New synthetic Lipid-A analogs based on monosaccharide (1) and disaccharide (2) derivatives were designed and prepared in the present invention. Both structures (1) and (2) incorporate novel lipid structures (3) and (4) that are not found in nature. Also, novel disaccharide Lipid-A structures (2) that incorporate novel contingents of uniform lipids and where R.sub.1, R.sub.4 and R.sub.5 are the same substitution group of structure (III) were synthesized. Liposome formulations containing totally synthetic components such as synthetic Lipid-A and synthetic lipopeptide derived from tumor-associated MUC1 mucin are described along with their therapeutic utility. Comparative test results of immunostimulating properties and toxicity of Lipid-A analogs (1) and (2) are included.

Description of the Invention

SUMMARY OF THE INVENTION

Though there are several publications detailing the minimal structure required for Lipid-A molecules for adjuvant activity with low toxicity, there has been no systematic study of structural features needed to maintain this activity.

A structurally defined molecule as an adjuvant is not commercially available for use with human therapeutic vaccines although some promising adjuvants are currently under clinical investigation. One example of such promising adjuvants is a natural Lipid-A product purified from bacterial cultures. The natural Lipid-A adjuvant product contains a mixture of several Lipid-A components with varying number of lipid chains. Lipid chain esters, which are attached to the carbohydrate core-, can be cleaved during controlled hydrolysis of cell wall, leading to the formation of many components. One of the major problems associated with these, preparations is the inconsistency in composition and performance as an adjuvant, the latter being highly critical to the effectiveness of vaccine based therapies. Other added factors such as high production costs and the difficulty in determining active ingredients in the final pharmaceutical composition render such adjuvants from natural sources commercially unattractive.

Synthetic Lipid-A analogs have several advantages over naturally derived adjuvant preparations. Synthetic compound is chemically defined with single structure and thus facilitates its tracking and control from manufacturing to final formulation. Synthetic product is cost effective and is easily adaptable for commercial scale-up while maintaining the consistency in both quality and performance.

Under the present invention novel Lipid-A analogs are designed, synthesized and finally incorporated into liposome vaccines containing the cancer associated mucin (MUC1) derived antigen, as a lipopeptide, to evaluate their adjuvanticity. Salient features of the present invention are described as follows.

New Lipid Structures

Though on any given natural Lipid-A structure the contingent of lipids are never of uniform length or structure, the most commonly found lipid in nature is (R)-3-hydroxy-tetradecanoic acid (3-hydroxy myristic acid) and its 3-O-acylated derivatives. Lipid diversity contributes to by far the most significant variations among natural Lipid-A structures. While they are all linked through ester and amide bonds to the hydroxy and amino groups of the sugar respectively, variations include the number of lipids attached, the length of each lipid chain and the functional groups contained within the lipid chains. It is believed that these variations contribute to various biological functions of the entire Lipid-A molecule and more importantly to its adjuvant properties. Chemically speaking, ester linkages are labile as they are vulnerable to hydrolysis under physiological conditions. Gradual loss of lipid chains may slowly reduce the activity of the adjuvant under long storage of the vaccines thus diminishing their shelf life. Introduction of unnatural but stable ether linkages in place of esters, or combinations of both may enhance the stability of the adjuvant and may result in the longer shelf life for the vaccine formulations. A major advantage in the synthesis of a Lipid-A analog is that a molecule may be designed to achieve effectiveness as an adjuvant, safety and stability using the diversity in lipid chains and their linkages.

Incorporating these features, we have designed new synthetic lipid acids with general formulae (3) and (4) (FIG. 2, see Original Patent) for building synthetic Lipid-A analogs. Compounds 5 and 23 were prepared in this invention as two specific examples of general structures (3) and (4). Compound 5 contains an aspartic acid moiety, which can be viewed as an b-amino acid. Its absolute configuration corresponds to (R)-3-hydroxy-tetradecanoic acid and thus compound 5 is considered to be a mimic of (R)-3-acyloxy-tetradecanoic acid. Compound 23 is a tri-lipid fatty acid containing an ether linkage, incorporated to enhance the stability of the whole molecule. Though ester based tri-lipid constructs have not so far been discovered among natural Lipid-A analogs, their presence may not be entirely excluded. The present invention also focuses on the synthesis of Lipid-A with a uniform lipid contingent in order to compare the adjuvant activities of those that exhibit lipid diversity.

Lipid-A Analogs with New Lipid Acid Attachments

Lipids of general formulae (3) and (4) (FIG. 2, see Original Patent) are of new design and thus the corresponding Lipid-A structures that incorporate them are all distinctive. Two types of Lipid-A analogs, monosaccharide (1) and disaccharide (2) (FIG. 3, see Original Patent), have been designed and synthesized as part of the invention.

The monosaccharide derivative of structure (1) (see Original Patent) where at least one of R.sub.1 and R.sub.2 is independently chosen from structures (I) and (II) (FIG. 3) features the non-reducing end sugar of the natural Lipid-A structure. Compound 33 (FIG. 9, see Original Patent) is an example of such structures.

And the disaccharide derivative of structure (2) (see Original Patent)where at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is independently chosen from structures (I) and (II) (FIG. 3), see Original Patent is a monophosphorylated analog of natural Lipid-A structure. Compounds 58 (FIG. 16, see Original Patent), 77 (FIG. 21, see Original Patent), 102 (FIG. 29, see Original Patent) and 104 (FIG. 30, see Original Patent) are some examples with such structural features.

Lipid-A Analogs with Uniform Lipid Contingent

The lipid diversity of Lipid-A renders the molecule too complex and impractical for large-scale preparation through chemical synthesis. One of the main features of compounds designed as part of this invention is that the lipid substituents on 2-amino, 2'-amino, and 3'-O positions of the disaccharide backbone are identical and are composed of either ether or ester-linked di-lipid structure fragments. Such features are summarized in FIG. 4 (see Original Patent). The compound has the general formula (2) where R.sub.1, R.sub.4 and R.sub.5 are identically having the di-lipid structure (III) (see Original Patent). Specific compounds are prepared in this invention as representative examples, such as structure 54 (FIG. 15, see Original Patent), see Original Patent, 70 (FIG. 19, see Original Patent), 86 (FIG. 24, see Original Patent) and 94 (FIG. 26, see Original Patent).

A New Process for the Preparation of Lipid-A Analogs

The invention includes new processes for the synthesis of Lipid-A analogs (1) and (2). Disclosed herein are general synthetic routes to prepare variously substituted Lipid-A analogs of the invention. Different analogs can be easily obtained by using alternative starting materials. Details are illustrated in drawing figures and examples.

The process for the synthesis of monosaccharide derivative 33 is illustrated in FIG. 9 and the disaccharide derivative 48 in FIG. 10-FIG. 13 (see Original Patent). An important feature of this new process is the general strategy of using combinations of different carbohydrate building blocks, protecting group strategies and reagents to accomplish specific structures. For example, the 4,6-benzylidene protection on glucosamine derivative offers the freedom of selective ring opening to free the 4-OH on which the phosphate group may be introduced. Benzyl ester protected phosphate group, which is introduced through a two-step procedure, also offers the advantage of being easily deprotected, together with other benzyl groups on the molecule, at the final stage of the synthesis through catalytic hydrogenation. More examples are described in FIG. 14-FIG. 21 (see Original Patent) for the synthesis of compounds 54, 58, 70 and 77.

This process has been further modified to provide a more efficient procedure, which is particularly useful for the preparation of compounds with identical substituents on both amino groups of the carbohydrate backbone. FIGS. 22-24 illustrate the synthesis of compound 86 using this modified process.

In this modified process the phosphate group is introduced on the monosaccharide derivative before the glycosidic linkage is formed, and the glycosylation acceptor, which has both 4-OH and 6-OH groups unprotected (e.g. compound 79 in FIG. 22), is prepared through a simplified pathway. The steps involved in the whole process are reduced, especially at the disaccharide stage at which the material becomes more expensive and practically more difficult to handle. The process is designed for large-scale production and has been proven to be very efficient at gram-scale synthesis of a Lipid-A analog. Additional examples of compounds 94, 102 and 104, prepared using this modified process, are described in FIGS. 25-30 (see Original Patent).

The strategic intermediates disclosed in this invention are used in the synthesis of Lipid-A analogs and are not known in the literature.

Liposome Formulations

Liposomes are globular particles formed by the physical self-assembly of polar lipids, which define the membrane organization in liposomes. Liposomes may be formed as uni-lamellar or multi-lamellar vesicles of various sizes. Such liposomes, though constituted of small molecules having no immunogenic properties of their own, behave like macromolecular particles and display strong immunogenic characteristics.

Taking advantage of the self-assembling properties of lipids, one or more immunogens may be attached to the polar lipids that in turn become part of the liposome particle. Each immunogen comprises one or more antigenic determinants (epitopes). These epitopes may be B-cell epitopes (recognized by antibodies) or T-cell epitopes (recognized by T-cells). The liposome can act to adjuvant the immune response elicited by the associated immunogens. It is likely to be more effective than an adjuvant that is simply mixed with an immunogen, as it will have a higher local effective concentration.

Moreover, a hapten may be attached in place of the aforementioned immunogen. Like an immunogen, a hapten comprises an antigenic determinant, but by definition is too small to elicit an immune response on its own (typically, haptens are smaller than 5,000 daltons). In this case, the lipid moiety may act, not only as an adjuvant, but also as an immunogenic carrier, the conjugate of the hapten and the lipid acting as a synthetic immunogen (that is, a substance against which humoral and/or cellular immune responses may be elicited).

Even if the lipid does not act as an immunogenic carrier, the liposome borne hapten may still act as a synthetic antigen (that is, a substance which is recognized by a component of the humoral or cellular immune system, such as an antibody or T-cell). The term "antigen" includes both haptens and immunogens.

Thus, the invention contemplates a liposome whose membrane comprises a Lipid A analogue as disclosed herein, and at least one B-cell or T-cell epitope.

We have designed several synthetic antigens in the form of lipo-peptides, glyco-lipids and glyco-lipo-peptides that form the liposome membrane. Similarly, synthetic Lipid-A molecules of well-defined structural characteristics can be anchored into the liposome membrane.

Unlike the bacterial adjuvant preparations, a synthetic Lipid-A analog contributes a structurally well-defined lipids to the liposome membrane. Such defined structures not only reduce the burden of re-affirming the `active` membrane components after formulation, but also contribute to the definition of liposome membrane. Such liposomes may be designated as totally synthetic vaccine formulations' containing synthetic Lipid-A analog as an adjuvant and a synthetic lipopeptide as an antigen.

Epitope

The epitopes of the present invention may be B-cell or T-cell epitopes, and they may be of any chemical nature, including without limitation peptides, carbohydrates, lipids, glycopeptides and glycolipids. The epitope may be identical to a naturally occurring epitope, or a modified form of a naturally occurring epitope.

B-cell peptide epitopes are typically at least five amino acids, more often at least six amino acids, still more often at least seven or eight amino acids in length, and may be continuous ("linear") or discontinuous ("conformational") (the latter being formed by the folding of a protein to bring noncontiguous parts of the primary amino acid sequence into physical proximity). T-cell peptide epitopes are linear and usually 8 to 15, more often 9-11 amino acids in length.

Epitopes of interest include those specific to or otherwise associated with a pathogen, or a tumor. An epitope may be said to be associated with a particular infectious disease if it is presented by an intracellular, surface, or secreted antigen of the organism which causes the disease, or in the case of a virus, if it is associated with viral particles or is specific to a cell infected by the virus.

It may be said to be associated with a particular tumor if it is presented by an intracellular, surface or secreted antigen of said tumor. It need not be presented by all cell lines of the tumor type in question, or by all-cells of a particular tumor, or throughout the entire life of the tumor. It need not be specific to the tumor in question. An epitope may be said to be "tumor associated" in general if it is so associated with any tumor (cancer, neoplasm).

The term "disease associated epitope" also includes any non-naturally occurring epitope which is sufficiently similar to an epitope naturally associated with the disease in question so that cytotoxic lymphocytes which recognize the natural disease epitope also recognize the similar non-natural epitope. Similar comments apply to "tumor associated epitope".

An epitope may be said to be specific to a particular source (such as a disease-causing organism or a tumor), if it is associated more frequently with that source than with other sources. Absolute specificity is not required, provided that a useful prophylactic, therapeutic or diagnostic effect is still obtained.

In the case of a "tumor-specific" epitope, it is more frequently associated with that tumor that with other tumors, or with normal cells. Preferably, there should be a statistically significant (p=0.05) difference between its frequency of occurrence in association with the tumor in question, and its frequency of occurrence in association with (a) normal cells of the type from which the tumor is derived, and (b) at least one other type of tumor. An epitope may be said to be "tumor-specific" in general is it is associated more frequently with tumors (of any or all types) than with normal cells. It need not be associated with all tumors.

The term "tumor specific epitope" also includes any non-naturally occurring epitope which is sufficiently similar to a naturally occurring epitope specific to the tumor in question (or as appropriate, specific to tumors in general) so that cytotoxic lymphocytes stimulated by the similar epitope will be essentially as specific as CTLs stimulated by the natural epitope.

In general, tumor-versus-normal specificity is more important than tumor-versus-tumor specificity as (depending on the route of administration and the particular normal tissue affected), higher specificity generally leads to fewer adverse effects. Tumor-versus-tumor specificity is more important in diagnostic as opposed to therapeutic uses.

The reference to a CTL epitope as being "restricted" by a particular allele of MHC, such as HLA-A1, indicates that such epitope is bound and presented by the allelic form in question. It does not mean that said epitope might not also be bound and presented by a different allelic form of MHC, such as HLA-A2, HLA-A3, HLA-B7, or HLA-B44.

The term "specific" is not intended to connote absolute specificity, merely a clinically useful difference in probability of occurrence in association with a pathogen or tumor rather than in a matched normal subject.

Pathogens may be submicrobial (e.g., viruses), microbial (e.g., fungi, protozoa), or multicellular (e.g, worms, arthropods, etc.). Tumors may be of mesenchymal or epithelial origin. Cancers include cancers of the colon, rectum, cervix, breast, lung, stomach, uterus, skin, mouth, tung, lips, larynx, kidney, bladder, prostate, brain, and blood cells.

Naturally occurring epitopes may be identified by a divide-and-test process. One starts with a protein known to be antigenic or immunogenic. One next tests fragments of the protein for immunological activity. These fragments may be obtained by treatment of the protein with a proteolytic agent, or, if the peptide sequence is known, one may synthetically prepare smaller peptides corresponding to subsequences of the protein. The tested fragments may span the entire protein sequence, or just a portion thereof, and they may be abutting, overlapping, or separated.

If any of the fragments are immunologically active, the active fragments may themselves be subjected to a divide-and-test analysis, and the process may be continued until the minimal length immunologically active sequences are identified. This approach may be used to identify either B-cell or T-cell epitopes, although the assays will of course be different. Geysen teaches systematically screening all possible oligopeptide (pref. 6-10 a.a.) abutting or overlapping fragments of a particular protein for immunological activity in order to identify linear epitopes. See WO 84/03564.

It is also possible to predict the location of B-cell or T-cell peptide epitopes if an amino acid sequence is available. B-cell epitopes tend to be in regions of high local average hydrophilicity.

See Hopp and Wood, Proc. Nat. Acad. Sci. (USA) 78: 3824 (1981); Jameson and Wolf, CABIOS, 4: 181 (1988). T-cell epitopes can be predicted on the basis of known consensus sequences for the peptides bound to MHC class I molecules of cells of a particular haplotype. See e.g., Slingluff, WO98/33810, especially pp. 15-16; Parker, et al., "Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side chains", J. Immunol. 152: 163 (1994).

Naturally occurring T-cell epitopes may be recovered by dissociating them from their complexes with MHC class I molecules and then sequencing them, e.g., by mass spectroscopic techniques.

Generally speaking, in addition to epitopes which are identical to the naturally occurring disease- or tumor-specific epitopes, the present invention embraces epitopes which are different from but substantially identical with such epitopes, and therefore disease- or tumor-specific in their own right. It also includes epitopes which are not substantial identical to a naturally occurring epitope, but which are nonetheless cross-reactive with the latter as a result of a similarity in 3D conformation.

An epitope is considered substantially identical to a reference epitope (e.g., a naturally occurring epitope) if it has at least 10% of an immunological activity of the reference epitope and differs from the reference epitope by no more than one non-conservative substitution.

If it is a CTL epitope, it may incorporate further nonconservative substitutions which are suggested by a known binding motif of the pertinent MHC molecule. Kast, et al., J. Immunol, 152:3904-12 (1994) sets forth HLA-A specific peptide binding motifs for the HLA molecules A1, A2.1, A3, A11 and A24. Engelhard, et al., in Sette, ed., Naturally Processed Peptides, 57:39-62 (1993) explored the features that determined binding to HLA-A2.1 and HLA-B7. See also Hobohim et al; Eur. J. Immunol., 23:127'-6 (1993); Kawakami, et al., J. Immunol., 154:3961-8 (1995). Based on these and other sources, the preferred and tolerated AAs for various HLA molecules include (but are not limited to) the following -- see Original Patent.

If a position is not listed, studies revealed a greater variability of AAs than for the listed positions. For listed positions, AAs not listed may be tolerated, especially if they are conservative or semi-conservative substitutions for "preferred" or "tolerated" AAs.

Conservative substitutions are herein defined as exchanges within one of the following five groups: I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly II. Polar, negatively charged residues: and their amides Asp, Asn, Glu, Gln III. Polar, positively charged residues: His, Arg, Lys IV. Large, aliphatic, nonpolar residues: Met, Leu, Ile, Val, Cys V. Large, aromatic residues: Phe, Tyr, Trp

Within the foregoing groups, the following substitutions are considered "highly conservative": Asp/Glu His/Arg/Lys Phe/Tyr/Trp Met/Leu/Ile/Val

Semi-conservative substitutions are defined to be exchanges between two of groups (I)-(V) above which are limited to supergroup (A), comprising (I), (II) and (III) above, or to supergroup (B), comprising (IV) and (V) above. Also, Ala is considered a semi-conservative substitution for all non group I amino acids.

It will be appreciated that highly conservative substitutions are less likely to affect activity than other conservative substitutions, conservative substitutions are less likely to affect activity than merely semi-conservative substitutions, and semi-conservative substitutions less so than other non-conservative substitutions. In addition, single substitutions are less likely to affect activity than are multiple mutations.

Although a substitution mutant, either single or multiple, of the peptides of interest may not have quite the potency of the original peptide, such a mutant may well be useful.

Substitutions are not limited to the genetically encoded, or even the naturally occurring amino acids. When the epitope is prepared by peptide synthesis, the desired amino acid may be used directly. Alternatively, a genetically encoded amino acid may be modified by reacting it with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.

A non-genetically encoded amino acid is considered a conservative substitution for a genetically encoded amino acid if it is more similar in size (volume) and hydrophilicity to the original amino acid, and to other amino acids in the same exchange group, than it is to genetically encoded amino acids belonging to other exchange groups.

Substantially identical peptide epitopes may be identified by a variety of techniques, some of which do not depend on preexisting knowledge of the binding motif. Thus, it is known in the art that one may synthesize all possible single substitution mutants of a known peptide epitope. For a nonpeptide, there are (20.times.9-1=179) such mutants. Geysen, et al., Proc Nat. Acad. Sci. (USA), 81:3998-4002 (1984). While the effects of different substitutions are not always additive, it is reasonable to expect that two favorable or neutral single substitutions at different residue positions in the epitope can safely be combined in most cases.

Both naturally occurring and non-naturally occurring peptide epitopes may be identified, if a suitable antibody or other receptor is available, by screening a peptide combinatorial library for peptides bound by the target. Humoral peptide epitopes may be identified by screening a combinatorial peptide phage library for specific binding to a target monoclonal antibody known to recognize the antigen of interest. Preferably, the library is prescreened to eliminate peptides which bind the antibody other than at the epitope binding site of the antibody; this can be done by eliminating phage which bind to a second, control antibody of the same isotype.

Similarly, to identify CTL peptide epitopes, one may synthesize a family of related single or multiple substitution mutants, present the mixture to the HLA-A2.1 positive lymphoblastoid cell line T2 (or other cell line capable of presenting specific CTL epitopes), and expose the T2 cells to CTLs of the desired specificity. If the T2 cells are lysed, the effective epitopes may be identified either by direct recovery from the T2 cells or by a progressive process of testing subsets of the effective peptide mixtures. Methods for the preparation of degenerate peptides are described in Rutter, U.S. Pat. No. 5,010,175, Haughten, et al., Proc. Nat. Acad. Sci. (USA), 82:5131-35 (1985), Geysen, et al., Proc. Nat. Acad. Sci. (USA), 81:3998-4002 (1984); WO86/06487; WO86/00991.

Multiple mutagenesis may be used to screen a few residue positions intensely or a larger number of positions more diffusely. One approach is to explore at least a representative member of each a.a. type at each position, e.g., one representative of each of exchange groups I-V as hereafter defined. Preferably, Gly and Pro are screened in addition to one other group I residue. Preferably, at least one screened residue is an H-bonding residue. If a positive mutant features a particular representative, like amino acids can be explored in a subsequent library. If, for example, a Phe substitution improves binding, Tyr and Trp can be examined in the next round.

The person of ordinary skill in the art, in determining which residues to vary, may also make comparisons of the sequences of the naturally processed MHC associated peptides, and may obtain 3D structures of the MHC: peptide: TCR complexes, in order to identify residues involved in MHC or TCR binding. Such residues may either be left alone, or judiciously mutated in an attempt to enhance MHC or TCR binding.

An extensive discussion of carbohydrate haptens appears in Wong, U.S. Pat. No. 6,013,779.

Adjuvanticity of Lipid-A Analogs

It is generally understood that a synthetic antigen of low molecular weight is weakly immunogenic, which is the biggest obstacle to the success of a fully synthetic vaccine. One way to improve the immunogenicity of such a synthetic antigen is to deliver it in the environment of an adjuvant. The primary target of those new synthetic Lipid-A analogs in the present invention is their adjuvant properties. An ideal adjuvant is believed to non-specifically stimulate the immune system of the host, which upon the subsequent encounter of any foreign antigen can produce strong and specific immune response to that foreign antigen. Such strong and specific immune response, which is also characterized by its memory, can be produced only when T-lymphocytes (T-cells) of the host immune system are activated. Here we choose T-cell blastogenesis and IFN-.gamma. production as two important parameters for measuring the immune response.

Experimentally T-cell blastogenesis measures DNA synthesis that directly relates to T-cell proliferation, which in turn is the direct result of the T-cell activation. On the other hand, IFN-.gamma. is a major cytokine secreted by T-cells when they are activated. There fore, both T-cell blastogenesis and IFN-.gamma. production indicate T-cell activation, which suggests the ability of an adjuvant in helping the host immune system to induce a strong and specific immune response to any protein-based antigen. By using a synthetic lipopeptide antigen, H.sub.2N-STAPPAHGVTSAPDTRPAPGSTAPPK(Pal)G-OH SEQ ID NO:1 (FIG. 34, single letter amino acid codes are defined in Table 8, see Original Patent), a modified 25-amino-acid sequence that is derived from tumor-associated MUC1 mucin, we were able to evaluate the adjuvant properties of the synthetic Lipid-A analogs disclosed in this invention. Based on the data of T-cell blastogenesis and IFN-.gamma. level (FIGS. 31-33, see Original Patent) obtained through preliminary in vivo/in vitro studies, it is amply demonstrated that synthetic Lipid-A structures 48, 54, 70, 86, 102 and 104 are as effective, as adjuvants, as the Lipid-A preparations of bacterial origin.

The compound is considered an adjuvant if it significantly (p=0.05) increases the level of either T-cell blastogenesis or of interferon gamma production in response to at least one liposome/immunogen combination relative to the level elicited by the immunogen alone. Preferably, it does both. Preferably, the increase is at least 10%, more preferably at least 50%, still more preferably, at least 100%.

Preliminary in vivo toxicity evaluation of synthetic Lipid-A analog 86 has shown that its toxicity is much lower than that of natural Lipid-A product obtained from bacteria Salmonella (Table 4, Example 99, see Original Patent). Thus, there are many advantages Associated with the totally synthetic and novel Lipid-A structures disclosed in this invention, in terms of efficacy, safety, stability and compliance of such vaccine formulations with regulatory guidelines.

Preferably, the toxicity of the lipid compounds of the present invention is not more than 50% that of said natural Lipid-A product; more preferably it is less than 10% that of the latter.

The in vivo studies of the synthetic compounds disclosed in this invention have been limited to the assessment of their effectiveness as adjuvant. But they may have broader applications in other areas such as anti-tumor agents, LPS/Lipid-A antagonists, inhibitors for Lipid-A biosynthesis and thus useful as novel antibiotics. Results of various biological activities will be disclosed in due course.

Pharmaceutical Methods and Preparations

Applicants hereby incorporate by reference the discussion at pp. 32-46 of WO98/33810.

The preferred animal subject of the present invention is a primate mammal. By the term "mammal" is meant an individual belonging to the class Mammalia, which, of course, includes humans. The invention is particularly useful in the treatment of human subjects, although it is intended for veterinary uses as well. By the term "non-human primate" is intended any member of the suborder Anthropoidea except for the family Hominidae. Such non-human primates include the superfamily Ceboidea, family Cebidae (the New World monkeys including the capuchins, howlers, spider monkeys and squirrel monkeys) and family Callithricidae (including the marmosets); the superfamily Cercopithecoidea, family Cercopithecidae (including the macaques, mandrills, baboons, proboscis monkeys, mona monkeys, and the sacred hunaman monkeys of India); and superfamily Hominoidae, family Pongidae (including gibbons, orangutans, gorillas, and chimpanzees). The rhesus monkey is one member of the macaques.

The term "protection", as used herein, is intended to include "prevention," "suppression" and "treatment." "Prevention" involves administration of the protein prior to the induction of the disease. "Suppression" involves administration of the composition prior to the clinical appearance of the disease. "Treatment" involves administration of the protective composition after the appearance of the disease.

It will be understood that in human and veterinary medicine, it is not always possible to distinguish between "preventing" and "suppressing" since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, it is common to use the term "prophylaxis" as distinct from "treatment" to encompass both "preventing" and "suppressing" as defined herein. The term "protection," as used herein, is meant to include "prophylaxis." It should also be understood that to be useful, the protection provided need not be absolute, provided that it is sufficient to carry clinical value. An agent which provides protection to a lesser degree than do competitive agents may still be of value if the other agents are ineffective for a particular individual, if it can be used in combination with other agents to enhance the level of protection, or if it is safer than competitive agents.

The composition may be administered parentally or orally, and, if parentally, either systemically or topically. Parenteral routes include subcutaneous, intravenous intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes. One or more such routes may be employed. Parenteral administration can be, e.g., by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration may be by the oral route. The immunization is preferably accomplished initially by intramuscular injection followed by intradermal injection, although any combination of intradermal and intramuscular injections may be used.

It is understood that the suitable dosage of a immunogen of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. However, the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. This will typically involve adjustment of a standard dose, e.g., reduction of the dose if the patient has a low body weight.

Prior to use in humans, a drug will first be evaluated for safety and efficacy in laboratory animals. In human clinical studies, one would begin with a dose expected to be safe in humans, based on the preclinical data for the drug in question, and on customary doses for analogous drugs (if any). If this dose is effective, the dosage may be decreased, to determine the minimum effective dose, if desired. If this dose is ineffective, it will be cautiously increased, with the patients monitored for signs of side effects. See, e.g., Berkow, et al., eds., The Merck Manual, 15th edition, Merck and Co., Rahway, N.J., 1987; Goodman, et al., eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y., (1990); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. (1987), Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985), which references and references cited therein, are entirely incorporated herein by reference.

The total dose required for each treatment may be administered in multiple doses (which may be the same or different) or in a single dose, according to an immunization schedule, which may be predetermined or ad hoc. The schedule is selected so as to be immunologically effective, i.e., so as to be sufficient to elicit an effective immune response to the antigen and thereby, possibly in conjunction with other agents, to provide protection. The doses adequate to accomplish this are defined as "therapeutically effective doses." (Note that a schedule may be immunologically effective even though an individual dose, if administered by itself, would not be effective, and the meaning of "therapeutically effective dose" is best interpreted in the context of the immunization schedule.) Amounts effective for this use will depend on, e.g., the peptide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.

Typically, the daily dose of an active ingredient of a pharmaceutical, for a 70 kg adult human, is in the range of 10 nanograms to 10 grams. For immunogens, a more typical daily dose for such a patient is in the range of 10 nanograms to 10 milligrams, more likely 1 microgram to 10 milligrams. However, the invention is not limited to these dosage ranges.

It must be kept in mind that the compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the peptides, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions.

The doses may be given at any intervals which are effective. If the interval is too short, immunoparalysis or other adverse effects can occur. If the interval is too long, immunity may suffer. The optimum interval may be longer if the individual doses are larger. Typical intervals are 1 week, 2 weeks, 4 weeks (or one month), 6 weeks, 8 weeks (or two months) and one year. The appropriateness of administering additional doses, and of increasing or decreasing the interval, may be reevaluated on a continuing basis, in view of the patient's immunocompetence (e.g., the level of antibodies to relevant antigens).

A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019369, incorporated herein by reference.

The appropriate dosage form will depend on the disease, the immunogen, and the mode of administration; possibilities include tablets, capsules, lozenges, dental pastes, suppositories, inhalants, solutions, ointments and parenteral depots. See, e.g., Berker, supra, Goodman, supra, Avery, supra and Ebadi, supra, which are entirely incorporated herein by reference, including all references cited therein.
 

Claim 1 of 69 Claims

1. A compound of the following formula -- see Original Patent.

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