WO1997043308A1 - Lipophile derivatives of muramylpeptides for treatment of retroviral infection and induction of chemokines - Google Patents

Abstract

The invention is directed to methods of inducing the release of at least one chemokine by administering an effective amount of a muramyl dipeptide compound ('MDP compound') to a mammal. Another aspect of the invention is directed to methods of treating retroviral infections, such as Human Immunodeficiency Virus (HIV) infections by administering an effective amount of a muramyl dipeptide compound to a mammal. The invention is also directed to a pharmaceutical composition for inducing the release of at least one chemokine and treating retroviral infections, such as Human Immunodeficiency Virus (HIV) infections, wherein the pharmaceutical composition includes an amide linked analog of N-Acetylmuramyl-L-Ala-D-IsoGlutamine. The invention may further include a method of inducing the release of at least one chemokine and a method of treating retroviral infection in a patient by administering an effective amount of a non-toxic enterotoxin such as O-TSST in combination with the MDP compound.

Description

LIPOPHI LE DERIVATIVES OF MURAMYLPEPTIDES FOR TREATMENT OF RETROVIRAL INFECTION AND INDUCTION OF CHEMOKINES

BACKGROUND OF THE INVENTION

The regulation of Human Immunodeficiency Virus replication involves a complex network of endogenous regulatory cytokines and chemokines. For example, interferon α (IFN-α) and interleukin-13 (IL-13) primarily down regulate virus replication while pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin-1 β, up regulate virus replication in-vitro (See, Kinter et al., Proc. Nat 7.

Acad. Sci. 93:14076 (1996)).

In addition to endogenous cytokines, CD8+ cells have been demonstrated to suppress Human Immunodeficiency Virus (HIV) replication, by direct cytotoxic mechanisms and the production and release of soluble factors. Soluble factors identified as major suppressor factors of macrophage tropic HIV include chemokines such as "regulated on activation, normal T cell expressed and secreted" (RANTES), macrophage inflammatory protein lα (MlP-lα ) and macrophage inflammatory protein 1 β (MIP- 1 β) (See, Cocchi et al., Science 270: 1181 (1975)).

The term chemokines is applied to a family of host-derived cytokines capable of stimulating chemotaxis in-vitro and eliciting accumulation of inflammatory cells in- vivo in response to infection, inflammation, and/or trauma (See, for review, Oppenheim et al., Ann Rev Immunol 9:617 (1991)). Chemokines are typically produced by monocytes and/or macrophages and T-cells. However, chemokines may also be produced by endothelial cells, fibroblasts, and keratinocytes. All members ofthe chemokine family have molecular weights of 8- 10 kilodaltons and are basic heparin-binding polypeptides. The family is divided into two subfamilies depending upon their amino acid sequence and chromosome location. Chemokines in the α (alpha) subfamily (CXC) have the first two cysteine residues separated by one amino acid. The genes for the α subfamily are located on region ql2- 21 ofthe human chromosome and share 30-50% homology. In the β subfamily (CC), the two cysteine residues are adjacent and the genes are located on the qll-21 gene of the human chromosome. The intraspecies homology ranges from 28 to 45%.

The natural ligands ofthe CCR-5 receptor are macrophage inflammatory protein lα (MlP-lα), macrophage inflammatory protein lβ (MlP-lβ) and regulated on activation, normally T cell expressed and secreted protein (RANTES). The natural ligand for the CXCR-4 receptor is pre-β-cell growth stimulating factor/stromal cell derived factor- 1 (PBSF/SDF-1).

Chemokine receptors, in particular, CXCR-4 and CCR-5, behave as co-ligands with the CD4 receptor for entry of Human Immunodeficiency Virus (HIV) into lymphocytes and monocytes, respectively. CCR-5 receptors are important in initial infection of monocytes (See, e.g., Alkhatib et al., Science 272:1955 (1996)), while CXCR-4 receptors function in later infection of lymphocytes associated with progression ofthe disease (See, Connor et al., J Exp Med 57:621 (1997); and Feng et al, Science 272:872 (1996)).

Because co-binding to chemokine receptors is required for HIV infection and progression, blocking chemokine receptors with a natural ligand or related analog provides a therapeutic target. In fact, the presence of 100 ng/ml of RANTES causes a 2-3 log decrease in HIV replication in-vitro (See, Oravecz et al., JI 157:1329 (1996)). A derivative of RANTES, created by a chemical modification at the amino terminus with amino-oxypentane (AOP-RANTES), inhibits macrophage tropic virus replication at 200 ng/ml (See, Simmons et al., Science 276:276 (1997)). Comparable inhibition using natural RANTES has also been observed at 800 ng/ml.

Muramyldipeptide (N-Acetylmuramyl-L-Alanine-D-IsoGlutamine), abbreviated MDP, is the minimal immunoadjuvant structure originally isolated from the cells walls of Bacillus Calmette-Guerin (BCG). MDP and related compounds augment both humoral and cell-mediated immunity by inducing cytokines and activating macrophages. Such activated macrophages produce cytokines, for example, interleukin-1 (IL-1), tumor necrosis factor (TNF), interleukin-10 (IL-10), and granulocyte-macrophage colony stimulating factor (GMCSF), and demonstrate enhanced anti-microbial activity as well as an enhanced ability to destroy malignant cells.

A large variety of MDP analogs have been synthesized and evaluated for immunological activity. Modifications include substitutions ofthe first amino acid (L- alanine) with other naturally occurring amino acids or enantiomorphs thereof, for example, L-serine, L-valine or L-threonine. Other modifications include addition of a lipophilic group, usually to the 6-0 position (e.g., 6-0-stearoyl MDP) or to the peptide (e.g., muramyltripeptide phosphatidylamine or muramyl dipeptide glycerol dipalmitate).

The ability of MDP and some related compounds to inhibit HIV in-vitro has been reported. Inhibition of HIV has been demonstrated in CD4+ H9 lymphocyte and U937 monocytoid cell lines using doses of MDP at 100 and 1000 μg, as determined by

P24 antigen production in-vitro. (Masihi et al., AIDS Research and Human Retrovirus 6:393 (1990)). A similar in-vitro inhibitory effect of muramyltripeptide phosphatidyl¬ ethanolamine (MTP-PE) is observed in HIV infected human monocytes at concentrations of 1 to 50 μg/ml (See, Lazdin et al., AIDS Research and Human Retrovirus 6: 1157 (1990)). Additionally, inhibition of HIV replication in human monocytes in-vitro has been observed in the presence of 10 to 100 μg/ml of murabutide (NAc-Mur-L-Ala-D-Gln-O_nC₄H₉) and murametide (NAc-Mur-L-Ala-D-Gln-OMe) (See, W0 96/09837).

Although MDP and several analogs have been shown to have an inhibitory effect on HIV production in-vitro, no studies establishing anti-viral efficacy in-vivo in either man or an acceptable experimental model (e.g., Friend Murine Leukemia or Feline Leukemia) have been reported. The absence of reports of MDP and MDP analogs as effective in-vivo may relate to a lack of evaluation of such factors as dose, dose schedule and to the induction of undesirable cytokines which may augment HIV replication.

Although in-vivo efficacy of MDP type compounds in retrovirus disease has not been demonstrated, several MDP type compounds have been demonstrated to protect against infectious disease. N-Acetylmuramyl-L-Ala-D-IsoGln-β,γ-dipalmitoyl-sn- glycerol has been shown to be effective in protecting against Klebsiella pneumonia when given 24 hours prior to infection (Philips et al., U.S. Patent No. 4,939, 122). Threonine derivatives of N- Acetylmuramyl-L-Thr-D-IsoGln-β,γ-dipalmitoyl-sn- glycerol has been shown to be effective in protecting mice against Klebsiella pneumonia when given 24 hours prior to infection (Chedid et al., U.S. Patent No. 5,210,072). Staphylococcus aureus enterotoxins are causative agents of Staphylococcal food poisoning. Ingestion of enterotoxin in contaminated food leads to rapid development of symptoms characteristic of Staphylococcal food poisoning, such as vomiting and diarrhea. The Staphylococcus aureus enterotoxins are a group of serologically distinct extracellular proteins, SEA, SEB, SEC,, SEC₂, SEC₃, SED, and SEE. Enterotoxins SEC,, SEC₂, and SEC₃ share approximately 95% sequence similarly. SEB and SEC are approximately 45-50% homologous. Other enterotoxins include exfoliating toxins ETA, ETB and ETC from 5". aureus, pyrogenic exotoxins PEA, PEB, and PEC from S. pyrogenes and M. arthritis mitogen from M. arthritis. Toxic shock syndrome toxin, TSST-1, another protein produced by S. aureus that is related to enterotoxins, is the classic agent responsible for toxic shock syndrome, although other staphylococcal enterotoxins may also cause toxic shock by inducing cytokines (predominantly interleukin-1 (IL-1) and tumor necrosis factor (TNF)). TSST-1 and streptococcal pyrogenic enterotoxin C (PEC) share only 20-30% primary sequence homology to SEC.

Despite these sequence differences, the tertiary structure ofthe various enterotoxins show nearly identical folds. Sequence data demonstrate a high degree of similarity in regions of the enterotoxins related to binding ofthe major histocompatibility binding region ofthe monocyte and T-cell receptor (related to T cell activation) (See, Hoffman et al., Infect Immun 62:3396 (1994)).

Mutant enterotoxins can be produced which retain biological activities, such as stimulation of T cells, but have a 100-fold decreased lethality. For example, delivery of 2 mg of a TSST-1 enterotoxin mutant with a change in a single amino acid at position

136 (from histidine to alanine) by miniosmatic pump over 7 days showed no lethality compared the native enterotoxin, which was lethal at a dose of 200 μg, administered over a period of 7 days. However, biological activity of this mutant, as demonstrated by splenomegaly in the animals, was retained (See, Murray et al., Infect Immunol 64:371 (1996)). Mutants of SEC, which are unable to form a disulfide bond are non- toxic (i.e., the specimens did not die) when given doses of up to 10 μg/kg in endotoxin primed rabbits, even though such mutants retain biological activity, as demonstrated by in-vitro mitogenicity (See, Hovde et al., Molecular Microbiol 13:897 (1994)). Ovine staphylococcal enterotoxin ("O-TSST"), which differs by 9 amino acids from TSST-1 (isolated from a human infection), was non-toxic at doses of 200 μg in the miniosmatic pump rabbit toxicity test while retaining mitogenicity (See, Lee et al, JInfDis 165:1056 (1992)). Lipopolysaccharides, abbreviated LPS, are an example of an endotoxin. LPS are the major constituent ofthe cell wall of gram negative bacteria and are potent macrophage activators, causing production and release of monokines responsible for toxic shock syndrome (i.e., tumor necrosis factor (TNF) and interleukin-1 (IL-1)). LPS, at concentrations of 10 pg/ml to 1 pg/ml induces significant levels of RANTES, MlP-lα and MlP-lβ in-vivo and inhibits HIV in-vitro in both macrophage and T-cells. Inhibition of HIV replication in T cells suggests that suppressor factors, other than RANTES, MlP-lα and MIP-1 β are induced by LPS (Verani et al., J Exp Med 185:805 (1997)). Other studies indicate that inhibition of HIV is observed more frequently with primary HIV isolates whereas laboratory strains and HIV from patients with progressive disease have a broader tropism predominantly for T-cells and are less successfully controlled by a single or cocktail of defined chemokines.

ABBREVIATIONS The following abbreviations are used in connection with the invention:

Lipophilic Disaccharides

1. GMTP-NHDPG: N-Acetylglucosaminyl-N-Acetylmuramyl-L-Ala-IsoGln-L-

Ala-Di(palmitoyloxy)propylamide 2. GMTP(Thr)-NHDPG: N-Acetylglucosaminyl-N-Acetylmuramyl-L-Thr-D-

IsoGln-L-Ala-Di(palmitoyloxy)propylamide

3. GMTP-GDP : N-Acetylglucosaminyl-N- Acetylmuramyl-L-Ala-D-IsoGln-L- Ala-Glyceroldipalmitoyl

4. GMTP(Thr)-GDP: N-Acetylglucosaminyl-N-Acetylmuramyl-L-Thr-D- IsoGln-L- Ala-Glyceroldipalmitoyl

5. GMDP: N-Acetylglucosaminyl-N-Acetylmuramyl-L-Ala-D-IsoGln

6. GMDPfThr) : N-Acetylglucosaminyl-N-Acetylmuramyl-L-Thr-D-IsoGln

7. GMTP(Thr)-DD-NHDGP: N-Acetylglucosaminyl-N-Acetylmuramyl-D-Thr- D-IsoGln-L-Ala-Di(palmitoyloxy)propylamide 8. GMTP(Thr)-LL-NHDPG: N-Acetylglucosaminyl-N-Acetylmuramyl-L-Thr-

L-IsoGln-L-Ala-Di(palmitoyloxy)propylamide 9. GMTP(Thr)-DL-NHDPG: N-Acetylglucosaminyl-N-Acetylmuramyl-D-Thr- L-IsoGln-L-Ala-Di(palmitoyloxy)propylamide 10. GMTP(Thr)-NHDLG : N-Acetylglucosaminyl-N- Acetylmuramyl-L-Ala-D- IsoGln-L-Ala-Di(lauroyloxy)propylamide

11. GMTP(Thr)-NHDEG: N-Acetylglucosaminyl-N-Acetylmuramyl-L-Thr-D- IsoGln-L-Ala-Di(eicosanoyloxy)propylamide 12. GMTP(Thr)-NHDTG: N-Acetylglucosaminyl-N-Acetylmuramyl-L-Thr-D-

IsoGln-L-Ala-Di(tetrasanoyloxy)propylamide

1. MDP: N-Acetylmuramyl-L-Ala-D-IsoGln 2. MDP-GDP: N-Acetylmuramyl-L-Ala-D-IsoGln-Glyceroldipalmityol

3. MTP-PE : N-Acetylmuramyl-L-Ala-D-IsoGln-L-Ala-Phosphatidylethanolamine

4. MDP-6-O-Stearoyl: 6-0-Stearoyl-N-Acetylmuramyl-L-Ala-D-IsoGln

5. MDP(Thr): N-Acetylmuramyl-L-Thr-D-IsoGln

6. MTP-NHDPG: N-Acetylmuramyl-L-Ala-D-IsoGln-L-Ala- Di(palmitoyloxy)propylamide

7. MDP(Thr)-NHDPG: N-Acetylmuramyl-L-Thr -D-IsoGln-L-Ala- Di(palmitoyloxy)propylamide

8. MDP-6-O-Stearoyl: N-Acetylmuramyl-6-0-Stearoyl-L-Ala-D-IsoGln

9. rvIDP-N-e-Stearoyl-Lys: N-Acetylmuramyl-L-Ala-D-IsoGln-N-e-Stearoyl- Lys

Enterotoxins

1. SEA: Staphylococcal Enterotoxin A

2. SEB: Staphylococcal Enterotoxin B 3. SECi: Staphylococcal Enterotoxin Ci

4. SEC₂: Staphylococcal Enterotoxin C₂

5. SEC₃: Staphylococcal Enterotoxin C₃

6. SED: Staphylococcal Enterotoxin D

7. SEE: Staphylococcal Enterotoxin E 8. TSST-1: Toxic Shock Syndrome Toxin

9. O-TSST: Ovine Toxic Shock Syndrome Toxin

10. ETA: Staphylococcal Exfoliating Toxin A SUMMARY OF THE INVENTION

The invention is directed to methods of inducing the release of chemokines by administering an effective amount of a muramyl dipeptide compound ("MDP compound") to a mammal. As used in connection with the invention, a MDP compound is a compound including a N-acyl muramyl dipeptide or tripeptide core structure where the dipeptide or tripeptide includes a glutamine residue. Typically, the MDP compound is a lipophilic mono- or disaccharide dipeptide or tripeptide. Examples of particularly suitable MDP compounds for use in the present method include GMTP- NHDPG, GMTP(Thr)-NHDPG, MTP-NHDPG and MTP(Thr)-NHDPG. The method may also include administering a non-toxic enterotoxin in combination with the MDP compound.

The MDP compounds may be used to treat retroviral infections, such as Human Immunodeficiency Virus (HIV) infections, in a mammal. The MDP compounds are typically administered to the mammal, optionally in combination with a non-toxic enterotoxin using an intermittent dosing regime.

The invention is also directed to a pharmaceutical composition which includes an amide linked analog of N-Acetylmuramyl-L-Ala-D-IsoGlutamine. The pharmaceutical composition is suitable for inducing the release of chemokines, such as RANTES, MlP-lα, and/or MlP-lβ, and treating retroviral infection such as Human Immunodeficiency Virus (HIV) in a patient. Optionally, the pharmaceutical composition also includes a non-toxic enterotoxin (such as O-TSST) or a non-toxic variant of a staphylococcal enterotoxin.

Another aspect ofthe invention is directed to a method of inducing the release of chemokines and a method of treating retroviral infection in a patient by administering an effective amount of a non-toxic enterotoxin such as O-TSST. In an alternative embodiment, the invention is directed to a pharmaceutical composition for inducing the release of chemokines and treating retroviral infection, wherein the pharmaceutical composition includes a non-toxic enterotoxin such as O-TSST.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect ofthe invention is directed to a method of inducing the release of chemokines in a mammal by administering an effective amount of an muramyl dipeptide compound ("MDP compound") to a patient. As used in connection with the invention, "inducing the release of chemokines" means stimulating cells of an organism, such as monocytes, macrophages and T-cells, to increase production and release of at least one chemokine. Typically, the administration of an MDP compound leads to an increase in the production and release of at least one chemokine by at least about 50% when compared to untreated cells (i.e., cells not exposed to an MDP compound and/or a non-toxic enterotoxin). Preferably, the level of one or more chemokine in the blood ofthe treated patient is increased to about 100 to about 1000 ng/ml. Preferably, the MDP compound induces the release RANTES, MlP-lα and/or MIP-1 β. The MDP compound is typically capable of inducing the release of chemokines which interact with CXCR-4 and CCR-5 receptors. These receptors appear to play a role in the entry of Human Immunodeficiency Virus (HIV) into lymphocytes and monocytes. Examples of chemokines which interact with these receptors include pre-β- cell growth stimulating factor (PBSF/SDF-1), macrophage inflammatory protein lα (MlP-lα), macrophage inflammatory protein 1 β (MIP- 1 β) and RANTES .

Another aspect ofthe invention is directed to a method of treating a retroviral infection in a mammal. Examples of retroviruses include leukoviruses such as Friend murine leukemia virus, feline leukemia virus, simian immunodeficiency virus and Human Immunodeficiency Virus (HIV). In particular, the invention is directed to a method of treating Human Immunodeficiency Virus Infection. According to the invention, "treatment" or "treating" includes ameliorating symptoms ofthe viral infection, preferably, reducing the virus load (viral titer) ofthe patient about 50-75% and increasing the CD₄ count to at least about 200 cells/μl, more preferably to at least about 400 cells/μl. According to the invention, "MDP compounds" are defined as a compound including a core N-acylmuramyl di- or tripeptide structure wherein the di- or tripeptide includes a glutamine residue. Preferably, the MDP compounds ofthe invention have a reduced toxicity and therefore have less undesirable side effects when compared to other known MDP compounds. In particular, threonine MDP analogs are non- toxic/non-pyrogenic.

The MDP compound ofthe invention typically has a lipophilic group attached to the core structure (e.g., attached to the dipeptide side chain or the 6-0 position ofthe muramyl ring). It is believed that the lipophilic group prevents the MDP compound from being rapidly excreted through the kidney and therefore increases the half-life of the compound in-vivo. Examples of suitable lipophilic groups include carbon chain amides formed from diacyloxypropyl amines having long chain fatty ester groups. The MDP compounds preferably include the lipophilic group linked to the core structure through an amide linkage. It is believed that the amide linkage is less prone to hydrolysis in-vivo, thereby retarding cleavage ofthe lipophilic group and prolonging the half-life ofthe compound.

Suitable MDP compounds for use in the present methods include compounds represented by the formula:

wherein the Lip group is one ofthe following structures (a)-(d): (a)

(b)

(c)

or

(d) -X-NHR₇ .

In the formula shown above, R, is a C_rC₉ alkyl group (i.e., saturated hydrocarbon), preferably CH₃ R₂, R₃, R^ and R₇ are, independently, a C₆-C₃₀ hydrocarbon having from 0 to about 4 double bonds, preferably, a C_]2-C₂₃ hydrocarbon having from 0 to about 1 double bonds. More preferably, R₂, R₃, Rg and R₇ are, independently, a C_I3 -C₁₇ alkyl group. R₅ is (CH₂)_nCH₃ wherein n is an integer between 0 and 22, preferably between 0 and 12.

R_<, is hydrogen or N-acylglucosaminyl attached to the N-acylmuramyl moiety. Preferably, the acyl functionality attached to the nitrogen ofthe muramyl group has about 2 to 6 carbons, preferably 2 carbons (acetyl).

X is a spacer that does not substantially adversely affect the activity or the toxicity ofthe MDP compound. X is typically a single bond or a peptidyl residue comprising about 1-10 amino acid residues. Preferably X is a single bond or a D or L naturally occurring amino acid residue. More preferably, X is an uncharged amino acid (as used herein an "uncharged" amino acid is defined as an amino acid having an uncharged side chain). Naturally occurring uncharged amino acids include neutral aliphatic, neutral aromatic, and neutral heterocyclic amino acids. Neutral aliphatic naturally occurring amino acids include: glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), valine (Val), leucine (Leu), isoleucine (Ile), cysteine (Cys), cystine (Cys-Cys), and methionine (Met). Neutral aromatic amino acids include phenylalanine (Phe) and tyrosine (Tyr). Neutral heterocyclic amino acids include: proline (Pro), hydroxyproline (Hyp), and tryptophan (Trp). Preferably, X is an uncharged aliphatic amino acid such as valine or alanine. Y can be a single bond or a peptidyl residue comprising about 1 to 10 amino acid residues. Preferably, Y is a D or L amino acid residue, preferably an uncharged aliphatic amino acid residue such as alanine, valine, serine or threonine.

Preferably the MDP compound is non-toxic. As used in connection with the invention, a "non-toxic MDP compound" does not induce significant side effects such as fever, chills, hypotension or increases in interleukin-1 (IL-1 ) and/or tumor necrosis factor (TNF) when administered intraveneously at a dose of up to 10 mg, more preferably when administered at a dose of up to 20 mg. It should be noted that some amount ofthe above-described side effects may be acceptable. The amount of side effects considered acceptable will vary among patients. Pharmaceutically acceptable salts of the MDP compounds and liposomes thereof are also suitable for use in the invention.

Preferably, the MDP compound is a lipophilic disaccharide di- or tripeptide. The disaccharide moiety of N-Acylglucosamine-N-Acylmuramate is bonded to the N- terminus of a peptidyl moiety ("-Y-IsoGln-X-") through an acetyl ether linkage at the number 3 position on the muramyl group. The peptidyl moiety includes an amino acid residue, "Y", bonded to D-isoglutamine (IsoGln), which is bonded through its C- terminus, to the group X.

Preferred peptidyl moieties include: L-Alanyl-D-IsoGlutaminyl-L-Alanine (L- Ala-D-IsoGln-L-Ala); L-Alanyl-D-IsoGlutaminyl-D-Alanine (L-Thr-D-IsoGln-D-Ala); L-Threonyl-D-IsoGlutaminyl-D-Alanine (L-Thr-D-IsoGln-D-Ala); and L-Threonyl-D-

IsoGlutaminyl-L-Alanine (L-Thr-D-IsoGln-L-Ala). The disaccharide tripeptide portion ofthe MDP compound may be referred to as an N-Acetyl-Glucosaminyl-L- Acetylmuramyl tripeptides.

The lipophilic end ofthe MDP compound includes an amino derivative of glycerol substituted with two acyl groups (i.e., a l-amino-2,3,-diacyloxypropyl group or a 2-amino-l ,3-diacyloxyρropyl group). The acyl groups can have about 7 to 31 carbons, preferably about 13 to 24 carbons (i.e., R₂, R₃, R^, or R₇ are, independently (C₁₂ and C₂₃) alkyl groups), and about 0 to 4 double bonds, preferably 0 to 1 double bond. Preferably both acyl groups have 16 carbons (C₁₆) (i.e., R₂, R₃, Rg, or R₇ are, independently (Cj₅) alkyl groups) to form a dipalmitoyl-glycerol (DPG) derivative.

The glycerol derivative is attached to the C-terminus ofthe terminal amino acid of X through an amide linkage attached to the number 2 carbon ofthe glycerol backbone. Preferred lipophilic disaccharide dipeptides, in which R₄ is N-

Acetylglucosaminyl, include: GMTP-NHDPG; GMTP(Thr)-NHDPG; GMTP-GDP;

GMTP(Thr)-LL-NHDPG; GMTP(Thr)-DL-NHDPG; GMTP(Thr)-NHDEG;

GMTP(Thr)-NHDLG. Preferably, the lipophilic disaccharide dipeptide has the formula N-Acetylglucosaminyl-N-Acetylmuramyl-L-Thr-D-Isoglutaminyl-L-Ala-NHDPG. Additionally, lipophilic monosaccharide di- or tripeptides, in which R₄ is hydrogen, having similar biological activity as the disaccharide lipophilic di- or tripeptides discussed above, are suitable for use in the invention. Examples of suitable monosaccharide lipophilic peptides include: MDP-6-0-stearoyl; MDP(Thr); MTP- NHDPG; MDP(Thr)-NHDPG; MDP(Thr)-N-e-Stearoyl-Lys-OMe; and MDP(Thr)-N-

G-Lys Decyl Ester. Preferably, the lipophilic monosaccharide tripeptide has the formula N-Acetylmuramyl-L-Thr-D-IsoGln-L-Ala-NHDPG.

In another embodiment, the method ofthe invention includes a non-toxic enterotoxin. Non-toxic enterotoxins include mutant enterotoxins and some native enterotoxins (e.g., ovine TSST). As used in connection with the invention, a "non-toxic enterotoxin" does not cause death in a primed rabbit at a dose below about 100 μg (as a comparison, one microgram (1 μg) of TSST-1 can induce lethal shock in man).

Mutant enterotoxins include deletion mutants of staphylococcal enterotoxins.

Examples of suitable enterotoxins include non-toxic mutants of SEA, SEB, SEC,, SEC₂, SEC₃, SED, and SEE. An example of a suitable non-toxic enterotoxin for use in mammals is O-TSST. Other preferred non-toxic enterotoxins include staphylococcal deletion mutants, preferably SEC deletion mutants.

Preferably, the MDP compound and the non-toxic enterotoxin are administered in a ratio from about 100:1 to about 5000:1, more preferably in a ratio of about 800:1 to about 1200: 1. Preferred combinations include GMTP(Thr)-NHDPG co-administered with O-TSST or a non-toxic SEC mutant. The MDP compound may be administered by a variety of routes including, but not limited to intramuscular, intravenous, subcutaneous, intracutaneous, or oral administration. The MDP compound is typically administered in combination with a pharmaceutically acceptable carrier such as a saline solution. The inventors discovered that timing and dose concentration significantly affects the efficacy of a MDP compound in its ability to induce the release of chemokines and to treat a retroviral infection. It appears that the MDP compounds of the invention can effectively induce the release of chemokines without inducing HIV. According to the invention, the MDP compound is preferably administered at a dose such that the concentration in the patient's blood remains below the critical micelle formation concentration.

It is believed that administration of a dose of an MDP compound stimulates the release of chemokines from macrophages. While not intending to be bound by theory, it is believed that an intermittent dose is most effective if given after the refractory period ofthe macrophage subsides. Therefore, the efficacy of an intermittent dosing schedule will vary depending on the amount of MDP compound administered. An optimal dosing schedule can be determined by monitoring the blood concentration of chemokines. Upon dosing, a spike or peak in the blood concentration of chemokines will appear. As used in connection with the invention "spike" or "peak" mean that there is at least about a 50% increase in the release of at least one of RANTES, MlP-lα or MlP-lβ. If no spike appears in connection with the subsequent dose, the dosing schedule should be altered. For example, an increased dosage may require a longer interval between doses.

The inventors have discovered that intermittent dosing, with too long an interval between doses may be ineffective because the overall levels of chemokines induced by the treatment are insufficient to cause a decrease in progression of a viral infection, as seen by a decrease in viral titer. "Effective" means that a spike in chemokine concentration is noted for both the first dose and each subsequent dose. Additionally, a significant decrease in viral infection progression is also noted. For intravenous administration of an MDP compound, a dose of about 1 to 20 mg, is typically administered every 2 to 7 days. For oral administration, it is anticipated that a dose of about 50 to 100 mg of a MDP compound, administered every 2 to 7 days would be effective. The optimum interval between doses will be a function ofthe amount of MDP compound per dose and can be determined by monitoring chemokine levels as described above.

For intravenous or intramuscular administration of a non-toxic enterotoxin, it is anticipated that a dose of 10 to 100 ng, administered every 2 to 7 days, will be effective to decrease virus load. Oral doses of a non-toxic enterotoxin are anticipated to be in the range of 1 to 10 μg, administered every 2 to 7 days.

EXAMPLE 1 Preparation of Human Peripheral Blood Mononuclear Cells

For evaluation of chemokine induction, human peripheral blood mononuclear cells were isolated from 30 to 60 ml of heparinized blood obtained by venipuncture. The blood was mixed 1 :1 with Dulbeccols Phosphate Buffered Saline (PBS) (without calcium or magnesium), layered onto Fico/Lite (density 1.079 g/ml, Atlanta Biologicals, Norcross, GA) and centrifuged at 400 x g for 20 minutes. The interface containing the mononuclear cells was removed and washed 3 times with PBS. After the last wash, the pellet was resuspended in Hank's Balanced Salt solution without calcium or magnesium, layered on Fetal Bovine Serum (Atlanta Biologicals, Norcross, GA) and centrifuged at 100 x g for 10 minutes to remove platelets. The cells were then washed in Hank's Balanced Salt solution (Cat. No. MI211 -021 -LV; MediaTech Inc.,

Atasca, IL). A cell count was done by Trypan Blue exclusion. An aliquot of cells were plated in 48-well microliter plates to provide 1 x IO⁶ cells and incubated for one hour at 37°C. Test compound was then added at an appropriate concentration. The final volume in the wells was 1 ml of RPMI 10% FBS. The plates were incubated for 18 to 96 hours after which supernatant fluid was harvested and assayed for chemokine production using commercially available ELISA kits (R&D Systems; Minneapolis, MN).

The ELISA assay uses a 96 well microtiter plate loaded with an antibody specific for the particular chemokine. One hundred microliters of supernatant fluid is added and the plates are incubated at 37°C for 75 or 120 minutes depending upon the particular assay. The plates are washed and a second antibody directed to a different epitope and conjugated to horseradish peroxidase is added and incubated at 37°C for an equal amount of time. The plates are washed and tetramethylbenzidine dihydrochloride is added. The plates are incubated for 20 minutes and the reaction is stopped by adding sulfuric acid. The plates are read on a spectrophotometer at 570 nm. Quantitative determinations are made by comparison to a standard curve.

EXAMPLE 2

Induction of Chemokines by a Variety of MDP-related Compounds

Analogs of N-Acetylmuramyl-L-Ala-D-IsoGlutamine were synthesized using standard chemistry (See, for example, U.S. Patent No. 5, 416, 070) and evaluated for their ability to induce chemokines. Human peripheral blood mononuclear cells were prepared as described in Example 1, plated at 1 x 10⁶ cells per well in a 48-well microliter plate and cultured for 48 hours. MDP analogs were used at a final concentration of 10 μg/ml. Culture fluid was obtained and chemokine levels were determined using commercially available kits (See, Example 1).

The results are given in Table 2-1. GMTP(Thr)-NHDPG and MDP-GDP showed the highest induction of chemokines.

Table 2-1. Induction of Chemokines (pg/ml) by a Variety of Muramyldipeptide Analogs at 10 μg/ml

RANTES MlP-lα MIP-1 β

Compound

Control 408 56 89

GMTP-NHDPG 529 573 733

GMTP(Thr)-NHDPG 1102 1763 1324

GMTP-GDP 521 486 565

GMTP(Thr)-GDP 627 751 853

GMDP 470 351 545

GMDP(Thr) 567 482 582

MDP-GDP 1334 1859 1510

MTP-PE 569 436 443

MDP-6-O-Stearoyl 531 651 694

MDP-N-G-Stearoyl 564 418 519

MDP(Thr) 641 738 857 EXAMPLE 3

Chemokine induction in Human Peripheral Blood Mononuclear Cells by a Variety of Mono and Disaccharide Peptides Human peripheral blood mononuclear cells were isolated as described in

Example 1 and plated at 1 X IO⁶ cells in a 48-well microtiter plate. The cells were cultured with a final concentration of 1 or lOμg/ml of a MDP analog. Various MDP analogs were used, having variations in the length ofthe lipophilic carbon chain. Chemokine levels were determined using commercially available ELISA kits (See, Example 1).

The results in Table 3-1 concur with the data in Table 2-1. Both indicate a high level of chemokine induction by MDP-GDP and the monosaccharide analog MTP(Thr)-NHDPG. Changes in the carbon chain length to GMTP(Thr)-NHDPG or GMTP(Thr)-NHDEG did not alter the activity ofthe compound.

Table 3-1. Chemokine Induction (pg/ml) by Various MDP Analogs at 1 μg/ml

Compound RANTES MIP- MIP- lα iβ

Control 1858 11 33

GMTP-NHDPG 1886 179 1136

GMTP(Thr)- 1623 30 132

NHDPG

GMTP(Thr)- 2058 142 789

NHDTG

GMTP(Thr)- 1578 92 586

NHDEG

MDP-GDP 2387 1606 3296

MTP(Thr)-NHDPG 2257 1642 3196

EXAMPLE 4

Efficacy of GMTP-NHDPG Combined with Enterotoxin to Arrest the Retrovirus Friend Erythroleukemia Virus infection in Mice -

A Model of AIDS

Among the models for screening of potential therapeutics for AIDS is the Friend Virus Complex consisting of helper Friend murine leukemia virus and defective spleen focus-forming virus. This model can be evaluated in either (B10.A X Al WySn)F, hybrids or Balb/c mice. (See, Sidwell et al., Ann NY Acad Sci 685:432 (1993)). Friend Virus Complex disease results in a large spleen and decreased red cell mass. Similar to AIDS in man, this disease in mice results in a defect in cellular immunity.

A combination of enterotoxin and GMTP-NHDPG was evaluated for its effect on Friend virus in female Balb/c mice. 18-21 gram mice were injected intraperitoneally

(i.p.) with 0.2 ml of a 1 :20 dilution of homogenized spleen cells from a mouse infected with the Friend Virus Complex. This dose typically results in 90% lethality (lethal dose 90%). Beginning 24 hours after injection, the mice were treated with saline; AZT, a known antiviral agent (25 mg/kg/d in the drinking water); or with a combination of SEC₂ and GMTP-NHDPG (50 μg GMTP-NHDPG to 5 ng of SEC₂ or half that dose).

Mice were treated every other day for twenty days. On day 21 , the mice were sacrificed. The spleen weight and hematocrit (a measurement of red cell mass) were determined.

The results in Table 4-1 indicate the combination of SEC₂ (50 ng/injection, every other day) and GMTP-NHDPG (50 μg injection, every other day) resulted in a significant decrease in the mean spleen weight. This result indicates that a combination of SEC₂ and GMTP-NHDPG is effective to reduce progression of Friend Virus Complex disease.

Table 4-1. Effect of i.p. Treatment with SEC₂ and GMTP-NHDPG on Friend Virus Infections in Mice

Compound Dose Surv/ MST^D Mean Mean

Total (days) Spleen Hematocrit

Weight (%)

(mg)

GMTP- 50 μg 9/10 21 979 ± 553** 55.2 ± 8.8

NHDPG + 5 ng/inj

+ SEC₂ ^a

GMTP- 25 μg 10/10 >21 1,611 ± 715 56.5 ±.8.1

NHDPG + 2.5

+ SEC₂ ^a ng/inj

AZT 0.2 10/10 >21 120 ± 20** 42.9 ± 1.6** mg/ml

Saline — 18/20 18 1,780 ± 710 60.2 ± 9.4

Normal — 3/3 >21 70 ± 40** 47.3 ± 0.6

' Every other day for a total of 10 treatments starting 24 h post-virus inoculation. ^b MST: Median Survival Time

**p < 0.01 compared to saline-treated controls.

EXAMPLE 5

Chemokine induction by Different Concentrations of Non-toxic Mutant Enterotoxin

Peripheral blood mononuclear cells were prepared as described in Example 1 and plated at 1 x IO⁶ cells per well in a 48-well microliter plate. O-TSST (Lot# 4299450, Toxin Technology of Sarasota, FL) was used added to the cell preparation at concentrations of 1 μg, 100 ng, 10 ng, 1 ng, 100 pg, and 10 pg per ml. Supernatant fluid was harvested after 48 hours and chemokine levels were determined using commercially available ELISA kits (R&D Systems; Minneapolis, MN).

The results in Table 5-1 indicate an increased induction of RANTES, MlP-lα, and MIP-1 β with increasing concentrations of O-TSST.

Table 5-1 Effect of Concentration on Chemokine Induction By a Ovine Toxic Shock Syndrome Toxin

O-TSST Concentration (ng/ml)

Chemokine Zero 0.01 0.1 1 10 100 1000

RANTES (pg/ml) 511 628 500 618 888 1225 1827

MIP- lα (pg/ml) 30 39 34 110 1062 2062 2824

MIP- l β (pg/ml) 197 310 307 514 1402 2141 2386 EXAMPLE 6 Effect of O-TSST and SEC₂on Friend Retroviral infection in Mice

The ability of O-TSST and SEC₂to reduce the effect of Friend Leukemia virus infection in mice, as shown by mean spleen weight and hematocrit, was evaluated. SEC₂ and O-TSST were obtained from Toxin Technology (Sarasota, FL).

Groups of female Balb/c mice were injected with Friend leukemia virus. 24 hours after inoculation, the mice were treated intraperitoneally with enterotoxin (dosage is indicated in Table 6-1). The mice received enterotoxin treatment every other day for ten total treatments (qod x 10). Spleen weights and hematocrit were determined on day 21.

The results are shown in Table 6-1. O-TSST at a dose of 2 μg per injection resulted in a significant decline in the progression ofthe virus infection as evidenced by a decreased spleen weight and decreased hematocrit when compared to saline treated controls. At 200 ng/injection, the effect of SEC₂ was insignificant.

Table 6-1. Effect of Treatment" with a Enterotoxins on a Friend Retroviral infection in Mice

Compound Dosage Surv/ Mean Spleen Mean

Total Wt. Hematocrit ± SD

(mg ± SD)

SEC₂ 200ng/inj 10/10 1970 ± 874 55.1 ± 12.0

O-TSST 2 μg/inj 10/10 1151 ± 949* 46.9 ± 5.2*

Zidovudine 0.2% 10/10 138 ± 20** 42.4 ± 1.6**

(in drinking water)

Saline Control — 19/20 2140 ± 799 56.1 ± 9.6

Normal Control* — 3/3 107 ± 12 46.0 ± 1.2

*P < 0.05

**P < 0.01 compared to saline-treated controls. ⁺Normal Control = mice not injected with Friend leukemia virus Saline control = mice injected with Friend leukemia virus and treated with saline

EXAMPLE 7 Induction of Chemokines in Patients Receiving GMTP-NHDPG

GMTP-NHDPG was formulated in 50 mg dextrose as a 2 wt% solution (1 mg GMTP-NHGDP/50 mg dextrose). The dextrose formulation was administered weekly, for at least five weeks, to a group of cancer patients with metastatic disease. Analysis of serum samples demonstrated that the patients inherently had high levels of RANTES. Therefore, the measurements of RANTES concentration must be discounted. However, the results indicate that GMTP-NHDPG induced increased levels of MlP-lα and MlP-lβ (See Table 7-1).

Table 7-1. Chemokine Induction in Patients Receiving GMTP-NHDPG

Patient Dose μglm* Time(hr) RANTES MlP-lα MD?-lβ

206912 1600 0 3810 0 345

4 3403 84 1547

206918 1600 0 3643 0.7 10.9

4 3656 24.5 374.6

206982 3200 0 3664 0 11.9

4 4199 164 2142

206978 3200 0 3470 0.3 21.9

4 2996 47.5 2002

206960 3200 0 3539 7.9 22.9

4 3275 140 764.9

*m of body surface area

EXAMPLE 8 Chemokine Induction by GMTP-NHDPG

With or Without Various Enterotoxins

The induction of chemokines by GMTP-NHDPG alone or in combination with a variety of enterotoxins was evaluated. Human PBMC were prepared as described in Example 1. The prepared cells were placed at 1 x IO⁶ cells per well in a 48-well microliter plate in Ruswell Park Memorial Institute (RPMI) media with 10% FBS, as described in Example 1. The cells were incubated for 48 hours with 0 or 10 μg/ml of GMTP-NHDPG, Lot #A008, alone or combined with 1 ng/ml ofthe following superantigen enterotoxins: SEA (Lot #71595A), SEB (Lot #112295B), SEC, (Lot #91495C1), SEC₂ (Lot #8295C2), SEC₃

(Lot #91195C3), SED (Lot #1129940), SEE (Lot #12195E), TSST-1 (Lot #9195ET), and ETA (Lot #111694ET). Culture fluid was harvested and assayed for RANTES, and MlP-lα. The results in Table 8-1 demonstrate a marked synergism in the induction of MlP-lα and RANTES when the cells were incubated with a combination of GMTP-NHDPG and an enterotoxin.

Table 8-1 Chemokine Induction by Various Enterotoxins (1 ng/ml) Alone or Combined with GMTP-NHDPG (GMTP) at

MIP- ^■lα(ng/ml) RANTES(ng/ml)

Enterotoxin Alone +GMTP Alone +GMTP

None (Control) 23.9 383.4 111 196

SEA 1101 1667 623 1001

SEB 1484 1783 742 1035

SEC 1 1500 1705 764 1069

SEC 2 1422 1811 666 1011

SEC 3 1217 1684 631 1005

SED 1285 1713 677 999

SEE 1324 1628 665 971

TSST-1 994 1743 559 940

ETA 709 1599 462 930

EXAMPLE 9 Chemokine induction by a Variety of Threonine MDP Analogs

Human peripheral blood mononuclear cells were prepared as described in Example 1 and plated at 1 x 10⁶ cells per well in a 48-well microtiter plate. The cells were exposed to a final concentration of 10 μg/ml of a monosaccharide or disaccharide threonine analog of MDP. The analogs had differences in the stereochemistry at the first amino acid and/or differences in the length ofthe lipophilic group carbon chain.

The results in Table 9-1 indicate that, among the analogs tested, MTP-NHDPG and MTP(Thr)-NHDPG demonstrated the heightened induction in chemokines compared to the control. Table 9-1. Chemokine Induction (pg/ml) by Various Threonine MDP Analogs

Compound MIP-1 a MIP-1/? RANTES

Control 76 298 706

GMTP-NHDPG 615 1194 995

GMTP(Thr)-LD-NHDPG 143 426 678

GMTP(Thr)-DD-NHDPG 129 493 757

GMTP(Thr)-LL-NHDPG 135 271 546

GMTP(Thr)-DL-NHDPG 104 299 643

GMTP(Thr)-NHDPG 334 780 732

GMTP(Thr)-NHDEG 525 1036 792

GMTP(Thr)-NHDTG 395 800 717

GMTP(Thr)-DD-NHDEG 958 1266 844

MTP-NHDPG 1427 1890 1137

MTP(Thr)-NHDPG 1065 1646 1197

MDP 482 1 167 1034

EXAMPLE 10

Effect of GMTP(Thr)-NHDPG at Two Different Dose Schedules on Friend Murine

Leukemia Virus infection in Mice

Mice were inoculated with Friend Murine Leukemia Virus, as described in Example 4, and treated with 50 μg of GMTP(Thr)-NHDPG every other day for 21 days or every third day for 21 days. Spleen weight, hematocrit and spleen virus titers were determined on the control group and group which demonstrated a significant decrease in spleen weight.

The individual spleen weight, hematocrit, and virus titer are shown in Table 10- 1. A significant decrease (p < 0.01 ) in mean spleen weight and decrease in a virus titer was seen when mice received a 50 μg intraperitoneal dose of GMTP(Thr)-NHDPG every other day (qod). In contrast, treatment at 3 day intervals (q3d) with a 50 μg intraperitoneal dose had no effect. Table 10-1. Effect of IP Treatment on Friend Murine Leukemia Virus Infection in Mice

Control GMTP(Thr)-NHDPG GMTPfThr)-

50 μg (qod) NHDPG

50 /Jg (q3d)

Spleen HCT Virus Spleen HCT Virus Spleen HCT

Weight (%) Titer Weight (%) Titer Weight (%)

(gms) (log (gms) dog (gms) dose) dose)

2.81 48 6.10 1.51 48 3.78 3.38 52

2.70 49 5.70 1.46 49 5.80 3.13 56

2.70 48 6.80 1.38 45 4.90 3.02 64

2.69 56 5.60 0.60 45 4.55 2.59 59

2.41 63 6.80 0.57 46 6.57 2.58 56

2.40 71 9.00 0.27 48 0.21 2.10 55

2.13 56 3.40 0.26 45 0.21 2.05 50

2.10 49 9.00 0.17 47 2.35 1.99 50

1.95 53 5.60 0.14 45 3.22 1.60 49

1.95 55 7.60 0.1 1 42 0.19 1.55 43 1.90 49 7.60 1.70 49 6.80 1.60 49 0.26 1.32 48 0.26 1.30 48 9.10 0.34 45 5.90 0.29 43 0.26 0.27 42 2.30 0.23 43 7.10 0.20 42 0.24

MEAN 1.650 50.30 5.271 0.647* 46.00 3.178 2.399 53.40

STD 0.902 7.002 2.984 0.549 1.949 2.254 0.607 5.589

'Significantly different at P< 0.01 compared to control.

EXAMPLE 11 Effect of MDP Analogs on Friend Leukemia Virus Infection in Mice

Mice were inoculated with Friend Murine Leukemia Virus and then injected with a variety of lipophilic monosaccharide or disaccharide peptides beginning 24 hours after infection, as described in Example 10. The efficacy of the analog was evaluated in relationship to the saccharide moiety, length ofthe lipophilic group, and dose schedule. Mice were sacrificed on Day 21 and the spleen weight and hematocrit were determined. The results Tables 11-1 and 11-2 demonstrate that both the disaccharide threonine compound, GMTP(Thr)-NHDPG, and the monosaccharide analog MTP(Thr)- N-DPG are active.

Table 11-1. Efficacy of Various MDP Analogs on Friend Murine Leukemia Virus Infection

Compound Dose Schedule Neg Spleen Hematocrit Plasma Virus tøs⁾ Spleen"/ Weight Mean Mean ± SD Titer^b

Total ±SD Mean ± SD

GMTP-NHDPG 50 qodx 10 2/10 1961 ± 1084 54.2 ± 7.6

GMTP(Thr)- 50 qod x 10 6/9** 1001 ±1133* 53.6 ±5.5 2.06 ±2.33^* NHDPG

GMTP(Thr)- 100 qod x 10 2/9 1682 ± 1003 56.4 ± 6.9 NHDPG

GMTP(Thr)- 100 q3dx7 3/9* 1692+1170 51.9±4.7 4.66 ±2.88 NHDPG

GMTP(Thr)- 50 qodx 10 1/10 1731 ±828 52.0 ±5.3 NHDEG

GMTP(Thr)- 100 qodx 10 2/10 1437 + 877 47.3 ± 7.9 NHDEG

MTP(Thr)- 50 qod x 10 5/10** 1057+1220 46.18±6.1 2.46 ±2.01^* NHDPG

MDP-GDP 50 qodx 10 1/10 1925 ±734 54.4 ± 7.0

AZT 2% oral qd 10/10** 1874 ±657 37.0 ±

2.2**

Saline Control qodx 10 20/20 1624 ±766 49.3 ± 6.7 5.15 ± 3.06'

Normal Control qodx 10 116±17 49.3 ± 6.7

^* Negative spleens considered those weighing < 400 mg ^b Virus titer is shown as log infectious dose

* P<0.05.

** P < 0.01 compared to saline treated controls.

Table 1 l-2a. Effect of Treatment with Various MDP Analogs at 50 μg on a Friend Retroviral infection in Mice*

GMTP- GMTP(Thr)- GMTP(Thr)- MTP(Thr)- MDP-GDP Saline

NHDPG NHDPG NHDEG NHDEG 50 μg qod x 10

50 μg 50 μg 50 μg 50 μg qod x 10 qod xlO qod : xlO qod: K 10 qod: it 10

Spleen HCT Spleen HCT Spleen HCT Spleen HCT Spleen HCT Spleen HCT

Weight (%) Weight (%) Weight (%) Weight (%) Weight (%) Weight (%)

(gms) (gms) (gms) (gms) (gms) (gms)

345 55 281 56 265 51 278 47 272 50 277 65

308 58 237 59 257 55 249 50 232 58 271 59

269 59 231 58 240 59 246 61 231 48 251 48

213 61 040 62 236 49 212 48 223 52 245 49

212 56 029 52 192 55 014 37 222 60 225 49

210 40 029 51 180 52 014 47 218 49 223 59

197 52 028 50 159 54 013 44 210 49 217 48

I 74 66 014 49 106 57 011 45 175 54 199 48

020 47 012 45 074 41 010 43 128 51 183 54

013 48 032 47 010 46 014 40 170 47 150 55 144 46 140 49 127 42 117 48 1 14 40 08 43 047 44 036 44 032 48 std 10 72 1 1 52 08 50 12 58 07 53 07 61 mean 542 10 536 17 520 14 468 19 511 23 493 493 ^kSpleen weight reported in mg, Hematocrit (HCT) reported in percentage (%)

Table 1 l-2b. Effect of Treatment with Various MDP Analogs at 100 μg on a Friend

Retroviral infection in Mice

GMTP(Thr)- GMTP(Thr)- GMTP(Thr)- Saline

NHDPG NHDPG NHDEG qod x lO

100 μg 100 μg 100 μg qod : < 10 q3d qod > : 10

Spleen Spleen Spleen Spleen

Weight HCT Weight HCT Weight HCT Weight HCT

2.77 62 2.74 56 2.60 49 2.77 65

2.65 48 2.59 61 2.31 36 2.71 59

2.57 61 2.53 48 2.25 54 2.51 48

2.18 53 2.46 51 1.97 50 2.45 49

2.07 59 2.43 50 1.60 60 2.25 49

1.29 69 2.02 50 1.36 54 2.23 59

1.19 53 0.19 56 1.34 48 2.17 48

0.29 55 0.15 47 0.45 36 1.99 48

0.13 48 0.12 48 0.27 42 1.83 54

0.22 44 1.70 47

1.50 55

1.44 46

1.40 49

1.27 42

1.17 48

1.14 40

0.8 43

0.47 44

0.36 44

0.32 48

std 0.9 6.5 1.1 4.5 0.8 7.5 0.7 61 mean 56.4 1.7 51.9 1.7 47.3 1.1 49.3 49.3

EXAMPLE 12

Chemokine Induction by N^c-Stearoyl-L-Lysine Analogs of GMTP

Human peripheral blood mononuclear cells were prepared as described in Example 1 and plated at 1 x IO⁶ cells per well in a 48-well microtiter plate. GMDP(Thr)- N^e-Stearoyl-L-Lys-OCH₃ and GMDP(Thr)- N^e-Stearoyl-L-Lys-O- (CH₂)₉-CH₃ were added to a final concentration of 10 μg/ml. Supernatant fluid was obtained after 48 hours and assayed for chemokines, as described in Example 1. The results in Table 12-1 indicate that N^e-Stearoyl-L-Lysine derivatives are as active as GMTP-NHDPG.

Table 12-1. Chemokine Induction (ng/ml) by N^c-Stearoyl-L-Lysine Analogs of GMTP at 10 μg/ml

Analog RANTES MlP-lα MIP-1 β

Saline (control) 907 513 1227

-L-Lys-O-CH₃ 1163 1094 2689

-L-Lys-O-(CH₂)₉-CH₃ 1210 1420 2460

GMTP-NHDPG 1018 1239 2040

EXAMPLE 13 Preparation of BOC-D-IsoGln-OBn Benzyl alcohol (245 mg, 2.26 mMol), DMAP (125 mg, 1.025 mMol) and BOC-

D-IsoGln-OH (Bachem, 505.0 mg, 2.05 mMol) were dissolved in 30 ml of anhydrous CH₂C1₂ and 5 ml of anhydrous DMF and cooled in an ice bath. EDCI (482 mg, 2.46 mMol) was added. The resulting solution was then stirred at 5°C for 1 hour, and at room temperature for 20 hours. After the solvents were removed on the rotary evaporator, the residue was partitioned between 50 ml of ethyl acetate and 30 ml of water. The layers were separated and the aqueous layer was extracted with 25 ml more EtOAc. The acetate solutions were combined and washed successively with saturated NaCHO₃ (3 x 30 ml) and water (3 x 40 ml). After drying over MgSO₄, the solvent was removed on the rotary evaporator. The solid residue was recrystallized from EtOAc/Hexane to yield 603 mg of BOC-D-lsoGln-OBn as a white solid.

EXAMPLE 14 Preparation of H-D-IsoGln-OBn

BOC-D-IsoGln-OBn (530 mg, 1.576 mMol) was dissolved in 15 ml of IN HCl/HOAc and the resulting solution allowed to stand at room temperature for 2 hours.

The solvents were removed on the rotary evaporator. The solid residue was further dried under high vacuum then recrystallized from methanol-diethyl ether to yield 404 mg of white, fluffy solid. EXAMPLE 15 Preparation of BOC-L-Thr-D-IsoGln-OBn

N-BOC-O-Benzyl-L-Threonine (Bachem, 336 mg, 1.086 mMol) in 8 ml of CH₂C1₂ was cooled to -15°C by an ice-salt bath and treated with N-methylmorpholine ( 120 μl, 1.092 mMol). Isobutyl chlorocarbonate ( 142.5 Ml, 1.091 mMol) was added dropwise over a period of three minutes. The resulting solution was stirred at -15°C for 15 minutes. A pre-cooled solution of D-IsoGln-OBN»HCl (297 mg, 1.089 mMol) and N-methylmorpholine (120 μl, 1.092 mMol) in 5 ml of anhydrous DMF was dropwise added over a period of 20 minutes through an addition funnel. The resulting mixture was stirred at -10°C for three hour, then at room temperature overnight. The solvents were removed on the rotary evaporator. The residue was partitioned between 60 ml of ethyl acetate and 30 ml of water. The layers were separated and the aqueous layer was extracted with 30 ml EtOAc. The organic solutions were combined and washed successively with 10% citric acid (3 x 30 ml), H₂O (40 ml), saturated NaHCO₃ solution (3 x 30 ml) and water (3 x 40 ml) then dried over MgSO₄. After the solvent was removed on the rotary evaporator, the solid residue was recrystallized from EtOAc- Hexane to yield 482 mg of product as a white solid.

EXAMPLE 16 Preparation of N^α-BOC-N^e-CBZ-L-Lys-OC_loH_2I:

N^α-BOC- N^e-CBZ-L-Lysine (Bachem, 348 mg, 0.915 mMol), decyl alcohol (Aldrich, 160 mg, 1.011 mMol) and DMAP (56 mg, 0.458 mMol) were dissolved in 5 ml of CH₂C1₂, and treated with EDCI (220 mg, 1.125 mMol). The resulting solution was stirred at room temperature for 24 hour. After removing the solvent on the rotary evaporator the oily residue was partitioned between 70 ml of ethyl acetate and 50 ml of water. The layers were separated and the aqueous layer was extracted with 30 ml of EtOAc. The organic solutions were combined and successively washed with 8% NaHCO₃ solution (2 x 50 ml) and water (3 x 50 ml), then dried over MgSO₄. The solvent was removed on the rotary evaporator. The residue was precipitated from methanolethyl ether at 2°C to yield 451 mg of a white solid.

For further purification, the entire crude product was dissolved in 2:1 (v/v) Hexane-EtOAc, then applied to a column of Biosil A (1.5 x 27 cm) and eluted with EtOAc/Hexane. The fractions were assayed by TLC (in CHCl₃:MeOH:HOAc=90:8:2, ninhydrin), and the appropriate fractions (R_f = 0.65) were combined and concentrated to dryness to afford 356 mg of a white solid.

EXAMPLE 17

Preparation of N^α-Boc-L-Lys-OC|₀H_2]

N^α-Boc-N^e-CBZ-Lysine decyl ester (330 mg, 0.655 mMol) was dissolved in 30 ml of methanol. 0.5 ml of HOAc was added followed by 5% palladium on activated carbon (Aldrich, 120 mg). The mixture was hydrogenated under 35 PSI at room temperature for 36 hour. The catalyst was removed by filtration and washed with methanol. The filtrate and the washings were combined and concentrated on the rotary evaporator to dryness. The product was further dried under high vacuum and yield a TLC-pure product, 257 mg, as a colorless oil.

EXAMPLE 18

Preparation of N^α-Boc-N^e-Stearoyl-L-Lysine Decyl Ester

N^α-Boc-L-Lysine decyl ester (150 mg, 0.387 mMol) and TEA (39.5 mg, 0.390 mMol) were dissolved in 2 ml of anhydrous CH₂C1₂ and 1 ml of anhydrous DMF, and cooled in an ice-bath. A solution of stearoyl chloride (129 mg, 0.425 mMol) in 3.0 ml of CH₂C1₂ was dropwise added. The mixture was stirred at 2°C f or 5 hour and at room temperature for 2 hours, then treated with 0.5 ml of 50% NH₄OH. The solvents were removed on the rotary evaporator and the residue was partitioned between 40 ml of ethyl acetate and 20 ml of water. The aqueous layer was separated from the acetate layer and extracted with 30 ml more acetate. The organic layers were combined and washed successively with: saturated NaHCO₃ (3 x 30 ml), H₂O (40 ml), 10% citric acid (3 x 30 ml), and water (3 x 40 ml). After drying over MgSO₄, the solvent was removed on the rotary evaporator. The residue was further dried under high vacuum to yield the crude product, 231 mg, as a solid.

For further purification, 180 mg of this product was dissolved in 8 ml of 5% i-ProH in hexane, then applied to a column of Biosil A (1.5 x 34 cm) and eluted by: (A)

40 ml of 1% i-ProH in hexane, (B) 100 ml of 2% i-ProH in hexane; 0 50 ml of 4% i- ProH in hexane. The fractions were assayed by TLC (in i-ProH:Hexane - 1 :7, ninhydrin). The appropriate fractions were combined and concentrated to dryness to yield 122 mg of pure product as a white amoφhous solid. FAB mass spectrometry found its molecular weight (M+H)⁺ at 653.

EXAMPLE 19

Preparation of N^e-StearoyI-L-Lysine Decyl Ester

102 mg (0. 156 mMol) of N^α-Boc-N^e-Stearoyl-L-LysOC₁₀H₂₁ was treated with 10 ml of IN HCl/HOAc and allowed to stand at room temperature for 2 hours. The solvent was evaporated on the rotary evaporator and co-evaporated with benzene to yield a glassy solid. This crude product was recrystallized from methanol-ethyl ether to afford 73 mg ofthe hydrochloride salt of N^e-stearoyl-L-Lysine decyl ester as a white, fluffy solid.

EXAMPLE 20 Preparation of N^α-CBZ-N^e-Stearoyl-L-Lys-OMe

N^α-CBZ-Lys-OMe hydrochloride (Bachem, 180 mg, 0.545 mMol) was dissolved in 3 ml of anhydrous CH₂C1₂ and cooled in an ice-bath, then treated with TEA (116 mg, 1.15 mMol). A solution of stearoyl chloride (177 mg, 0.584 mMol) in 3 ml of anhydrous CH₂C1₂ and 1 ml of DMF was dropwise added. The resulting solution was stirred at 25°C for 16 hours then treated with 0.5 ml of 50% NH₄OH. The solvents were removed on the rotary evaporator and the residue was partitioned between 50 ml of ethyl acetate and 25 ml of water. The layers were separated and the aqueous layer was extracted with 30 ml more acetate. The organic solutions were combined and successively washed with: 8% NaHCO₃ (3 x 40 ml), H₂0 (50 ml), 10% citric acid (3 x 30 ml), and water (3 x 40 ml). After drying over MgSO₄, the solvent was removed on the rotary evaporator, and the residue was dried under high vacuum to yield 298 mg of a white solid.

The entire crude product was further purified by a column of BioSil A (1.5 x 23 cm), and eluted by (A) 50 ml of 2% i-ProH in Hexane; (B) 50 ml of 3% i-ProH in Hexane; (C) 100 ml of 4% i-ProH in Hexane. The appropriate fractions (R_f = 0.72 on

TLC, CHC1₃, MeOH:HoAC=90:8:2) were combined and concentrated to dryness on the rotary evaporator to yield 233 mg of N^α-CBZ-N^c-stearoyl-L-Lysine methyl ester as a white, fluffy solid with a melting point at 95-96°C.

EXAMPLE 21 Preparation of N^e-stearoyl-L-Lysine Methyl Ester

N^α-CBZ-N^e-stearoyl-Lys-OMe (200 mg, 0.357 mMol) was dissolved in 15 ml of isopropanol and 10 ml of ethanol, 0.5 ml of 50% HOAc was added, followed by 5% Pd-C (Aldrich, 92 mg). The mixture was hydrogenated under 37 PSI for 28 hours. The catalyst was removed by filtration and washed with ethanol. The filtrate and washings were combined and concentrated on the rotary evaporator to dryness. The residue was further dried under high vacuum at room temperature for 24 hours to yield 147 mg of the crude product as a solid. 100 mg of this product was reprecipitated from methanol and ethyl ether at 2°C and collected on a filter, washed with ether:Hexane=l:l (v/v); then dried under high vacuum to yield 81 mg of N^e-stearoyl-L-Lysine methyl ester as a white, fluffy solid. FAB Mass spectrometry found its molecular weight (M+H)⁺ at 427.

EXAMPLE 22

Coupling of Benzyl-4,6-O-Benzylidene-N-Acetyl Muramic Acid with L-Ala-D-IsoGln-OBn A slurry of Woodward's reagent K (155 mg, 0.612 mMol) and benzyl-4,6-O- benzylidene-N-acetylmuramic acid (Sigma, 250 mg, 0.477 mMol) in 6 ml of anhydrous acetonitrile was cooled in an ice-bath. TEA (54.5 mg, 0.539 mMol) was added and the resulting slurry was stirred at 2°C for 1 hour. A solution of 184 mg (0.535 mMol) of HCl-H-L-Ala-D-IsoGln-OBn and TEA (54.5 mg, 0.539 mMol) in 4 ml of acetonitrile and 1 ml of DMF was dropwise added over a period of 15 minutes at 2°C. The resulting solution allowed to stir at room temperature for 44 hours. The solvents were removed on the rotary evaporator, first by an aspirator then under high vacuum. After further drying under high vacuum, the residue was stirred with 40 ml of warm water for 30 minutes then filtrated. The solid was collected, washed with water, and dried in air to yield 378 mg ofthe crude product as a solid.

The entire crude product was then taken up in 100 ml of hot ethanol, filtered from the insoluble material, then concentrated to about 20 ml. After standing at 2°C overnight, the precipitate was collected on a filter, washed with 40% ethanol then dried in air to yield 283 mg ofthe product as a white solid, which was further purified by recrystallized from ethanol- water to yield 235 mg of TLC-pure product.

EXAMPLE 23

Preparation of Mur(NAc)-L-Ala-D-IsoGln

175 mg (0.230 mMol) of Benzyl-4,6-O-benzylidene-N-acetyl-Mur-L-Ala-D- IsoGln-OBn ("N-acetyl-Mur-" is used herein as an abbreviation for N-acetyl-muramic acid or N-acetylmuramyl) was dissolved in 20% acetic acid-ethanol and treated with 200 mg of 10% Pd-C (Aldrich), then hydrogenated under 35 PSI for 72 hours at room temperature in a Parr apparatus. After 72 hours, the catalyst was removed by filtration through a celite pad, and washed with methanol. The filtrate and washings were combined and concentrated to dryness on the rotary evaporator at 30-35°C. The residue was taken up in 30 ml of methanol and concentrated to dryness to yield a glassy solid which was dissolved in 40 ml of water and lyophilized to yield a white, fluffy solid

(109 mg). 100 mg of this product was re-precipitated from ethanol-ethyl ether to afford 78.5 mg of Mur(NAc)-L-Ala-D-IsoGln as the TLC-pure (in CHCl₃:MeOH:NHeOH:H₂0=50:25:2:4; 5% H₂SO₄ in C₂H₅OH, 115°C) product.

EXAMPLE 24

Preparation of Mur(NAc)-L-Ala-D-IsoGln-L-Ala-NHDPG

72 mg (0.145 mMol) of Mur(NAc)-L-Ala-D-IsoGln was dissolved in 4.0 ml of anhydrous DMF (distilled from ninhydrin). To this was added 22 mg of 1-Hydroxylbenzotriazole (HOBT) (0.147 mMol) and 33 mg of 1 -(3 -Dimethylaminopropyl)-3 -ethylcarbodiimide hydrochloride (EDCI) (0.168 mMol).

The mixture was stirred at room temperature for 20 minutes.

1 1 1 mg (0.147 mMol) of L-Ala-NHDPG^«TFA was dissolved in 2 ml of anhydrous CH₂C1₂ and added TEA (15.1 mg, 0.149 mMol). This solution was cooled at 2°C for 10 minutes, then added dropwise to the MDP/EDCI solution over a period of 10 min. The reaction mixture was stirred at room temperature for 24 hours; and analyzed by TLC (in CHCl₃:MeOH:NH₄OH:H₂0=50:25:2:4; 5% H₂SO₄-C₂H₂OH, 115°C). After 24 hours, 10 mg of EDCI was added, and the reaction solution allowed to stand at room temperature for another 24 hours. The solvents were removed on the rotary evaporator and the oily residue was dried under high vacuum overnight. The resulting yellow oil was washed three times with about 10 ml of ethyl acetate, then centrifuged at 1,500 RPM to generate a slightly yellow precipitate. This precipitate was taken up in 20 ml of sterile water and dialyzed (MWCO 3,500) against 4 x 1 L of sterile water. The inner dialysate was then lyophilized to yield 113 mg of a white solid.

For final purification, 60 mg ofthe dialyzed product was dissolved in 2.0 ml of CHCl₃:MeOH:H₂O=2:3:l, and subjected to sephadex LH-20 (Pharmacia) and cellulose DE-52 (Whatman) chromatography. Following the column treatments and lyophilization, MTP-NHDPG was obtained as a white electrostatic powder.

EXAMPLE 25

Coupling of Benzyl-4,6-O-Benzylidene-N-Acetyl Muramic Acid with L-Thr-D-IsoGln-OBn A slurry of Woodward's reagent K (95 mg, 0.356 mMol) and benzyl-4,6-O- benzylidene-N-acetylmuramic acid (Sigma, 153 mg, 0.292 mMol) in 5 ml of anhydrous acetonitrile was cooled in an ice-bath. TEA (33.5 mg, 0.331 mMol) was added and the resulting slurry was stirred at 2°C for 1 hour. A solution of 153 mg (0.330 mMol) of HCl^»H-L-Thr-D-IsoGln-OBn and TEA (33.5 mg, 0.331 mMol) in 3 ml of acetonitrile and 1 ml of DMF was dropwise added over a period of 10 minutes at 2°C. The resulting solution allowed to stir at room temperature for 60 hours. The solvents were removed on the rotary evaporator first by an aspirator, then under high vacuum. After further drying under high vacuum, the residue was stirred with 50 ml of warm water for 30 minutes then filtrated. The solid was collected, washed with water, and dried in air to yield 257 mg ofthe crude product as a solid.

The entire crude product was then taken up in 70 ml of hot ethanol, filtered from the insoluble material, then concentrated to about 15 ml. After standing at 2°C overnight, the precipitate was collected on a filter, washed with 40% ethanol then dried in air to yield 194 mg ofthe product as a white solid, which was further purified by recrystallized from ethanol-water to yield 147 mg of TLC-pure product. EXAMPLE 26 Preparation of Mur(NAc)-L-Thr-D-IsoGln

147 mg (0.167 mMol) of Benzyl-4,6-O-benzylidene-N-acetyl-Mur-L-Thr-D- IsoGln-OBn was dissolved in 20% acetic acid-ethanol and treated with 130 mg of 10% Pd-C (Aldrich), then hydrogenated under 35 PSI for 72 hours at room temperature in the Parr apparatus. After 72 hour, the catalyst was removed by filtration through a celite pad, and washed with methanol. The filtrate and washings were combined and concentrated to dryness on the rotary evaporator at 30-35°C. The residue was taken up in 35 ml of methanol and concentrated to dryness to yield a glassy solid which was dissolved in water and lyophilized to yield a white, fluffy solid (88 mg). This product was reprecipitated from ethanol-ethyl ether to afford 66 mg of Mur(NAc)-L-Thr-D- IsoGln as the TLC-pure (in CHCl₃:MeOH:NH₄OH:H20=50:25:2:4; 5% H₂SO₄ in C₂H₅OH, 115°C) product.

EXAMPLE 27

Preparation of Mur(NAc)-L-Thr-D-IsoGln-L-Ala-NHDPG

51 mg (0.0976 mMol) of Mur(NAc)-L-Thr-D-IsoGln was dissolved in 3.0 ml of anhydrous DMF (distilled from ninhydrin), To this was added 13 mg of HOBT (0.098 mMol) and 23 mg of EDCI (0.1 18 mMol). The mixture was stirred at room temperature for 20 minutes.

75 mg (0.0997 mMol) of L-Ala-NHDPG^«TFA was dissolved in 2 ml of anhydrous CH₂C1₂ and added TEA (10.5 mg, 0.104 mMol). This solution was cooled at 2°C for 10 minutes, then added dropwise to the Thr-MDP/EDCI solution over a period of 10 min. The reaction mixture was stirred at room temperature for 24 hour; and analyzed by TLC (in CHCl₃:MeOH:NH₄OH:H20=50:25:2:4; 5% H₂SO₄-C₂H₅OH,

115°C). After 24 hour, 10 mg of EDCI was added, and the reaction solution allowed to stand at room temperature for 36 more hours. The solvents were removed on the rotary evaporator and the oily residue was dried under high vacuum overnight. The resulting yellow oil was washed three times with about 10 ml of ethyl acetate, then centrifuged at 1 ,500 RPM to generate a slightly yellow precipitate. This precipitate was taken up in

15 ml of sterile water and dialyzed (MWCO 3,500) against 4 x IL of sterile water, The inner dialysate was then lyophilized to yield 82 mg of a white solid. For final purification, 50 mg ofthe dialyzed product was dissolved in 2.0 ml of CHCl₃:MeOH:H₂0=2:3:l, and subjected to sephadex LH-20 (Pharmacia) and cellulose DE-52 (Whatman) chromatography. Following the column treatments and lyophilization, Thr-MTP-NHDPG was obtained as a white electrostatic powder.

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope ofthe invention.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a retroviral infection in a mammal comprising administering an effective amount of a MDP compound having the formula

wherein Lip is

(a)

(b)

(c)

or

(d)

-X-NHR 7

wherein R) is (C_rC₉) alkyl;

R₂, R₃, Rβ, and R₇ are independently (C₆-C₃₀) hydrocarbons having about 0-4 double bonds;

R^ is hydrogen or N-acylglucosaminyl; R₅ is (CH₂)_nCH₃ wherein n is an integer between 0 and 22;

X is a single bond or a peptidyl residue comprising 1-10 amino acid residues; and Y is an amino acid residue.

2. The method of claim 1 wherein R,, is N-Acetylglucosaminyl.

3. The method of claim 1 wherein R, is hydrogen.

4. The method of claim 1 wherein R, is -CH₃.

5. The method of claim 1 wherein X is an amino acid residue.

6. The method of claim 5 wherein X is valine or alanine.

7. The method of claim 1 wherein Y is selected from the group consisting of threonine, alanine, valine, and serine.

8. The method of claim 7 wherein Y is L-alanine or L-threonine.

9. The method of claim 1 wherein R₂, R₃, R^ and R₇ are independently C_n-C₂₃ hydrocarbons having 0-1 double bonds.

10. The method of claim 9 wherein R₂, R₃, Rg, and R₇ are independently C_I3-C₁₇ alkyl groups.

1 1. The method of claim 1 wherein: Lip is

R, is -CH₃;

R₂ and R₃ are C₁₃ to C_I7 hydrocarbons; X is L-Ala; and Y is L-Ala or L-Thr.

12. The method of claim 1 1 wherein:

R₂ and R₃ are -(CH₂)|₄CH₃ alkyl groups; R₄ is acetylglucosaminyl; X is L-Ala; and

Y is L-Ala.

13. The method of claim 1 1 wherein:

R₂ and R₃ are -(CH₂)|₄CH₃ alkyl groups; R^ is acetylglucosaminyl;

X is L-Ala; and

Y is L-Thr.

14. The method of claim 1 1 wherein: R, is -CH₃;

R₂ and R₃ are -(CH₂)ι₄CH₃ alkyl groups; R₄ is hydrogen; X is L-Ala; and

Y is L-Ala.

15. The method of claim 1 1 wherein: R, is -CH₃;

R₂ and R₃ are -(CH₂)_]4CH₃ alkyl groups; R₄ is hydrogen; X is L-Ala; and Y is L-Thr.

16. The method of claim 1 wherein

Lip is

R₄ is acetylglucosaminyl;

R₅ is -(CH₂)_nCH₃ wherein n is an integer from 0-15; R_t is a Cj , to C₂₃ alkyl group; X is a single bond; and

Y is L-Ala or L-Thr.

17. The method of claim 1 further comprising administering a non-toxic enterotoxin.

18. The method of claim 17 wherein the enterotoxin is ovine TSST.

19. The method of claim 17 wherein the enterotoxin is a deletion mutant of staphylococcal enterotoxin C.

20. The method of claim 17 wherein the MDP compound and the non-toxic enterotoxin are co-administered in a ratio of about 100:1 to about 5000:1.

21. The method of claim 1 wherein the retroviral infection is Human Immunodeficiency Virus infection.

22. A method of inducing the release of a chemokine in a mammal comprising administering an effective amount of a MDP compound having the formula

C— Lip

wherein Lip is

(a)

CH₂0 _C- -R2

/

— X-NH— CH 0

\ CH₂0 _C-

(b)

(c)

or

(d)

-X-NHR₇ wherein R_{ is (C_rC₉) alkyl; R₂, R₃, R_f,, and R₇ are independently (C₆-C₃₀) hydrocarbons having about 0-4 double bonds;

R₄ is hydrogen or N-acylglucosaminyl; R₅ is (CH₂)_πCH₃ wherein n is an integer between 0 and 22; X is a single bond or a peptidyl residue comprising 1-10 amino acid residues; and Y is an amino acid residue.

23. The method of claim 22 comprising inducing the release of at least one chemokine selected from the group consisting of RANTES, MlP-lα, or MlP-lβ.

24. A method of treating a retroviral infection in a mammal comprising administering an effective amount of a non-toxic enterotoxin.

25. The method of claim 24 wherein the enterotoxin is O-TSST.

26. The method of claim 24 wherein the enterotoxin is a deletion mutant of staphylococcal enterotoxin C.

27. A method of inducing the release of a chemokine in a mammal comprising administering an effective amount of a non-toxic enterotoxin.

28. The method of claim 26 wherein the enterotoxin is O-TSST.

29. A MDP compound having the formula

wherein Lip is (a)

or

(b)

wherein R_t is (C_rC₉) alkyl;

R₂ and R₃ are independently (C₆-C₃₀) hydrocarbons having about 0-4 double bonds; R₄ is hydrogen;

X is a single bond or a peptidyl residue comprising 1-10 amino acid residues; and Y is an amino acid residue.

30. A pharmaceutical composition for treatment of a retroviral infection comprising an effective amount ofthe MDP compound of claim 29 and a pharmaceutically acceptable carrier.

31. The pharmaceutical composition of claim 29 further comprising an non-toxic enterotoxin.

32. A MDP compound having the formula

wherein Lip is

wherein R, is (C,-C₉) alkyl;

R₄ is hydrogen or N-acylglucosaminyl;

R₅ is -(CH₂)_nCH₃ wherein n is an integer between 0 and 22;

R-s is a (C₆-C₃₀) hydrocarbon having about 0-4 double bonds;

X is a single bond or a peptidyl residue comprising 1-10 amino acid residues; and Y is an amino acid residue.

33. A medicament for the treatment of a retroviral infection in a mammal comprising an effective amount of a MDP compound.