WO2018031782A1 - Nanoparticle compositions and methods for enhanced stability and delivery of glycopeptide drugs - Google Patents

Abstract

Drug delivery systems such as Solid Lipid Nanoparticles (SLNs), Microemulsions (MEs), or Self-Microemulsifying Drug Delivery Systems (SMEDDS), that can deliver glycopeptide drugs in vivo. The systems incorporate a glycopeptide drug Into a lipid nanoparticle, wherein the lipid may help to protect labile peptide chains from peptidase activity in the bloodstream until the particles can arrive at the BBB where the peptide can cross and then bind to its receptors.

Description

NANOPART1CLE COMPOSITIONS AND METHODS FOR ENHANCED STABILITY AND DELIVERY OF GLYCOPEPTIDE DRUGS

FIELD OF THE INVENTION

{ i I The present invention relates to methods and lipid nanopartscie compositions for enhancing the stability and delivery of glycopeptide drugs.

GOVERNMENT SUPPORT

0002) This Invention was made with government support under Grant No. R01 NS052727 awarded by NIH and Grant No. CHE1339597 awarded by SF. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

{00031 Applicant asserts that the information recorded in the form of an Annex C ST.25 text file submitted under Rule 13?er.1(a), entitled UNIA_„16_„40_„PCT_Sequ©nc8_„Listin8_„ST25.txt_t is identical to that forming part of the international application as fifed. The content of the sequence listing is incorporated herein by reference In its entirety.

BACKGROUND OF THE INVENTION

fiHMMJ Over 250 peptide neurotransmitters are produced in the human brain. These endogenous neuropeptides have th molecular features to bind and activate G-Protein Coupled Receptors (GPCRs) in the brain with exquisite specificity, potency, and minimal, predictable side effects, Clinically relevant targets include acute and/or chronic pain, inflammation, cognition and neuroprotection. Native peptides typically have poor metabolic stability, pharmacokinetics (PK) and pharmacodynamics (PD). The PK/PD properties have been considerably improved by glycosyiation of the peptides. However: the pharmaceutical industry has been reluctant to adopt glycopeptide drugs due to their non-linear PK. It was surprisingly discovered by using in vivo microdsalysis coupled with mass spectrometry (MS² and S^S) detection that even though glycopeptide drugs are essentially absent from serum 20— 30 minutes following Ay. administration, they can persist in the brain well beyond 60 minutes. |088S] The present invention features drug delivery systems, e.g., Solid Lipid Nanoparticies (SL s), Microemuisions (MEs), or Se!f-Microemu!sifying Drug Delivery Systems (S EDDS), that can deliver glycopeptide drugs in vivo (see review article by Lawrence &. Rees, 2012, Advanced Drug De!ivery Reviews 64; 175-193). Numerous colloidal delivery systems have been used in the delivery of lipophilic drugs; including liposomes, microspheres, niosomes, polymeric nanoparticies, as well as SLNs. Typically, the drugs formulated in this fashion are lipophilic in nature and may be regarded as "oils." Microemuisions are clear, stable isotropic mixtures comprised by oil, water and a surfactant; sometimes a co-surfactant is also added. Self-Microe ulslfying Drug Delivery Systems (SMEDDS) are isotropic mixtures of oil and a surfactant co- surfactant, but require mild agitation in the presence of water to form the microemulsion. The glycopeptide drugs in the present application may be regarded as "co-surfactants," not "oils." By incorporating the glycopeptide drug into a lipid nanopartieSe, the lipid can protect labile peptide chains from peptidase activity in the bloodstream until the particles can arrive at the BBS where the glycopeptide can cross and then bind to its receptors. Because glycopeptides are incorporated into micelles, the system mimics the way lipoproteins transport material in the bloodstream. While SLNs and SMEDDS have been used previously to deliver lipophilic drugs with poor wafer solubility, their use has been unexplored for the delivery of surface-active drugs such as glycopeptides, which can function as co-surfactants in the delivery process. Without wishing to limit the present invention to any theory o mechanism, it was surprising that the drug delivery systems of the present Invention were able to he produced given the technical difficulties associated with the glyeolipid's solubility in buffer (e.g., more ionic solvents make them less soluble) and the difficulties making sure the solution is homogenous and dissolved. Lipid based warm microemuisions (WMEs) have been successfull employed in the preparation of drug-loaded SLNs for drug delivery.. The SIN characteristics vary with the W E microstructure and the quenching process. Quenching is typically achieved by rapidly dropping the temperature from about 80— 7CTC to ~4 C by dispersing the microemulsion in -¼0 under continuous mixing to form aqueous dispersions of particles with diameter In the order of 100—300 nm. imnwi Since there are hundreds of GPCR targets available, this invention could be transformative In terms of producing new drugs for the treatment of manifold pathologies, including neurodegenerative diseases such as Alzheimer's and Parkinson's, as well as pain, depression and behavioral disorders. The present invention also features glycopepiide drugs based on endogenous peptides in the areas of acute pain and chronic pain (opioids), post-surgical dementia (angiotensin), and Parkinson's Disease (PACAP and VIP),

B mY

0Θ 7| The present invention features glycopeptide drug delivery systems. In some embodiments, the glycopeptide drug delivery system comprises a glycopeptide drug; and a micelle formed from a glycolipid surfactant, wherein the glycopeptide drug is integrated into a lipid portion of ihe micelle and therein is resistant to peptsdases. In some embodiments, the glycopeptide drug delivery system has a ratio of giycopeptides:micelies from about 1 :1 to 10:1. In some embodiments, the Is from about 25 to 85. In some embodiments, the concentration of glycopeptide is from about 1 mlvl to 30 mM,

In some embodiments, the glycopeptide drug delivery system further comprises an additional lipid component, the additional ilpid component is within the micelle, in some embodiments, the additional lipid component comprises a triglyceride. In some embodiments, the glycopeptide drug can cross the blood brain barrier, In some embodiments, the glycopeptide drug functions as a co-surfactant. In some embodiments, the glycopeptide drug deliver system Is from about 350— 800 nm in size across its largest dimension, In some embodiments, the glycopeptide drug delivery system comprises a naturall occurring lecithin surfactant. In some embodiments, the naturally occuning lecithin surfactant comprises 1 ,2-dspa!mstoyi~sn-glycero-3-phospbo~ choline (OPPC). in some embodiments, the triglyceride comprises glyceryl trioctanoate or glyceryl tripalmifate,

\itt¼B} The present invention also features a method of delivering a glycopeptide drug in a subject, in some embodiments, the method comprises introducing into the subject a glycopeptide drug delivery system of the present invention, wherein the glycopeptide drug delivery system delivers the glycopeptide drug within the glycopeptide drug delivery system to a location of interest. In some embodiments, the location of interest is the blood brain barrier (BBB), In some embodiments, the location of interest is a brain ceil via the biood brain barrier (BBB). In some embodiments, the method is used for administering a drug to the subject to treat or manage a pathological condition. f #10 Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill In the art. Additional advantages and aspects of the presen invention are apparent in the following detaiied description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

l&bi i] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which: filil2.| FIG, 1A shows a schematic view of giycopeptides acting as cosurfacianis to inser into micelles formed by glycoiipids (surfactants), producing Solid Liquid Nanopariicles (SLNs).

Ϊ0β13| FIG. 18 shows a schematic view of in vivo injection of SLNs. The fully loaded SLNs retain the amphipathic drug in water, but can transfer the giycopeptides from one amphipathic environments (e.g., glycolipid micelle) to another similar environment (e.g., one with reduced membrane curvature) in vivo. In some embodiments, the micelle merges into the lipid biiayer.

6 1 j FIG. iC shows a possible pathway of entry of the glycopeptide-drug mixed micelles.

1 1 f FIG. 2A shows examples of giycopeptides: LY 147 (Tyr-D-Ala-Gly-Phe- Ser(hefa-D-Glc)- H_2! SEQ ID NO: 1), MMP2200 (Tyr-D- hr-Gly-Phe-Leu-Ser(beta~ Lact}~NH₂, SEQ ID NO: 2), AT5 (Asp~Arg~Val--Tyr-ISe~H!S-Ser(beta~D~G!c)~NH_2> SEQ ID NO: 3), LYX4200 (YtGFL-gamma-NLBEioKAL ~S(beta-D-Gic)-L-NH₂, SEQ ID NO: 4), and EMJ1285 (HSDAVFTDNYi_flT L KQLAVK2oKYLNSiL-S(beta- ct)-NH_2> SEQ ID NO: 5).

0016j FIG. 28 shows examples of glycoiipids,

(0017} FIG. 3A shows the stability of AT5 in vitro,

FIG, 3B shows serum stability and brain levels of Angiotensin^? after .v. injection (1 mg/Xg),

j0 59j FIG, 3C shows serum stability and brain levels of ATS after Lv, injection (1 mg Kg).

}¾ 26] FIG. 4 shows a non-limiting example of synthesis of glycolipids and O-ii.nked serine gSycopeptides,

[0 2i ) FIG. 5A shows a schematic view of a 2~corriponent micelle.

1 221 FIG. 58 shows a schematic view of a 3-component micelle.

1 0 1 FIG, 8A is related to HMR Studies in d&ut&rat@d SDS Micelles in 02G. FIG. 8A shows two-component micelles in D2O.

[0024] FIG. 6B shows three distinct binding modes observed; 1. ionic binding for the short QAiMGO-reiated glycopeptide LYM147. 2, ionic + Lipophilic binding for the Leu- enkephalin related glycopeptide MP2200, and 3. Ionic + Amphipathic Helix binding for the endorphin\dynorphin related helices such as LYX4200.

DETAILED DESCRIPTION OF THE INVENTION

f0ft:2S| The present invention features drug delivery systems, e.g., Solid Lipid Nanoparticies (SLNs), Microemuisions (MEs), or Self- icroemulsifying Drug Delivery Systems (S EDDS), that can deliver glycopeptide drugs in vivo.

[0026] FIG. 1A shows a schematic view of giycopeptldes acting as cosurfactants to insert into micelles formed by glycolipids (surfactants), producing Solid Liquid Nanoparticies (SLNs). FIG. 18 shows a schematic view of in vivo injection of SLNs. The fully loaded SLNs retain the amphipathic drug in water, but can transfer the giycopeptldes from one amphipathic environments (e.g., g!ycoiipid micelle) to another similar environment (e.g., one with reduced membrane curvature) in vivo, in some embodiments, the micelle merges into the lipid bl!ayer, FIG. 1C shows a possible pathway of entry of the glyeopeptide-dnsg mixed micelles.

[0027] By incorporating the glycopeptide drug into a lipid nanoparticSe, the lipid can protect labile peptide chains from peptidase activity in the bloodstream until the particles can arrive at the BBB where the peptide can cross and then hind to its receptors. Because giycopeptldes are incorporated into micelles, the system mimics the way lipoproteins transport material in the bloodstream. Without wishing to limit the present invention to any theory or mechanism, it is believed that incorporation of the glycopeptides drugs info gtycopeptide-giycolipkJ nanoparilcles may mitigate potential non-linearity in drug distribution, as the drug may be delivered to the site of action from a protected, particle-bound reservoir of drug in serum, rather than being rapidly degraded as the free giycopeptide. And, it is possible that giycopeptide-lipid particles ma help prevent premature membrane insertion. For reference, FIG, 2A (Table 1 ) and FIG, 28 (Table 2) show non-limiting examples of glycopeptides and giycolipids, respectively. Others may include but are not limited to: Giycolipids: The octyl glycoside of glucose, melibiose, and ceilobiose, with possible addition of the lacioside and ma!toside; Glycopeptides: Lactomophin, etc.

\W28 The following is a non-limiting example of preparation of 2-component mixed micelle: A 1G0mM phosphate buffer at pH 7.4 was prepared by dissolving 309 mg of NaHS0₄, 1102 mg Na₂S0 , and 900 mg NaCi in 100 ml D₂0. A 100 mSVl solution of octyl meiibioside was prepared by diluting 1136 mg (2.5 mmol) of the g!yco!ipid in less than 25 ml of the D^O phosphate buffer and stirred until homogeneous. Minimal heating (5Q°C) was required to aid in dissolution. Once the solution had cooled to room temperature, it was diluted to 25ml total volume. For a two-component solution containing -5 lactomorphin molecules per micelle (100 m ociyl meiibioside, estimated at 30 molecules per micelle), 89 mg of the giycopeptide drug Lacfomorphin was dissolved in less than 5 ml of the glycolipid/phosphate buffer solution by stirring and minima! heat. At room temperature, the solution was diluted to 5mL total volume. β0291 The following is a non-limiting example of preparation of a 3-component mixed micelle: A 100mM phosphate buffer at pH 7.4 was prepared by dissolving 309 mg of NaHSO», 1102 mg NazSO*, and 900 mg NaCI in 100 ml D₂0. A 100 mM soiution of octyl meiibioside was prepared by diluting 1136 mg (2.5 mmol) of the glyco!ipid in less than 25 ml of the D^O phosphate buffer and stirred until homogeneous. Minimal heating (50 °C} was required to aid in dissolution. Once the solution had cooled to room temperature, it was diluted !o 25mL total volume. For a three component solution containing 5 mo!% tricaprylin (C8 triglyceride} -5 lactomorphin molecules per micelle (100 mM ocfyi meiibioside, estimated H at 30 molecules per micelle), 89 mg of the giycopeptide drug Lactomorphin and 11.8 mg of tricaprylin were dissolved in less than 5 mL of the glycolipid/phosphate buffer solution by stirring and minimal heat. At room temperature, the solution was diluted to 5mL total volume. 1θβ; | In some embodiments, the systems of the present invention (e.g., glycopeptide" glycolipid nanoparticles, etc.) comprise synthetic analogues of high-density lipoproteins {HDL}, low-density lipoproteins (LDL), intermediate-density lipoproteins (IDL), very low- density lipoproteins {VLDL), ultra low-density lipoproteins (ULDL), or a combination thereof, 0 3l| FIG. 3A_S FIG, 3B, and FIG. C show microdialysis-MS³ quantification of brain levels of ATS in mice (ATS is shown in FIG. 2A). For example, FIG, 3A shows the native compound has a half-life (t1/2) of 14 min. Arnidation extends the tl/2 to 21 min, and glucosylstlon of the amide extends the t1/2 to 82 min. The Saotoside has a tl/2 of 350 min, a 25-fold improvement in serum stability, FIG. 3B shows serum stability and brain levels of Angiotensin after ίν. injection (1 mg/Kg}, and FIG. 3C shows serum stability and brain levels of ATS after Lv. injection (1 mg/Kg). Angiotensini_™? reached lower eoneentrations in serum than the glycoside, and glucoside ATS persisted longer in the CSF compared to the native peptide. 0032| Studies wer done for analgesics related to enkephalins embedded in deuierated SDS and DPPC micelles using high field NMR, as well as the larger endorphin-like glycopeptides. In addition to conformational analyses using COSY/NOSY/ROSY experiments, the orientation of the glycopeptide with respect to the micelle surface with spin labels (unpaired electrons ^♦) was determined. Lipophilic 5-DOXYL-stearate was used to promote relaxation of amino acid hydrogen atoms "buried" in the micelles, and H2 O-soluble Mrt++ ions were used to relax hydrogen atoms In the aqueous environment. Furthermore, unpublished DOSY experiments have been used to measur diffusion rates of micelles; the "unloaded" micelles diffuse significantly faster than micelles ^''loaded" with glycopeptides. These studies suggest that glycopeptides may "recruit" additional SDS molecules into the SDS micelle to increase Nagg, although other interpretations are possible- e.g., th glycopeptides could induce a change in shape, invalidating the Stokes-Einstein assumptions.

[0833] Glycopeptides and glycoiiplds of the present invention may be synthesized using methods known to one of ordinary skill in the art. A non-limiting example is shown in FIG. 4. In some embodiments, "Minimally Competent Catalysis" provides the desired glycosides, which are converted to the corresponding glycopeptide drug candidates (Co Surfactants), or simply deprotecied via Zemp!en deacylation to provide the desired glycoiipids (Surfactants). In some embodiments, glycopeptides are synthesized from endogenous peptide neurotransmitters such as the opioids enkephalins, endorphins and dynorphlns, as well as non-opiolds related to angiotensin, PACAP and VIP, however the present invention is not limited to these examples. 0 3 | As previously discussed, regarding the formation of St s and SMEDOS, the giycopepikle drug itself can serve as a surfactant of cosurfactant

[0035} In some embodiments, the compositions of the present invention are binary (2- component) or ternary (3-component) systems (FIG. 5A shows a smaller, 2-component micelle formed from glycoiipid surfactant and giycopeptide cosurfactant; FIG. 58 shows a larger, 3-component micelle formed from glycoiipid surfactant, giycopeptide co- surfactant, and a third lipid component e.g., a triglyceride or other appropriate lipid component).

[0836] The following is a non-limiting example of synthesis of a drug delivery system of the present invention: 100mM phos buffer at pH-7.0~7.4 with 0.9% NaCI in D2O (for N R) was used to dissolve the solid/powder glycoiipld, diluting to a given concentration (e.g., brought to volume). Octyl giucoside 40Mm (major solubility issues in buffer), octyl eellohioside up to 100-150mM» octyl melibioside up to ~250m . Giycopeptide was weighed and dissolved in above surfactant/buffer solution, again brought to volume depending on the desired peptide:mieeSie. For three component systems, the peptide was weighed, the lipid of choice was added, then it was diluted in surfactant solution. Without wishing to limit the present invention to any theory or mechanism, it is believed that the system of the present invention presents challenges with respect to balancing ratios and the use of heat (e.g., mild heat may sometimes be added but it may result in precipitate/cloudy solutions when cooled to rt).

|ββ37;| In some embodiments, the glycopepfide-glycoSipid mixtures of the present invention are evaluated using NMR studies and/or surface tensiometry (e.g., for determining the critical micelle concentration (CMC). According to regular solution theory, for a mixture of surfactants A and 8, the CMC behavior changes according to:

where γ is a function of an interaction paramete β that is 0 for ideal mixing (ίβ, no interaction) and takes on negative values for synergistic interactions and positive values for antagonistic values. According to this theory, the micelle composition can thus he predicted according to:

£0038] Extreme repulsive interactions can result in phase separation with extremely large and positive values of the interactio parameter β. Without wishing to limit the present invention to any theory or mechanism, it is believed that such interactions are not expected in these mixtures since both the giycolipids and the glyeopeptides are largely neutral in aqueous madia at neutral H, or have a small positive charge. The emergence of lyotropic phases of order higher than mieelSar (e.g., lamellar, hexagonal, cubic, etc.) as indicated by the formation of solution aggregates of sizes comparable to or larger than the wavelength of light (which will increase light scattering and hence, apparent absorbance) may be Investigated using titration with UV-Vis turhidimetric detection as a function of composition and temperature {25\ 50° and 75^*0}. These regions of the phase diagrams ma help move toward 3- component systems that will lead to oiMn-water emulsion-based SL s. 6 39j In some embodiments, the glycope tide/glycolipid ratio is small in SLNs used for drug delivery. If so, initial estimates of micelle size and shape may be ascertained. For example, the aggregate shape may be predicted by comparing the volume of the hydrocarbon chain (v«) to the volume determined by multiplying a by the length of the hydrocarbon: chain (y, A packing parameter (P_e) determined by this ratio (P_;; - v {a₀l_c)) predicts spherical aggregates if P_c < 1/3, ellipsoidal aggregates if 1/3 < P_c < 1/2, and lamellae or vesicles if 1/2 < P_c < 1.48 The aggregation number of a spherical micelle is calculated from the volume of a sphere with a radius equal to the length of the hydrocarbon chain divided by the volume of a hydrocarbon chain as determined above (M ~ ((4 3)ni_c ³)/v_c}, This value may he compared to experimental H values to assess shape. Also, values increase with afkyl chain length, n_c. homologous series; so experimental A ₈._3S/n_c values may predict micelle shape as well. Experimentally determined growth may be compared with the geometrically predicted growth for various shapes (sphere, oblate, prolate} and e!Hpticities (ratio of semsmajor axis to semi-minor axis) calculated using approximations described by Tanford. Dynamic light scattering (DLS) based on photon correlation spectroscopy may be used to measure true micelle sizes of these two-component systems as a function of mixture composition and temperature. The apparent hydrodynamic radius (RH) may he calculated from the intensity-averaged translational diffusion coefficients (D) and the Stokes-Einstein relationship extracted from the autocorrelation functions with a shape parameter of one (/.a spherical). RH is the apparent hydrodynamic radius that is equal to the radius of a spherical particle with the same D as the aggregate measured; therefore, if is minimally sensitive to growth in a single axial direction, i.e. minimal anisotropy does not significantly affect measured RH,

I *H?4 I in some embodiments, the ratio of giycopeptides to micelles is 1 :1 , in some embodiments, the ratio of gSycopeptides to micelles is 2:1. in some embodiments, the ratio of giycopeptides to miceiies is 3:1. In some embodiments, the ratio of giycopeptides to micelles is 4:1. In some embodiments, the ratio of giycopeptides to micelles is 5: 1. In some embodiments, the ratio of giycopeptides to micelles is 6:1 . In some embodiments, the ratio of giycopeptides to micelles is 7:1. in some embodiments, the ratio of giycopeptides to micelles is 8:1. In some embodiments, the ratio of giycopeptides to micelles is 9:1. In some embodiments, the ratio of giycopeptides to micelles is 10:1. In some embodiments, the ratio of giycopeptides to miceiies greater than 10:1. 04ij In some embodiments, the peptide concentration is from about 1 mM to 5 mM (e.g., 2,5 mM). in some embodiments, the peptide concentration is from about 1 mM to 10 mM. in some embodiments, the peptide concentration is from about 5 mM to 10 mM In some embodiments, the peptide concentration is from about 1 m to 15 mivL In some embodiments, the peptide concentration Is from about 10 mM to 15 mM. In some embodiments, the peptide concentration is from about 1 mM to 30 mM (e.g., 25 mM). In some embodiments, the peptide concentration is from about 1 mM to 10 mM. In some embodiments, the peptide concentration is less than 1 mM. In some embodiments, the peptide concentration is greater than 30 mM

}0642J Without wishing to limit the present invention to any theory or mechanism, it is believed that a larger headgroup and shorter tail may be easier because of better solubility.

[0043 | DOSY M R allows the measurement of diffusion rates of the surfactants as free molecules in solution and as aggregates (mixed micelles). "Empty micelles" have been observed to migrate at significantly faster rates than "loaded micelles" that carry a glycopeptide cosurfactani. Since it is possible to also measure the diffusion rate of the free glycopepiides, the efficiency of Drug Loading for each giycoiiptd-glycopeptide combination may be estimated. Binding coefficients may be on the order of _on ** 10² for glycosylated enkephalins and endorphins. However, the present invention is not limited to m * 2 X i0², e.g., binding coefficients may be much greater. The binding coefficient is generally such thai the micelle is able to reiease the compound in order to delive it. In some embodiments, * is from about 1 x 10² to 5 x 1Q², from about 1 x 10³ to 5 x 10³, from about 1 x 10^s to 1 x 10^s, from about 1 x 10¹ to 5 x 10⁴, etc. Without wishing to limit the present invention to any theory or mechanism, it is believed that this binding should not necessarily be too tight or the micelle will not want to "deliver" the glycopeptide to a biological membrane (e.g., as shown in FIG. 18). The diffusion rates for inverse micelles formed by giycolipids in CDC may be measured, and thi information may be useful in the prediction of Loading\Delivery rates.

{W44\ FIG. 6A and FIG. 68 show NM studies in deuierai&d SDS micelles in D₂0. Two- component micelles {cfe-SDS + glycopepiides, e.g. LYM147 / MMP2200 / LYX4200) in

DaO are shown in FIG, 6A, Low tumbling rates (e.g., high τγ, with effective fvlVV, *

2x10⁶) for the glycopepiides show they are locked" to the micelle. Spin labels 5- DOXYL-stearate and MnCbwere used to characterize the orientation of glycopepiides in ihe deul&ratetl micelles. FIG. 68 shows three distinct binding modes that were observed: 1. Ionic binding for the short DA GOreiated glycopeptide LYM147, 2. Ionic + Lipophilic binding for the Leu-enkephalin related glycopeptide P2200. 3. Ionic + Amphipathic Helix binding for the endorphin\dynorphin related helices such as LYX4200, Classical lipophilic drugs (e.g., morphine) are completely buried In the micelles as observed by fast tumbling independent of the micelles in the lipophilic interior {i.e. low τ with normal fVl.W,}.

[fl045| Various forms of fluorescence spectroscopy and microscopy may be used for characterization studies. For example, it is possible that a more accurate estimate of 2- component aggregate size may be obtained through determination of the aggregation number (N_ag8), the number of monomers in the average micelle at any given concentration , A non-limiting example of a range of N_agg is 25— 85 fo spherical or slightly elliptical micelles. Fluorescence quenching spectroscopy, both steady-state and time resolved using time correlated single photon counting (TCSPC), may b used to determine aggregation number. Aggregation number may be determined based on probe fluorescence decay by fluorescence quenchers distributed within micelles. Probe fluorescence decays at a rate of 1/TQ + n k_q where ¾ is the fluorescence lifetime, k_q is the quenching rat fo one quencher and one probe within a micelle, and n Is the number of quenchers within a micelle. Micelles act as discrete microenvironmenis within a solution resulting in a distribution of quenchers across the micelles such that the probability of n quenchers in a micelle with one probe (P) follows a Poisson distribution. Therefore, a time-resolved fluorescence intensity, l(t), versus time (t) curve represents all micelles with various n and is modeled by the reduced Infelta-Tachiya equation;

/ (£) - A_t exp [~A_tt -A₃ (1 - exp(-4₄t))J where A-; ~ i(0). As ~ 1/¾ + [Q (k A ){k~ [ ]), for which [O] is the quencher concentration and M] is the concentration of micelles, As ~ (1¾ Α«) [0|/|Μ), and A* ~ k_¾ k- wher k- is the quencher exit rate out of a micelle. A Time-Resolved Fluorescence Quenching (TRFQ) curve is fit to this equation to solve for fitting parameters At, Aa, A3 and A4. The fluorescence lifetime To is determined by collecting a TRFQ curve without quencher (A3 is zero). Then, τ₀ is used in calculating k_q, k-_s and |Q] j ] from fits to a TRFQ curve with known |Q|. The average aggregation number, U_$ is calculated from [t¾/[M], the known [S]_« and the CMC using: [M] - ( [S] ~ CMC)!N_m. 80 6_.1 Fluorescence spectroscopy may also be used to determine micro-polarity of these mixed micelles at different temperatures, e.g., using both pyrena and prodan fluorescence probes. Both probes are known to partition into hydrophobic regions of surfactant aggregates. One common measure of microenvironment polarity is the intensity ratio of the third (ill) to first (I) vibronic bands of pyrene; this ratio ranges from values >1 in more nonpolar environments to -0.5 in extremely polar environments. Prodan exhibits a biue shift in its A_m8x of emission as Its environment become increasingly more nonpolar. Additionally, confeeal fluorescence microscop can be used to visualize the presence of iyofropic phases of various sorts through partitioning of a polarity sensitive dye into the aggregate structures. Polarized fluorescence microscopy also a!!ows insight into the presence of lyotropic phases.

|0β47;| In some embodiments, in order to determine the partition coefficient for a given giycopeptide into the g!ycoiipid surfactant, a hydrophobic residue of the giycopeptide with a polarity sensitive fluorescent dye that will report its microenvironment may be synthetically modified, it may be that using the change in fluorescence signal and lifetime as a function of giycopeptide mole fraction, the partition coefficient can be quantitatively determined using previously reported approaches.

[Θ648) Fluorescence spectroscopy using micropolarity probes may also be used to assess lifetimes of these aggregate structures after rapid mixing. This property may be important to assess for consideration of such materials fo drug delivery applications. Recent literature reports micelle lifetimes from common nonionic surfactants ranging from about 10's to 100^:s of seconds. The present invention features rapidly mixing a mtceliar solution containing a fluorescence probe that strongly partitions into the micelles (e.g. pyrene, merocyanine, eosin, rhodamine sudan) with aqueous in vitro models of plasma (e.g. saline solution, saline solution doped with plasma proteins, etc.) and monitoring the fluorescence behavior with time. Given the network of intermolecuiar hydrogen bonding between sugar headgroups that is expected to stabilize these structures, it is possible that the lifetimes will be towards the upper end of the times previously reported or even exceeding those values fo these glycofipid-based systems, ¾ ^s) in some embodiments, the ability of these systems to form reverse micelles, oil- in-water (o w) and water-in-oii (w/o) rnicroemuisions {/.©. three-component systems) may be determined first using a series of simple nonpolar solvents as the oil phase. Measurements described above (e.g., surface fens ometry, DLS, UV-VIs turbidlmetry and fluorescence spectroscopy and microscopy, etc.) may be used to study the reverse micelles at 25*, 50° and 75 *C. For example, a gSycoiip!d/glycopeptide mixture may be first be dissolved in hexane with water titrated In to form reverse micelles. These reverse micelles may also be characterized by DLS and fluorescence spectroscopy. j 11 01 Three component o/w and w/o rnicroemu!sions may be fabricated and characterized using oil phases compatible with drug delivery vehicles, such as the triglycerides and long chain fatty acids. In some embodiments, in order to create systems from which SLNs can be fabricated by quenching in aqueous media, the system may need to exist in an isotropic region of the pseudo-ternary phase diagram. The full phase diagrams for these systems may be explored at 25°, 60^* and 75 in orde to determine suitable compositions that fall in this Isotropic regime. From these regions, SLNs may be fabricated by dropping the o/w micro-emulsion into a cooled aqueous medium under continuous stirring. The SNLs so formed may then be characterized as to their size and shape using DLS and oonfoca! fluorescence microscopy, and their microenvironments may be probed using polarity sensitive fluorescent dyes. The lifetimes of these SLNs may also foe characterized by rapid mixing using similar polarity sensitive fluorescent dyes.

[Q05i] Stable SLNs of various sizes can be formulated for drug delivery. Extremely small 2-component micelles -4-8 nm can be produced, or very large 3-component micelles with an oil "core" resembling VLDL particles that can reach -400-BOO nm can be generated with a non-toxic oil. Naturaiiy-occurring lecithin surfactants, such as 16:0 PC (a.k.a. DPPC or 1 ,2-dip3imitoyl-s ?-giycero-3-phospho~choiine), have been used in conjunction with triglycerides such as glyceryl trioctanoaie [a.k.a. TG(10:0/10:0/10:0)] or glyceryl tripaimitata {s,k.a. TG(16:0/16/:0/16/0}^'). The present invention is not limited to the aforementioned: materials,

|Θ052] The present invention also features methods for determining effectiveness of the SMEODS and/or SLNs In drug delivery. The following methods and protocols are examples only and do not limit the present invention in any way. The effectiveness of the SMEDDS and/or SLNs In drug delivery may be examined in vivo in mic using M P2200. The antinociceptive (analgesic) efficacy of unformulated MMP2200 has been well-characterized In mice and rats following Lv or s.c. administration. The 55°C tail flick assay may be used (a modified version of the classic tail-flick test adapted for mice instead of rats), wherein mice are lightly but firmly grasped by the nape of the neck with the evaSuaiors thumb and fingers, and the distal half of the tail is then dipped into a bath of circulating water thermostatically controlled at 56^*0, Latency to respond to the heat stimulus wit vigorous flexion of the tail may be measured to the nearest 0,1 see, A baseline determination may he made, followed by testing at various times (e.g., 10, 20, 30, 45, 80, 90 & 120 mln) after administration of the gSycopeptides at 3 doses. A 10 sec cut-off may be used to prevent tissue damage to the tall, Antinoeieeption (EDSO or ED90) may be calculated by the formula: % Antinociceptlon - [(Test ™ Baseline Latency ) {1Q ~ Baseline Latency)] x 100, Since latencies In this test may he affected by tail skin temperature careful attention may be paid to ensure that the ambient temperature is maintained at 22— 23X. The EDSO and ED90 of the new formulation of MMP220G may be compared In producing acute antinociceptlon in mouse hot water (55 ) tail Hick lest to those of unformulated form. The ED90 dose may be tested further to determine the potential antinociceptive tolerance following repeated drug administration (s,c, 2X daily for 3 days) using tail flick test The same mice may be injected with opioid antagonist naloxone (1 mg/kg, i.p.) to compare the propensity of the two formulations in inducing dependence. The respiratory depression activity of the two formulations at 3 doses may be assessed to obtain their LD50 and therapeutic index. The efficacy of MMP2200 in alleviating chronic bone cancer pain may be determined, of which opioids are the mainstay of the treatment options, Mice may be subject to inoculation of 100 NCTC2472 sarcoma ceils into the femur and the spontaneous and movement-evoked pain may be assessed at 12 days post-surgery before and after M P2200 administration:. The present invention is not limited to the application and methods previously described. fili53| Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.

[Θ054| Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled In the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. Reference numbers recited in the ciaiims are exemplary and for ease of review by the patent office only, and are not limiting in any way. In some embodiments, fhe figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. in some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase "comprising" includes embodiments that could be described as "consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase "consisting of is met. fytSSj The reference numbers recited In the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended In any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings.

Claims

WHAT IS CLAIMED iS:

1, A glycopeptide drug delivery system, said glycopeptide drug delivery system comprising: a. a glycopeptide drug; and b. a micelle formed from a glycoilpld surfactant, wherein the glycopeptide drug is integrated into a !lpid portion of the micelle and therein is resistant to peptidases; wherein the gfycopepiide drug delivery system has a ratio of glycopeptides:micelles from about 1:1 to 10:1 , wherein the is from about 25 to 85, wherein a concentration of glycopeptide Is from about 1 mM to 30 mM.

2, The glycopeptide drug delivery system of claim 1 further comprising an additional lipid component, the addiiionai lipid component is within the mscelie,

3, The glycopeptide drug delivery system of claim 2, wherein the additional lipid component comprises a triglyceride.

4, The glycopeptide drug delivery system of claim 1, wherein the glycopeptide drug can cross the blood brain barrier.

5, The glycopeptide drug delivery system of claim 1 , wherein the glycopeptide drug functions as a co-surfactant β. The glycopeptide drug delivery system of claim 1, wherein the glycopeptide drug delivery system is from about 350— 800 nm in size across its largest dimension.

7, The glycopeptide drug delivery system of claim 1 , wherein the glycopeptide drug delivery system comprises a naturall occurring lecithin surfactant.

8. The glycopeptide drug delivery system of claim 7, wherein the naturally occurring lecithin surfactant comprises 1 ,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC).

9. The glycopeptide drug delivery system of claim 3, wherein the triglyceride comprises glyceryl irioctanoate or glyceryl tripa imitate.

10. A method of delivering a glycopeptide drug in a subject, said method comprising introducing into the subject a glycopeptide drug delivery system of any of Claims 1-9, wherein the glycopeptide drug delivery system delivers the glycopeptide drug within the glycopeptide drug delivery system to a location of interest.

11. The method of claim 10, wherein the location of interest is the blood brain barrier (SBB),

12. The method of claim 10, wherein the location of interest Is a brain cell via the blood brain barrier (BBB).

13. The method of claim 10, wherein the method is used for administering a drug to the subject to treat or manage a pathological condition.

1 . A glycopeptide drug delivery system, said glycopeptide drug delivery system comprising: a. a glycopeptide drug; and b. a micelle formed from a glyoolipid surfactant, wherein the glycopeptide drug is integrated into a lipid portion of the micelle and therein is resistant to peptidases; wherein the glycopeptide drug delivery system has a ratio of glycopeptides:micelles from about 1 :1 to 10:1 and wherein the H_m is from about 25 to 85.

15. The glycopeptide drug delivery system of claim 14 further comprising an additional lipid component, the additional lipid component Is within the micelle,

18. The glycopeptide drug delivery system of claim 15, wherein the additional lipid component comprises a triglyceride. 7. The glycopeptide drug delivery system of claim 14, wherein the glycopeptide drug can cross the blood brain barrier.

18. The giycopeptide drug delivery system of claim 14, wherein the giycopeptide drug functions as a co-surfactant

19. The glyoopeptide drug deliver system of claim 14, wherein the giycopeptide drug delivery system is from about 350— 800 nm in size across its largest dimension.

20. The glyoopeptide drug deliver system of claim 14. wherein the giycopeptide drug delivery system comprises a naturally occurring lecithin surfaciant.

21. The glyoopeptide drug delivery system of claim 20. wherein the naturally occurring lecithin surfactant comprises 1 ^-dipaSrnitoyl-sn-glycero-S-phospbo- choline (DPPC).

22. The glyoopeptide drug delivery system of claim 16, wherein the triglyceride comprises glyceryl frioctanoate or glyceryl tnpalmitate.

23. A glyoopeptide drug delivery system, said glyoopeptide dmg delivery system comprising: a. a giycopeptide drug; and b. a micelle formed from a gSycoSipid surfaciant, wherein the glyoopeptide drug is integrated into a lipid portion of the micelle and therein is resistant to peptidases; wherein the giycopeptide drug delivery system has a ratio of glycopeptides:micelies from about 1 :1 to 10:1.

24. The giycopeptide drug deliver system of claim 23 further comprising an additional lipid component, the additional lipid component is within the micelle.

25. The giycopeptide drug delivery system of ciaim 24, wherein the additional lipid component comprises a triglyceride,

28. The giycopeptide drug deliver system of claim 23, wherein the giycopeptide drug can cross the blood brain barrier.

27. The glycopeptsde drug delivery system of claim 23, wherein the glycopeptide drug functions as a co-surfactant.

28. The glycopeptide drug delivery system of claim 23, wherein the glycopeptide drug delivery system is from about 360— 800 nm in size across its largest dimension.

29. The glycopeptide drug delivery system of claim 23, wherein the glycopeptide drug delivery system comprises a naturally occurring lecithin surfactant.

30. The glycopeptide drug delivery system of claim 29, wherein the naturally occurring lecithin surfactant comprises 1 ,2-dipalmitoyl-sn-glycero-3~phospho- choline (DPPC).

31. The glycopeptide drug delivery system of claim 25, wherein the triglyceride comprises giyoeryl triocfanoate or glyceryl tripalmitate.