required to achieve the desired effect in terms of treating a disease, condition, or achieving a biological occurrence. The pharmaceutical compositions can act as an adjuvant when co-administered with an antigen.
Compositions of this invention include both compositions that are formulated for direct administration of the active compounds to patients without dilution, either in conjunction with a vaccine or other active agent, or alone, as well as more concentrated compositions of the compounds that may be formulated for later dilution, so as to avoid shipment and/or storage of large amounts of diluent (e.g. water, saline or aqueous materials). In general, pharmaceutical compositions of this invention that are intended for direct or immediate administration to a subject (that is, without dilution) will contain one or more of the compounds, in a therapeutically effective amount. This amount will vary both based on the particular therapeutic compound or compounds and on the therapeutic effect desired. More concentrated compositions will contain amounts of the compound or compounds of the invention as may be appropriate for such compositions.
For preparing pharmaceutical compositions, the pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
Solid forms of the compositions also can be prepared by spray-drying aqueous formulations of the active adjuvants (e.g. in the form of a salt) or by lyophilizing and milling with excipients.
Suitable carriers for the solid compositions of this invention include, for instance, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term xe2x80x9cpreparationxe2x80x9d is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included.
Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution. In certain embodiments, the pharmaceutical compositions are formulated in a stable emulsion formulation (e.g., a water-in-oil emulsion or an oil-in-water emulsion)or an aqueous formulation that preferably comprise one or more surfactants. Suitable surfactants well known to those skilled in the art may be used in such emulsions. In one embodiment, the composition is in the form of a micellar dispersion comprising at least one suitable surfactant. The surfactants useful in such micellar dispersions include phospholipids. Examples of phospholipids include: diacyl phosphatidyl glycerols, such as: dimyristoyl phosphatidyl glycerol (DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoyl phosphatidyl glycerol (DSPG); diacyl phosphatidyl cholines, such as: dimyristoyl phosphatidylcholine (DPMC), dipalmitoyl phosphatidylcholine (DPPC), and distearoyl phosphatidylcholine (DSPC); diacyl phosphatidic acids, such as: dimyristoyl phosphatidic acid (DPMA), dipalmitoyl phosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); and diacyl phosphatidyl ethanolamines such as: dimyristoyl phosphatidyl ethanolamine (DPME), dipalmitoyl phosphatidyl ethanolamine (DPPE), and distearoyl phosphatidyl ethanolamine (DSPE). Other examples include, but are not limited to, derivatives of ethanolamine (such as phosphatidyl ethanolamine, as mentioned above, or cephalin), serine (such as phosphatidyl serine) and 3xe2x80x2-O-lysyl glycerol (such as 3xe2x80x2-O-lysyl-phosphatidylglycerol).
Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
Thus, the adjuvant systems of the invention are particularly advantageous in making and using vaccine and other immunostimulant compositions to treat or prevent diseases, such inducing active immunity towards antigens in mammals, preferably in humans. Vaccine preparation is a well developed art and general guidance in the preparation and formulation of vaccines is readily available from any of a variety of sources. One such example is New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., U.S.A. 1978.
In one illustrative embodiment, the antigen in a vaccine composition of the invention is a peptide, polypeptide, or immunogenic portion thereof. An xe2x80x9cimmunogenic portion,xe2x80x9d as used herein is a portion of a protein that is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor. Such immunogenic portions generally comprise at least 5 amino acid residues, more preferably at least 10, and still more preferably at least 20 amino acid residues of an antigenic protein or a variant thereof.
Immunogenic portions of antigen polypeptides may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones. As used herein, antisera and antibodies are xe2x80x9cantigen-specificxe2x80x9d if they specifically bind to an antigen (i.e., they react with the protein in an ELISA or other immunoassay, and do not react detectably with unrelated proteins). Such antisera and antibodies may be prepared as described herein, and using well known techniques. An immunogenic portion of a protein is a portion that reacts with such antisera and/or T-cells at a level that is not substantially less than the reactivity of the full length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Such immunogenic portions may react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide. Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, 125I-labeled Protein A.
Peptide and polypeptide antigens are prepared using any of a variety of well-known techniques. Recombinant polypeptides encoded by DNA sequences may be readily prepared from isolated DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells, such as mammalian cells and plant cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO.
Portions and other variants of a protein antigen having less than about 100 amino acids, and generally less than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, Calif.), and may be operated according to the manufacturer""s instructions.
Within certain specific embodiments, a polypeptide antigen used in the vaccine compositions of the invention may be a fusion protein that comprises two or more distinct polypeptides. A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the protein.
Fusion proteins may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3xe2x80x2 end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5xe2x80x2 end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.
A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
Within preferred embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids), and a protein D derivative may be lipidated. Within certain preferred embodiments, the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer). The lipid tail ensures optimal presentation of the antigen to antigen presenting cells. Other fusion partners include the non-structural protein from influenzae virus, NS 1 (hemagglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.
In another embodiment, the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E. coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 10:795-798, 1992). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion protein. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.
In another embodiment of the invention, the adjuvant system described herein is used in the preparation of DNA-based vaccine compositions. Illustrative vaccines of this type contain DNA encoding one or more polypeptide antigens, such that the antigen is generated in situ. The DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope. In one preferred embodiment, the DNA is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which typically involves the use of a non-pathogenic (defective), replication competent virus. Illustrative systems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art.
Alternatively, the DNA may be xe2x80x9cnaked,xe2x80x9d as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells. It will be apparent that a vaccine may comprise both a polynucleotide and a polypeptide component if desired.
Moreover, it will be apparent that a vaccine may contain pharmaceutically acceptable salts of the desired polynucleotide, polypeptide and/or carbohydrate antigens. For example, such salts may be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).
The adjuvant system of the present invention exhibits strong adjuvant effects when administered over a wide range of dosages and a wide range of ratios.
The amount of antigen in each vaccine dose is generally selected as an amount which induces an immunoprotective response without significant adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Generally, it is expected that each dose will comprise about 1-1000 xcexcg of protein, most typically about 2-100 xcexcg, preferably about 5-50 xcexcg. Of course, the dosage administered may be dependent upon the age, weight, kind of concurrent treatment, if any, and nature of the antigen administered.
The immunogenic activity of a given amount of a vaccine composition of the present invention can be readily determined, for example by monitoring the increase in titer of antibody against the antigen used in the vaccine composition (Dalsgaard, K. Acta Veterinia Scandinavica 69:1-40 (1978)). Another common method involves injecting CD-1 mice intradernally with various amounts of a vaccine composition, later harvesting sera from the mice and testing for anti-immunogen antibody, e.g., by ELISA. These and other similar approaches will be apparent to the skilled artisan.
The antigen can be derived and/or isolated from essentially any desired source depending on the infectious disease, autoimmune disease, condition, cancer, pathogen, or a disease that is to be treated with a given vaccine composition. By way of illustration, the antigens can be derived from viral sources, such as influenza virus, feline leukemia virus, feline immunodeficiency virus, Human HIV-1, HIV-2, Herpes Simplex virus type 2, Human cytomegalovirus, Hepatitis A, B, C or E, Respiratory Syncytial virus, human papilloma virus rabies, measles, or hoof and mouth disease viruses. Illustrative antigens can also be derived from bacterial sources, such as anthrax, diphtheria, Lyme disease, malaria, tuberculosis, Leishmaniasis, T. cruzi, Ehrlichia, Candida etc., or from protozoans such as Babeosis bovis or Plasmodium. The antigen(s) will typically be comprised of natural or synthetic amino acids, e.g., in the form of peptides, polypeptides, or proteins, can be comprised of polysaccharides, or can be mixtures thereof. Illustrative antigens can be isolated from natural sources, synthesized by means of solid phase synthesis, or can be obtained by way of recombinant DNA techniques.
In another embodiment, tumor antigens are used in the vaccine compositions of the present invention for the prophylaxis and/or therapy of cancer. Cancer cells often have distinctive antigens on their surfaces, such as truncated epidermal growth factor, folate binding protein, epithelial mucins, melanoferrin, carcinoembryonic antigen, prostate-specific membrane antigen, HER2-neu, which are candidates for use in therapeutic cancer vaccines. Because tumor antigens are normal or related to normal components of the body, the immune system often fails to mount an effective immune response against those antigens to destroy the tumor cells. To achieve such a response, the adjuvant systems described herein can be utilized. As a result, exogenous proteins can enter the pathway for processing endogenous antigens, leading to the production of cytolytic or cytotoxic T cells (CTL). This adjuvant effect facilitates the production of antigen specific CTLs which seek and destroy those tumor cells carrying on their surface the tumor antigen(s) used for immunization. Illustrative cancer types for which this approach can be used include prostate, colon, breast, ovarian, pancreatic, brain, head and neck, melanoma, leukemia, lymphoma, etc.
In another embodiment of the invention, the adjuvant system of the present invention can be administered alone, i.e., without a co-administered antigen, to potentiate the immune system for treatment of chronic infectious diseases, especially in immune compromised patients. Illustrative examples of infectious diseases for which this approach may be employed for therapeutic or prophylactic treatment can be found in U.S. Pat. No. 5,508,310. Potentiation of the immune system in this way can also be useful as a preventative measure to limit the risks of nosocomial and/or post-surgery infections.
In another embodiment, the antigen present in the vaccine compositions is not a foreign antigen, rather it is a self antigen, e.g., the vaccine composition is directed toward an autoimmune disease such as type 1 diabetes, conventional organ-specific autoimmune diseases, neurological diseases, rheumatic diseases, psoriasis, connective tissue diseases, autoimmune cytopenias, and other autoimmune diseases. Such conventional organ specific autoimmunity may include thyroiditis (Graves+Hashimoto""s), gastritis, adrenalitis (Addison""s), ovaritis, primary biliary cirrhosis, myasthenia gravis, gonadal failure, hypoparathyroidism, alopecia, malabsorption syndrome, pernicious anemia, hepatitis, anti-receptor antibody diseases and vitiligo. Such neurological diseases may include schizophrenia, Alzheimer""s disease, depression, hypopituitarism, diabetes insipidus, sicca syndrome and multiple sclerosis. Such rheumatic diseases/connective tissue diseases may include rheumatoid arthritis, systemic lupus erythematous (SLE) or Lupus, scleroderma, polymyositis, inflammatory bowel disease, dermatomyositis, ulcerative colitis, Crohn""s disease, vasculitis, psoriatic arthritis, exfoliative psoriatic dermatitis, pemphigus vulgaris, Sjogren""s syndrome. Other autoimmune related diseases may include autoimmune uvoretinitis, glomerulonephritis, post myocardial infarction cardiotomy syndrome, pulmonary hemosiderosis, amyloidosis, sarcoidosis, aphthous stomatitis, and other immune related diseases, as presented herein and known in the related arts.
While any suitable carrier known to those of ordinary skill in the art may be employed in the vaccine compositions of this invention, the type of carrier will typically vary depending on the desired mode of administration. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, intradermal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier will often comprise water, saline, alcohol, a fat, a wax or a buffer. For oral administration, the above carriers are often used, or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, can also be employed. Biodegradable microspheres (e.g., polylactate polyglycolate) may also be employed as carriers for the compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252, the disclosures of which are incorporated herein by reference in their entireties. Modified hepatitis B core protein carrier systems are also suitable, such as those described in WO/99 40934, and references cited therein, all incorporated herein by reference. One can also employ a carrier comprising particulate-protein complexes, such as those described in U.S. Pat. No. 5,928,647, the disclosure of which is incorporated herein by reference in its entirety, which are capable of inducing a class I-restricted cytotoxic T lymphocyte responses in a host.
In one illustrative embodiment, the vaccine formulations are administered to the mucosae, in particular to the oral cavity, and preferably to a sublingual site, for eliciting an immune response. Oral cavity administration may be preferred in many instances over traditional parenteral delivery due to the ease and convenience offered by noninvasive administration techniques. Moreover, this approach further provides a means for eliciting mucosal immunity, which can often be difficult to achieve with traditional parenteral delivery, and which can provide protection from airborne pathogens and/or allergens. An additional advantage of oral cavity administration is that patient compliance may be improved with sublingual vaccine delivery, especially for pediatric applications, or for applications traditionally requiring numerous injections over a prolonged period of time, such as with allergy desensitization therapies.
The vaccine compositions can also comprise buffers (e.g., neutral buffered saline, phosphate buffered saline or phosphate buffers w/o saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate. The compositions can also be encapsulated within liposomes using well known technology.
Therefore, in one embodiment, the vaccine compositions are aqueous formulations comprising an effective amount of one or more surfactants. For example, the composition can be in the form of a micellar dispersion comprising at least one suitable surfactant, e.g., a phospholipid surfactant. Illustrative examples of phospholipids include diacyl phosphatidyl glycerols, such as dimyristoyl phosphatidyl glycerol (DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoyl phosphatidyl glycerol (DSPG), diacyl phosphatidyl cholines, such as dimyristoyl phosphatidylcholine (DPMC), dipahnitoyl phosphatidylcholine (DPPC), and distearoyl phosphatidylcholine (DSPC); diacyl phosphatidic acids, such as dimyristoyl phosphatidic acid (DPMA), dipalmitoyl phosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); and diacyl phosphatidyl ethanolamines such as dimyristoyl phosphatidyl ethanolamine (DPME), dipalmitoyl phosphatidyl ethanolamine (DPPE) and distearoyl phosphatidyl ethanolamine (DSPE).
Typically, a surfactant:adjuvant molar ratio in an aqueous formulation will be from about 10:1 to about 1:10, more typically from about 5:1 to about 1:5, however any effective amount of surfactant may be used in an aqueous formulation to best suit the specific objectives of interest.
In another embodiment, the composition is an emulsion, such as a water-in-oil emulsion or an oil-in water emulsion. Such emulsions are generally well known to those skilled in this art.
The adjuvant system of the present invention can be employed as the sole adjuvant system, or alternatively, can be administered together with other adjuvants or immunoeffectors. By way of illustration, such adjuvants can include oil-based adjuvants (for example, Freund""s Complete and Incomplete), liposomes, mineral salts (for example, AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3, Ca3(PO4)2, kaolin and carbon), polynucleotides (for example, poly IC and poly AU acids), polymers (for example, non-ionic block polymers, polyphosphazenes, cyanoacrylates, polymerase-(DL-lactide-co-glycoside), among others, and certain natural substances (for example, lipid A and its derivatives, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella), bovine serum albumin, diphtheria toxoid, tetanus toxoid, edestin, keyhole-limpet hemocyanin, Pseudomonal Toxin A, choleragenoid, cholera toxin, pertussis toxin, viral proteins, and eukaryotic proteins such as interferons, interleukins, or tumor necrosis factor. Such proteins may be obtained from natural or recombinant sources according to methods well known to those skilled in the art. When obtained from recombinant sources, the adjuvant may comprise a protein fragment comprising at least the immunostimulatory portion of the molecule. Other known immunostimulatory macromolecules which can be used in the practice of the invention include, but are not limited to, polysaccharides, tRNA, non-metabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4xe2x80x2,4-diaminodiphenylmethane-3,3xe2x80x2-dicarboxylic acid and 4-nitro-2-aminobenzoic acid (See Sela, M., Science 166:1365-1374 (1969)) or glycolipids, lipids or carbohydrates.
In one embodiment, the adjuvant system is preferably designed to induce an immune response predominantly of the Th1 type. High levels of Th1-type cytokines (e.g., IFN-xcex3, TNFxcex1, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes Th1- and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Th1-type, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffinan, Ann. Rev. Immunol 7:145-173, 1989.
For example, additional adjuvants for use in eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, such as 3-de-O-acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt. MPL adjuvants are available from Corixa Corporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immnunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996. Other illustrative adjuvants that can be included in the vaccine compositions include Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), Detox(trademark) adjuvant (Corixa, Hamilton, Mont.).
The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule, sponge or gel (composed of polysaccharides, for example) that effects a slow release of compound following administration). Such formulations may generally be prepared using well known technology (see, e.g., Coombes et al., Vaccine 14:1429-1438, 1996) and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. Such carriers include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). The amount of active compound contained within a sustained release formulation will vary depending upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
Any of a variety of known delivery vehicles may be employed within pharmaceutical compositions and vaccines to facilitate production of an antigen-specific immune response that targets cells. Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-target effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
Certain preferred embodiments of the present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within a vaccine (see Zitvogel et al., Nature Med. 4:594-600, 1998).
Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNFA to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFxcex1, CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.
Dendritic cells are conveniently categorized as xe2x80x9cimmaturexe2x80x9d and xe2x80x9cmaturexe2x80x9d cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcxcex3 receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).
APCs may generally be transfected with a polynucleotide encoding an antigen polypeptide (or portion or other variant thereof) such that the antigen polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a composition or vaccine comprising such transfected cells, and the adjuvants described herein, may then be used for therapeutic purposes. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997. Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the antigen polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.
Treatment of Nitric Oxide Related Disorders
In one aspect, the present invention provides methods for treating diseases or conditions mediated by nitric oxide, particularly ischemia and reperfusion injury. The methods comprise administering to a subject in need of such treatment an effective amount of a compound of the present invention. It is generally agreed that inducers of iNOS gene transcription and protein synthesis are proinflammatory and consequently somewhat xe2x80x9ctoxicxe2x80x9d or poorly tolerated in animals and humans. Endotoxin (LPS) and proinflammatory cytokines such as IL-1, TNF-xcex1 and IFN-xcex3 are known inducers of iNOS. All are inherently toxic and capable of inducing a systemic inflammatory response, adult respiratory distress syndrome, multiple organ failure and cardiovascular collapse when administered to animals.
Investigation of the cardioprotective activity of MPL(copyright) immunostimulant demonstrated that induction of nitric oxide synthases (iNOS) is important in the delayed cardioprotective effect of the compound. Additionally, nitric oxide (NO) signaling, presumably through constitutive pools of NOS, is important in the acute cardioprotective effect of the compound. In view of the residual endotoxic-like activity of MPLO immunostimulant, it is not surprising that the compound could be capable of inducing nitric oxide signaling. Still further, nitric oxide signaling has been suggested as a potential pathway by which ischemic preconditioning elicits cardioprotection. This observation in combination with the fact that nitric oxide donors are cardioprotective provides further support for the NOS/NO pathway as the route for MPL(copyright) immunostimulant cardioprotection.
The compounds of the present invention are useful in methods for treating diseases or conditions modulated or ameliorated by nitric oxide, particularly ischemia and reperfusion injury (see, U.S. patent application Ser. No.: 09/808,669, filed Mar. 14, 2001, for a description of the cardioprotective properties of aminoalkyl glucosaminide phosphates and methods for assaying cardioprotective properties).


A process for the oligomerization of ethylene to a mixture of olefinic products having high linearity is provided, by using a catalyst comprising a reaction product of a simple divalent nickel salt; a boron hydride reducing agent; a water soluble base; a ligand selected from the group consisting of an o-dihydrocarbylphosphinobenzoic acid and alkali metal salts thereof; and a phosphite.

This invention relates to a certain nickel-containing catalyst and a process for the oligomerization of ethylene to a mixture of olefinic products having high linearity using such catalyst.
The production of a mixture of olefinic products which are substantially alpha-olefins and which have a high degree of linearity are known. Such olefins comprise for example, those of the C4-C10 range, useful as comonomers for LLDPE or as synthetic lubricants; those of the C12-C20 range, useful as detergents; and higher olefins. The lower molecular weight olefins can be converted to sulfonates or alcohols by known commercial processes. The C12-C20 olefins find use in the detergent-products area. Lower molecular weight alcohols can be esterified with polyhydric acids, e.g., phthalic acid to form plasticizers for polyvinylchloride.
U.S. Pat. No. 3,676,523, herein incorporated by reference, discloses the use of an ethylene oligomerization catalyst in the production of such olefinic products which comprises (1) a divalent nickel salt, (2) a boron hydride reducing agent, and (3) an o-dihydrocarbylphosphinobenzoic acid or alkali metal salt thereof.
One drawback to the use of this catalyst, however, is expense. There exists a need for a lower cost catalyst in the production of such olefinic products.
This invention relates to a process for the oligomerization of ethylene to a mixture of olefinic products having high linearity by using a catalyst comprising a simple divalent nickel salt; a boron hydride reducing agent; a water soluble base; a ligand selected from the group consisting of o-dihydrocarbyl-phosphinobenzoic acids and alkali metal salts thereof; and a trivalent (three-coordinate) phosphite.
It has been found that the use of a certain ligand provides for a cost effective catalyst useful in the production of olefinic products.
Nickel Salts:
In general, any simple divalent nickel salt can be employed for preparing the catalyst composition of the invention provided the nickel salt is sufficiently soluble in the reaction medium. By the term xe2x80x9csimple divalentxe2x80x9d nickel salt is meant a nickel atom having a formal valence of +2 and bonded through ionic or electrovalent linkages to two singly charged anionic groups (e.g., halides) or to one doubly charged anionic group (e.g., carbonate) and not complexed with or coordinated to any other additional molecular or ionic species. Simple divalent nickel salts therefore do not encompass complex divalent nickel salts which are bonded to one or two anionic groups and additionally complexed or coordinated to neutral chelating ligands or groups such as carbon monoxide and phosphines. However, simple divalent nickel salts are meant to include nickel salts containing water of crystallization in addition to one or two anionic groups.
In most instances, a simple divalent nickel salt with a solubility in the reaction diluent or solvent employed for catalyst preparation of at least 0.001 mole per liter (0.001M) is satisfactory for use as the nickel catalyst precursor. A solubility in the reaction diluent or solvent of at least 0.01 mole per liter (0.01M) is preferred, and a solubility of at least 0.05 mole per liter (0.05M) is most preferred. Reaction diluents and solvents suitably employed for catalyst preparation are the polar organic solvents suitably employed for the oligomerization process which solvents are defined below.
Suitable simple divalent nickel salts include inorganic as well as organic divalent nickel salts. Illustrative inorganic nickel salts are nickel halides such as nickel chloride, nickel bromide and nickel iodide, nickel carbonate, nickel chlorate, nickel ferrocyanide, and nickel nitrate. Illustrative organic divalent nickel salts are nickel salts of carboxylic acids such as nickel alkanoates of up to ten carbon atoms, preferably of up to six carbon atoms, e.g. nickel formate, nickel acetate, nickel propionate, nickel hexanoate and the like; nickel oxalate; nickel benzoate and nickel naphthenate. Other suitable organic salts include nickel benzenesulfonate, nickel citrate, nickel dimethylglyoxime and nickel acetylacetonate.
Nickel halides, especially nickel chloride, and nickel alkanoates, in part because of their availability at low cost and solubility in polar organic solvents are preferred nickel salts.
Dihydrocarbylphosphinobenzoic Acid:
The o-dihydro-carbylphosphino-benzoate ligands employed in the preparation of the catalyst composition of the invention generally have from eight to 30 carbon atoms, but preferably from 14 to 20 carbon atoms, and are preferably represented by the formula (I):
wherein R is a monovalent hydrocarbyl group and M is hydrogen or an alkali metal. The M group is preferably hydrogen, sodium or potassium. Illustrative examples of R groups are hydrocarbon alkyl groups such as methyl, ethyl, isobutyl, lauryl, stearyl, cyclohexyl, and cyclopentyl; hydrocarbon alkenyl R groups having aromatic substituents such as benzyl, phenylcyclohexyl, and phenylbutenyl. Aromatic R groups such as phenyl, tolyl, xylyl and p-ethylphenyl. Preferred R groups are aromatic groups of six to ten carbon atoms, especially phenyl, and cycloalkyl of five to ten carbon atoms, especially cyclohexyl.
Illustrative examples of o-dihydrocarbyl-phosphinobenzoate ligands of formula (I) are o-diphenylphosphinobenzoic acid, o-(methylphenyl-phosphino)benzoic acid, o-(ethyltolylphosphino)benzoic acid, o-dicyclohexylphosphinobenzoic acid, o-(cyclohexyl-phenylphosphino)benzoic acid, o-dipentylphosphinobenzoic acid and the alkali metal salts thereof.
Preferred benzoate ligands of formula (I) are those wherein the R groups are aromatic or cycloalkyl of six to ten carbon atoms, particularly diarylphosphinobenzoic acids, arylcycloalkylphosphinobenzoic acids and the alkali metal salts thereof. Such aryl- and cycloalkyl-substituted phosphino-benzoate ligands are preferred largely because catalyst compositions prepared therefrom catalyze the oligomerization of ethylene to a product mixture containing a high proportion of oligomers in the useful C12-C20 carbon range.
Although the o-dihydrocarbylphosphinobenzoate ligands are suitably employed as the free acid, better results are occasionally obtained with the alkali metal salts of the o-dihydrocarbylbenzoic acid. The alkali metal salts are suitably preformed from the benzoic acid by treatment with an alkali metal hydroxide or oxide solution prior to catalyst preparation or, alternatively, the carboxylic acid salt is generated in situ by the reaction of equimolar amounts of the carboxylic acid and an alkali metal hydroxide during catalyst preparation.
When preparing the catalyst, the molar ratio of nickel salt to benzoate ligand (free acid or salt thereof) is at least 1:1, i.e., at least one mole nickel salt is provided for each mole of benzoate ligand. Suitable molar ratios of nickel salt to benzoic acid ligand (or salt thereof) range from about 1:1 to about 10:1, although molar ratios of about 1:1 to about 3:1 are preferred.
Boron Hydride Reducing Agent:
In general, any boron hydride salt reducing agent of reasonable purity is suitable for use in the process of the invention. Specific examples include alkali metal borohydrides such as sodium borohydrides, potassium borohydride and lithium borohydride; alkali metal alkoxyborohydrides wherein each alkoxy has one to four carbon atoms, such as sodium trimethoxyborohydride and potassium tripropoxyborohydride and tetraalkylammonium borohydrides wherein each alkyl has one to four carbon atoms, such as tetraethylammonium borohydride. Largely because of commercial availability, alkali metal borohydrides are preferred and especially preferred is sodium borohydride.
When preparing the catalyst, the molar ratio of boron hydride salt to nickel salt is at least about 0.2:1. There does not appear to be a definite upper limit on the boron hydride/nickel ratio, but for economic reasons it is especially preferred that the molar ratio be not greater than about 15:1. The preferred molar ratio of boron hydride to nickel salt is usually between about 0.25:1 and about 5:1; more preferred is a ratio between about 0.5:1 and about 2:1. Best results are often obtained when the molar ratio is about 2:1.
Water Soluble Base:
Any water soluble base may be used for pH adjustment purposes. Examples include potassium bicarbonate, potassium methoxide, potassium ethoxide, potassium isopropoxide, potassium hydroxide, and potassium tert-butoxide as well as the corresponding sodium compounds.
When preparing the catalyst, the molar ratio of water soluble base to boron hydride salt ranges from about 0:1 to about 5:1. The preferred molar ratio of water soluble base to boron hydride is usually between about 0.25:1 and about 2:1.
Phosphite:
Any trivalent phosphite can be used, however, alkyl phosphites are preferred and linear alkyl phosphites are most preferred. Examples of suitable phosphites are triisopropyl-, triisobutyl-, tri-sec-butyl-, trimethyl-, triethyl-, tri-n-propyl-, and tri-n-butylphosphite. When preparing the catalyst, the molar ratio of benzoate ligand to phosphite can range from about 50:1 to about 1000:1, preferably in the range of from about 100:1 to about 300:1.
Catalyst Preparation:
The catalyst composition of the present invention is suitably preformed by contacting the catalyst precursors, i.e., the nickel salt, the benzoic acid ligand, the phosphite, the water soluble base and the boron hydride reducing agent, in the presence of ethylene in a polar organic solvent (or diluent), e.g., polar organic diluents or solvents employed for the oligomerization process which are not reduced by the boron hydride reducing agent. In a preferred modification, the nickel salt, borohydride and base are contacted under an ethylene atmosphere. The benzoic acid ligand and the trivalent phosphite are then added. Generally, the catalyst precursors are contacted under about 10 to about 1,500 psig of ethylene.
The catalyst is generally prepared at temperatures of about 0xc2x0 C. to about 50xc2x0 C., although substantially ambient temperatures, e.g. about 10xc2x0 C. to about 30xc2x0 C., are preferred. Contact times of about 5 minutes to 1 hour are generally satisfactory, but can be longer.
Reaction Conditions:
The ethylene is contacted with the catalyst composition in the liquid phase in the presence of a reaction solvent or diluent or solvent of up to about 30 liters per mole of ethylene are satisfactorily employed. Generally, the concentration of the catalyst, calculated as nickel metal, in the solvent or diluent is at least 0.001M, but preferably from about 0.002M to about 0.01M.
Suitable solvents (or diluents) are polar organic compounds such as organic compounds containing atoms such as oxygen, sulfur, nitrogen and phosphorus incorporated in functional groups such as, for example, hydroxy, alkoxy, aryloxy, carbalkoxy, alkanoyloxy, cyano, amino, alkylamino, diakylamine, amide, N-alkylamide, N,N-dialkylamide, sulfonylalkyl and like functional groups. Illustrative oxygenated organic solvents are fully esterified polyacyl esters of polyhydroxy alkanes such as glycerol triacetate, tetracyl esters of erythritol, diethylene glycol diacetate; monoesters such as ethyl acetate, butyl propionate and phenyl acetate; cycloalkyl ethers, e.g., dioxane, tetrahydropyran; acyclic alkyl ethers, e.g., dimethoxyethane, diethylene glycol dimethyl ether and dibutyl ether, aromatic ethers such as anisole, 1,4-dimethoxybenzene and p-methoxytoluene; aliphatic alcohols such as methanol trifluoroethanol, hexafluoroethanol, trifluoropropanol, sec-butanol, perfluorobutanol, octanol, dodecanol, cycloalkanols, e.g., cyclopentanol, and cyclo-hexanol, polyhydric acyclic hydroxyalkanes such as glycerol and trimethylene glycol, alkanediols of two to ten carbon atoms such as ethylene glycol, propylene glycol, 1,4-butanediol and 2,5-hexanediol; phenols, such as cresol, p-chlorophenol, m-bromophenol, 2,6-dimethylphenol, p-methoxyphenol, 2,4-dichlorophenol; and alkylene carbonates such as ethylene carbonate, propylene carbonate and butylene carbonate. Illustrative examples of nitrogen-containing organic solvents are nitriles, e.g., acetonitrile and propionitrile; amines, e.g., butylamine, dibutylamine, trihexylamine, N-methylpyrolidine, N-methylpiperidine, and aniline; N,N-dialkylamides, e.g., N,N-dimethylformamide and N,N-dimethylacetamide. Illustrative examples of sulfur-containing solvents are sulfolane and dimethylsulfoxide and illustrative phosphorus-containing solvents are trialkylphosphate, e.g., trimethylphosphate, triethylphosphate and tributylphosphate and hexaalkylphosphoramides such as hexamethylphosphoramide.
Preferred reaction diluents and solvents are oxygenated organic solvents. Especially preferred are alkanediols of four to six carbon atoms, e.g., 1,4butanediol and 2,5-hexanediol. Polar organic solvents and diluents are preferred for use in the process in part because the ethylene oligomerization product mixture is essentially insoluble in such solvents and diluents. For example, when a polar organic solvent such as an alkanediol is employed, a two phase reaction mixture is formed, i.e., one phase comprising the ethylene oligomerization product mixture, i.e., the alpha-olefins, and a second phase comprising the nickel catalyst and the reaction diluent of solvent. Where a two phase reaction is formed, the ethylene oligomerization product phase is separated and the catalyst containing diluent or solvent phase is utilized for further ethylene oligomerization. Polar organic solvents are also preferred in part because the same solvents are employed in catalyst preparation as defined above.
The precise method of establishing ethylene/catalyst contact during the oligomerization reaction is not critical. In one embodiment, the catalyst composition and the solvent are charged to an autoclave or similar pressure reactor, the ethylene is introduced, and the reaction mixture is maintained with agitation at reaction temperature and pressure for the desired reaction period. In the modification wherein a polar organic solvent is employed and a two phase reaction is formed, ethylene is passed in a continuous manner into a reaction zone containing the catalyst composition and the diluent while ethylene oligomerization product mixture which is produced is concomitantly withdrawn from the reaction zone.
In general, the oligomerization process is conducted at moderate temperatures and pressures. Suitable reaction temperatures vary from about 0xc2x0 C. to about 200xc2x0 C. The reaction is conducted at or above atmosphere pressure. The precise pressure is not critical so long as the reaction mixture is maintained substantially in a liquid phase. Typical pressures can vary from about 10 psig to about 5,000 psig with the range from about 400 psig to about 1,500 psig being preferred.
The oligomerization products are separated and recovered from the reaction mixture by conventional methods such as fractional distillation, selective extraction, adsorption and the like. The reaction solvent, catalyst and any unreacted ethylene can be recycled for further utilization. Spent catalyst, i.e., catalyst no longer active for ethylene oligomerization, can be regenerated for example, by reacting with additional boron hydride reducing agent and nickel salt in the molar ratios (based on benzoic acid ligand) hereinbefore defined. Additional benzoic acid ligand can be added to the regenerated catalyst but it is not required to regenerate the spent catalyst.
During the oligomerization process ethylene is converted to dimer, trimer, tetramer, and larger oligomers. The products are characterized by a high proportion (greater than about 95%) of linear terminal olefins with high linearity (greater than about 90%). The particular product composition generally depends upon the catalyst of the invention employed, the solvent employed, the reaction conditions, particularly reaction temperatures and diluent and whether the catalyst is used in the homogeneous or heterogeneous state. Depending upon the desired product mixture, the optimized components and conditions can readily be determined by one skilled in the art.
The ethylene oligomer products are materials of established utility and many are chemicals of commerce. The products can be converted by conventional catalysts to the corresponding alcohols.
The instant invention will be illustrated by the following illustrative embodiments which are provided for illustration only and are not to be construed as limiting the invention.
A series of ethylene oligomerization reactions was conducted with a nickel catalyst prepared by reacting nickel chloride hexahydrate (NiCl2xe2x80xa26H2O), potassium hydroxide, a dihydrocarbylphosphinobenzoic acid, sodium borohydride and optionally triethylphosphite in a reaction medium of 1,4-butanediol and ethylene. In this set of examples, Example 1 illustrates the effect of triethylphosphite addition in conjunction with reducing the o-dihydrocarbylphosphinobenzoic charge. Example 2 illustrates the effect of reducing the o-dihydrocarbylphosphinobenzoic charge only. Example 3 represents the normal mode of operation and serves as the control experiment.

Nickel-containing ethylene oligomerization catalyst and use thereof