WO2012097255A2 - Effet thérapeutique de protéines de choc thermique utilisées pour la prévention de l'agrégation de l'amyline dans le diabète sucré de type 2 - Google Patents

Effet thérapeutique de protéines de choc thermique utilisées pour la prévention de l'agrégation de l'amyline dans le diabète sucré de type 2 Download PDF

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WO2012097255A2
WO2012097255A2 PCT/US2012/021246 US2012021246W WO2012097255A2 WO 2012097255 A2 WO2012097255 A2 WO 2012097255A2 US 2012021246 W US2012021246 W US 2012021246W WO 2012097255 A2 WO2012097255 A2 WO 2012097255A2
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cells
antibody
nanoparticle
carrier
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WO2012097255A3 (fr
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Alexzander Asea
Paola ROSAS
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Scott & White Healthcare
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention generally concerns at least the fields of molecular biology, cell biology, and medicine.
  • the field concerns treatment of type 2 diabetes using heat and/or heat shock proteins.
  • Proteins that fold into a compact structure contain a large number of hydrophobic residues in their sequences. In the correctly folded structure, these residues are buried inside the protein core and are protected against non-native interactions. The presence of hydrophobic residues makes these proteins very susceptible to aggregation when unfolded or partially folded (Chiti et ah, 2002). An increasing number of neurodegenerative disorders are associated with the existence of misfolded proteins and the presence of non-native structures described as oligomers, protein aggregates, fibrils, or plaques.
  • HSPs or molecular chaperones many of which are ATP-dependent. HSPs are in contact with most cellular proteins from the moment of their production to assist with proper folding and later on to ensure the maintenance of native protein conformation during stress.
  • Islet amyloid polypeptide also called amylin
  • IAPP Islet amyloid polypeptide
  • pancreatic ⁇ -cells in a 1: 10 ratio. It is normally released in response to meals and appears to lower glucagon secretion and the rate of appearance of glucose after a meal.
  • Human, feline and non-human primate forms of amylin are the only ones with the capacity to oligomerize and form aggregates due to the hydrophobicity of amino acid residues 20-29.
  • Janson et al. (1999) demonstrated that amylin aggregates containing less than 6000 molecules (called intermediate- sized amyloid particles) are toxic to pancreatic islet cell membranes. Their cytotoxicity is mediated through disruption of cell membrane
  • amylin aggregates Once amylin aggregates become larger (> 10 6 molecules) the hydrophobic regions of amylin are hidden inside the core of the aggregate and their capacity to interpolate within the membrane is reduced (Janson et ah, 1999). However, accumulation of mature islet amyloid is responsible for space- occupying lesions associated with secretory and absorptive defects (Hayden et ah, 2005). Cells exposed to "aged" human IAPP (h-IAPP) solutions were surrounded and encased by these mature amyloid fibrils with no effect on the adjacent cell membranes (Janson et ah, 1999).
  • Pancreatic ⁇ -cells respond to the accumulation of unfolded or misfolded proteins by increasing their degradation through the ubiquitin-proteasome system. After existing proteases have been consumed, the cell will respond with survival and/or apoptotic pathways, depending on the nature of the stress. Survival responses include the up-regulation of genes encoding endoplasmic reticulum chaperones or HSPs in order to increase protein folding activity and prevent more protein aggregation. The second response includes the reduction of new protein synthesis in order to prevent further accumulation of unfolded proteins. Finally, transcriptionally-activated apoptosis occurs when the ER is chronically overwhelmed and can no longer deal with the overload of unfolded proteins (Hayden et al., 2005). The ability of HSPs to protect pancreatic ⁇ -cells against cell damage makes them an attractive therapeutic target in the treatment of T2DM.
  • HSPs were highly conserved during evolution and are essential for cell survival. They are primarily found intracellularly, but they are also found in the extracellular compartment.
  • the major heat shock protein families are HSP 60, 70 and 90.
  • Hsp72 also called Hsp70
  • Kurucz et al. (2002) reported a decreased expression of Hsp72 in skeletal muscle of T2DM patients that correlated with insulin resistance (Kurucz et ah, 2002).
  • pancreatic islets from T2DM patients have greater immuno staining for Hsp70 than the islets from non-diabetic humans (Laybutt et al., 2007).
  • the present invention is directed to methods and compositions that regard the therapeutic effect of heat shock proteins in T2DM in a mammal, including a human, dog, cat, or horse, for example.
  • the present invention is directed to methods and compositions that regard the therapeutic effect of heat shock proteins in preventing amylin aggregation in T2DM in a mammal.
  • the invention concerns a particular problem in T2DM, which is pancreatic Beta cell death due to amylin toxicity. No therapies are available yet to target this problem; therefore, the use of heat and/or heat shock proteins is an innovative therapeutic approach.
  • augmented levels of Hsp prevent misfolding and toxicity of amylin oligomers, including further misfolding and toxicity of amylin oligomers, thus conserving ⁇ -cell mass and ultimately improving insulin secretion.
  • compositions are employed to enhance levels of heat shock proteins in pancreatic B cells.
  • the compositions may be comprised of one or more components, including but not limited to heat shock proteins, antibodies, and/or heatable carriers.
  • the composition may include a heat shock protein, a nucleic acid encoding a heat shock protein, an agent that enhances the expression of a heat shock protein, or a
  • the heatable carriers may comprise one or more of nanoparticles, chitosan, and liposomes, for example.
  • the heatable carriers are employed alone for delivery to the pancreas, and upon their heating they increase the levels of Hsps in the pancreas.
  • the heatable carriers are used for delivering one or more Hsps to the pancreas and the heatable carrier then may or may not be heated upon direct or indirect localization to the pancreas.
  • the heatable carrier may comprise one or more tissue targeting moieties that target the heatable carrier to a pancreatic B cell in a subject.
  • the tissue targeting moiety is an antibody that binds to a pancreatic B cell.
  • the antibodies may be employed in the invention to specifically target the compositions to pancreatic B cells. Any method of delivery to a subject is contemplated. In one embodiment, the antibody is laparoscopically delivered to the pancreas.
  • the invention regards the use of heat shock proteins and/or thermo stress in the treatment of T2DM.
  • thermo stress is achieved by utilizing gold nanoshells delivered to pancreatic Beta cells (and optionally specifically targeted thereto) that are then irradiated with near-infrared pulsed-laser.
  • nanocarriers based on chitosan (Guo et ah, 2010) targeted to pancreatic B-cells, which in certain cases also carry gold nanorods to serve as a NIR thermotherapy device to induce the synthesis of HSPs; they may also include a fluorescent probe for real-time B-cell imaging, in specific embodiments. This not only would help to prevent B-cell death but would also allow the evaluation of B-cell mass prior and after treatment, for example.
  • nanoparticles conjugated to polyclonal/monoclonal antibodies and delivery such as
  • NIR near infrared laser
  • the present invention provides a novel use of nanoparticles, including gold nanoshells, because most of their current uses are reported in the treatment of cancer by inducing localized hyperthermia to kill cancer cells.
  • these nanoshells increase the levels of chaperones that will assist with folding and prevent aggregation of unfolded amylin. This ultimately improves pancreatic Beta cell survival and should be clinically used as a preventive and therapeutic intervention for T2DM.
  • gold nanoshells are generated to specifically target human pancreatic Beta cells, and in certain cases the nanoparticles are administrated intravenously to patients.
  • a particular time and amount of laser energy is utilized for the heatable carriers to produce thermal stress with minimum of toxicity. It has been demonstrated that NIR energy has the highest tissue penetration because water, blood and other tissues are relatively transparent in this spectrum; therefore, in certain embodiments this type of energy is used to heat the nanoshells.
  • a wavelength of 808 nm at a power intensity of 4W/cm may be employed, in certain aspects, although surrounding ranges of these parameters may also or alternatively be employed. Animal studies are useful to determine intensity and time of exposure of the pancreatic tissue, for example.
  • multifunctional nanocarriers for example based on chitosan, that are provided to pancreatic B- cells and, in certain cases will comprise gold nanorods to serve as a NIR thermotherapy device and a fluorescent probe for real-time B-cell imaging.
  • the toxicity of amylin in pancreatic Beta cells comprises an IC50 of amylin between 8 and 11 uM. It is determined that pancreatic Beta cells exposed to heat at 43°C for 30 minutes have better survival rates than the controls.
  • a transgenic mouse model is employed that accumulates human amylin in the pancreas.
  • these hIAPP mice develop diabetes with a deficit in B-cell mass due to B-cell apoptosis (Huang et ah, 2007).
  • this animal model is used to characterize the exemplary nanoshells and/or chitosan nanocarriers.
  • the rod shape and size is optimized and the required concentration range is determined of rods for sufficient heating to induce stress response without toxicity. Also, the duration, frequency and intensity of infrared irradiation is determined.
  • the present invention is employed in conjunction with another therapy for the individual, such as one that includes treatment of T2DM and/or symptoms thereof.
  • the nanoparticle is a rod, sphere, ellipsoid, elliptical disk, rectangular disk, or square, for example.
  • the carrier comprises nanoparticles, chitosan, poly(amidoamine) dendrimer or a lipid-based carrier.
  • the carrier is delivered directly to the pancreas.
  • the carrier further comprises an antibody that binds to a target polypeptide on the B cells and the direct delivery comprises targeting of the particle to pancreatic B cells using the antibody.
  • the target polypeptide may comprise Na-K-ATPase, in certain embodiments, and the antibody binds the FXYD2 domain of the regulatory subunit of Na-K-ATPase, in some cases. In specific embodiments, the antibody binds to a region in the N-terminal 1-20, 1-15, or 1-11 amino acids of the FXYD2 domain.
  • Direct delivery comprises delivery of the carrier by laparoscopy to the pancreas, in some cases, although in certain embodiments the carrier is delivered indirectly to the pancreas.
  • Nonlimiting examples of delivery methods include intravenous administration to the individual, oral administration, or via endoscopic retrograde cholangiopancreatography (ERCP).
  • the nanoparticle is a rod, sphere, ellipsoid, elliptical disk, rectangular disk, or square, and in specific embodiments the nanoparticle is a gold nanoparticle.
  • the carrier comprises one or more Heat Shock Proteins (HSPs) or fragments thereof, or a nucleic acid encoding one more more HSPs or fragments thereof.
  • HSPs Heat Shock Proteins
  • the carrier comprises HSP72, HSP27, HSP78, GRP78, fragments thereof, or a combination thereof.
  • the carrier further comprises a label, such as fluorescent, radioactive, or a nanoparticle.
  • a method of treating an individual for Type II diabetes comprising delivering to the pancreas of the individual a therapeutically effective amount of a composition comprising: a) HSP72, HSP27, HSP78, GRP78, fragments thereof, or a combination thereof; and b) one or more moieties selected from the group consisting of an antibody that binds a target polypeptide on pancreatic B cells, a nanoparticle, a lipid-based carrier, chitosan, or poly(amidoamine) dendrimer.
  • the composition is delivered to the pancreas directly, such as by targeting of the composition to pancreatic B cells using the antibody, for example. Direct delivery may comprise laparoscopy, although the composition may be delivered to the pancreas indirectly, such as by intravenous administration.
  • methods include the step of heating the pancreas.
  • compositions comprising: a) HSP72, HSP27, HSP78, GRP78, fragments thereof, or a combination thereof; and b) one or more moieties selected from the group consisting of antibody that binds a target polypeptide on pancreatic B cells, a nanoparticle, a lipid-based carrier (such as a liposome), chitosan, and poly(amidoamine) dendrimer.
  • the antibody is directed to the FXYD2 domain of the regulatory subunit of Na-K-ATPase.
  • the nanoparticle is a rod, sphere, ellipsoid, elliptical disk, rectangular disk, or square.
  • the composition comprises the nanoparticle and the antibody, such as wherein the nanoparticle is conjugated to the antibody, for example.
  • the nanoparticle is a nanorod that is conjugated with an antibody to the FXYD2 domain of the regulatory subunit of Na-K-ATPase.
  • the antibody binds to a region in the first 1-20, 1-15, or 1-11 amino acids of the FXYD2 domain.
  • the composition may be further defined as comprising the nanoparticle and chitosan, such as wherein the nanoparticle is seeded in the chitosan.
  • the nanoparticle is a gold nanoparticle.
  • it comprises a detectable label, such as one that is fluorescent, radioactive, or nanop articles.
  • kits comprising the composition of the invention, said composition housed in a suitable container.
  • the kit further comprises an additional therapy for diabetes.
  • FIG. 1 h-IAPP toxicity in Beta-TC-6 cells determined by trypan blue exclusion assay in which live cells exclude trypan blue, while dead cells that take up trypan blue appear blue when observed under white light. Data are representative of three independently performed experiments with similar results.
  • FIG. 2 h-IAPP IC50 determined in the Beta-TC-6 cell line using the MTS cell proliferation assay. Different cell concentrations were used. The mean h-IAPP IC50 was calculated at 10.43 + 1.92 ⁇ . Data are the sum of four independently performed experiments with similar results.
  • FIG. 3 provides a western showing that thermal stress activates the expression of Hsp72 and Hsp25.
  • Beta-TC-6 cells were heat shocked at 43°C for 30min, 45min or 60min or maintained at 37°C for 30min or 60min (control). Twenty-four hours later, cells were lysed and equal concentrations of cell lysate (3C ⁇ g) was added to a 10% SDS-PAGE and probed with anti-Hsp72 (top panel) or anti-Hsp25 (bottom panel). Data are representative of three independently performed experiments with similar results.
  • FIG. 4 demonstrates that heat shock treatment reduces h-IAPP toxicity against Beta-TC-6 cells.
  • Beta-TC-6 cells were heat shocked at 43°C for 30 min (HS) or maintained at 37 °C (Ctrl). Twenty-four hours later, cells were treated with various
  • FIG. 5 demonstrates that h-IAPP transgenic but not m-IAPP C. elegans develop toxic aggregates.
  • IAPP plasmids expressing in pharynx, body wall muscles and neurons were co-injected at a concentration of 20ng ⁇ l each.
  • A Body wall muscle expression; h-IAPP show more fluorescence and more aggregates than m-IAPP model.
  • B Pharynx expression; h- IAPP model show high fluorescence in the pharynx tissue that could correspond to the presence of protein aggregates.
  • C h-IAPP expressed in head muscles is distributed into discrete foci, while m-IAPP has a more soluble distribution pattern.
  • D h-IAPP show more intense
  • FIG. 6 Injection of h-IAPP transgenic C. elegans with Hsp72 promoter significantly reduces the appearance of toxic h-IAPP aggregates.
  • h-IAPP transgenic mice were injected with control plasmid (left panel) or Hsp72 plasmid (right panel) and the appearance of h-IAPP aggregates was observed using a 60x microscope. Data are representative of at least eight independently performed experiments with similar results.
  • FIG. 7 Transfection of Beta-TC-6 cells with Hsp72 significantly reduces h-IAPP-induced toxicity.
  • Beta-TC-6 cells were transfected with Hsp72 plasmids and incubated for 72 h in a 37°C incubator. Cells were then treated with h-IAPP. After a further 24 h of incubation the number of live cells were counted using a fluorescence microscope. Data are the mean number of live cells (+ SD) from 5 different areas under 60x magnification, and is a representative of three independently performed experiments with similar results.
  • thermoelectric refers to a carrier that is able to enter a tumor and deliver a specific amount of heat.
  • nanoparticle refers to particles sized between 1 and 100 nanometers, in certain embodiments.
  • terapéuticaally effective amount refers to an amount that results in an improvement or remediation of one or more symptoms of T2DM.
  • heat shock treatment is used as a therapy for the treatment of diabetic patients, including T2DM.
  • T2DM type 2 diabetes mellitus
  • the misfolded islet amyloid polypeptide hormone also called amylin
  • forms toxic aggregates that destroy pancreatic ⁇ -cells Hayden et ah, 2005.
  • HSPs molecular chaperones
  • increased levels of HSPs for example induced by heat shock therapy, improve pancreatic ⁇ -cell survival in T2DM.
  • heat shock treatment is used as a therapy, including a concomitant therapy, for the treatment of diabetic patients in which loss of ⁇ -cells worsens insulin resistance.
  • HSPs Heat Shock Proteins
  • one, two, three, four, or more HSPs are provided to an individual for the treatment of Type II diabetes, such as provided to the pancreas in the individual.
  • the HSPs are provided as proteins or protein fragments, although in alternative embodiments the HSPs are provided in nucleic acid form.
  • the HSPs are provided in a composition comprising an antibody, nanoparticle, liposome, chitoson, or a combination thereof.
  • one or more of a variety of HSPs are provided to an individual with Type II diabetes.
  • the one or more HSPs are capable of acting as chaperones on amylin in pancreatic B cells, thereby reducing amylin aggregation in the pancreatic B cells. In certain cases, the one or more HSPs are capable of inhibiting apoptosis of a pancreatic B cell.
  • the one or more HSPs are selected from the group consisting of HSP72 (also called Hsp72, Hspal, or Hsp70 1); HSP27 (having alternate names of 28 kDa heat shock protein, estrogen regulated 24 kDa protein; heat shock 27kDa protein 1; heat shock protein beta 1; HSP28; SRP27, and stress responsive protein 27); GRP75 (having alternate names of Heat shock 70 KD protein 9; Heat shock 70 KD protein 9B; HSPA9; Mortalin, perinuclear; MOT; MOT2; p66 mortalin; PBP74; Peptide binding protein 74; and Stress 70 protein, mitochondrial); and GRP78 (having alternate names of Endoplasmic reticulum lumenal Ca 2+ binding protein grp78; glucose related protein 78KD; heat shock 70kD protein 5; and immunoglobulin heavy chain binding-protein).
  • HSP72 also called Hsp72, Hspal, or Hsp70 1
  • HSP27 having alternate names
  • Heat shock protein 70 KD 1A (HSP72) protein sequence is provided in SEQ ID NO: 1 and the mRNA sequence is provided in SEQ ID NO:2 (open reading frame (ORF) is 244-2169 nt).
  • HSP 27 protein sequence is provided in SEQ ID NO:3, and the mRNA sequence is provided in SEQ ID NO:4 (ORF is 156-773 nt).
  • GRP75 protein sequence is provided in SEQ ID NO:5 (NP_004125.3) and the DNA sequence is provided in SEQ ID NO:6 (ORF is 109-2148; NM_004134.
  • GRP78 protein sequence is provided in SEQ ID NO:7
  • a fragment of one or more particular HSPs is employed in the invention.
  • the fragment may include the amylin-binding site and/or chaperone activity domains, in specific embodiments.
  • the fragment may include the C-terminus or the N- terminus of the HSP.
  • the fragment may comprise at least 5-20 consecutive amino acids of the corresponding HSP sequence, at least 5-25, at least 5-30, at least 5-35, at least 5-40, at least 5- 45, at least 5-50, at least 5-55, at least 5-60, at least 5-65, at least 5-70, at least 5-75, at least 10- 20, at least 10-25, at least 10-30, at least 10-35, at least 10-40, at least 10-45, at least 10-50, at least 10-55, at least 10-60, at least 10-65, at least 10-70, at least 10-75, at least 15-20, at least 15- 25, at least 15-30, at least 15-35, at least 15-40, at least 15-45, at least 15-50, at least 15-55, at least 15-60, at least 15-65, at least 15-70, at least 15-75, at least 20-25, at least 20-30, at least 20- 35, at least 20-40, at least 20-45, at least 20-50, at least 20-55, at least 20-60, at least 20-65, at least 20-70, at
  • the fragment may comprise between 5-20 consecutive amino acids of the corresponding HSP sequence, between 5-25, between 5-30, between 5-35, between 5-40, between 5-45, between 5-50, between 5-55, between 5-60, between 5-65, between 5-70, between 5-75, between 10-20, between 10-25, between 10-30, between 10-35, between 10-40, between 10-45, between 10-50, between 10-55, between 10-60, between 10-65, between 10-70, between 10-75, between 15-20, between 15-25, between 15-30, between 15-35, between 15-40, between 15-45, between 15-50, between 15-55, between 15-60, between 15-65, between 15-70, between 15-75, between 20-25, between 20-30, between 20-35, between 20-40, between 20-45, between 20-50, between 20-55, between 20-60, between 20-65, between 20-70, between 20-75, between 30-35, between 30-40, between 30-45, between 30-50, between 30-55, between 30-60, between 30-65, between 30-70, between 30-75, between 35, between 30
  • the fragment is identical in sequence to the corresponding wild-type HSP sequence, although in specific embodiments the fragment has between 70% and 75% identity to the corresponding wild-type sequence, between 75% and 80%, between 80% and 85%, between 85% and 90%, between 90% and 95%, or between 95% and 99% identity to the corresponding wild-type HSP sequence.
  • the fragment has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding wild-type HSP protein.
  • the HSP employed comprises HSP70 or HSP73 or fragments thereof. HSP70 and/or HSP73 may be utilized in conjunction with one or more of HSP72, HSP27, GRP75, GRP78, or as an alternative to one or more of HSP72, HSP27, GRP75, GRP78.
  • the carrier comprises one or more molecules of an HSP, and in cases where there are two or molecules of an HSP with the carrier, the two or more molecules may be different HSPs.
  • the levels and/or activity of HSPs are increased in pancreatic B cells of diabetic individuals.
  • endogenous HSPs have levels and/or activity increased in pancreatic B cells, whereas in some cases one delivers exogenous HSPs to the pancreas to thereby increase the level in pancreatic B cells.
  • HSP levels in pancreatic B cells are increased by both increasing endogenous levels and adding exogenous HSPs to the cellular level of the pancreatic B cells.
  • pancreatic B cell HSP levels are increased by delivering a moiety capable of being heated at the pancreas and applying heat to the pancreas.
  • the moiety is capable of being heated at the pancreas by near infrared (NIR) light, for example. NIR energy is useful in certain aspects because of its high penetration and high absorbance by gold nanoshells.
  • the moiety exhibits enhanced near infrared light absorption.
  • the moiety is a nanoparticle that strongly absorbs near infrared (NIR) light and locally heats the pancreatic cells, including pancreatic B cells.
  • pancreatic B cells Such local heating of pancreatic B cells will increase HSP levels (including HSP72, HSP27, GRP75, and/or GRP78, for example) therein, which will act as chaperone to amylin and will prevent or reduce amylin aggregation toxicity.
  • HSP levels including HSP72, HSP27, GRP75, and/or GRP78, for example
  • compositions comprise nanoparticles, lipid-based compounds, and/or chitosan, for example.
  • Such compositions act as carriers for the HSPs, in certain cases, although in other cases the compositions lack HSPs and themselves are delivered to the pancreas and heated.
  • Examples of chitosan-based nanoparticles are known in the art, such as are described in Liu et al. (2007).
  • Nanoparticles may be assembled by standard means in the art, although in specific embodiments they are assembled by the methodology of Prevo et al. (2008); Huang et al. (2008); Adegboyega et al. (2007); Cherukuri and Curley (2010); Gobin (2007); or Guo (2010), for example.
  • moieties that may be included in the invention are antibodies, which may be employed to directly target the compositions to pancreatic B cells of the diabetic individual.
  • Physiological media such as water, blood and tissue
  • Noble metal nanoparticles due to the phenomenon of surface plasmon resonance, exhibit an enhanced visible and near-infrared light absorption.
  • the use of these plasmonic nanoparticles that strongly absorb NIR light has introduced a more specific and efficient cancer therapy strategy to locally heat and kill cancer cells within tumors located deep within body tissue. This type of treatment is called photothermal heating or plasmonic photothermal therapy (PPTT) (Huang et al., 2008).
  • PPTT plasmonic photothermal therapy
  • PPTT is employed in T2DM not to kill pancreatic ⁇ -cells, but to stress them and stimulate their heat shock response in order to elucidate the potential role of thermal stress in the protection of pancreatic ⁇ -cells against amylin deposition.
  • pancreatic ⁇ -cells not to kill the cells but instead to stress them in order to increase the levels of HSPs (the molecular chaperones that will assist with folding and prevent aggregation of unfolded amylin). This would ultimately improve pancreatic ⁇ -cell survival and is clinically useful as a preventive and/or therapeutic intervention for T2DM.
  • HSPs the molecular chaperones that will assist with folding and prevent aggregation of unfolded amylin.
  • a marker on pancreatic B cells is employed such that the carrier composition is targeted to the pancreatic B cells.
  • Ay marker that is specific for pancreatic B cells may be employed in the invention.
  • Antibodies recognizing the first 11 amino acids from the amino terminal extracellular domain of mouse FXYD2ya (which differs from humans in four amino acids) are conjugated to nanorods allowing their binding to ⁇ -cells. Absorption of NIR pulsed laser energy by antibody-bound nanorods will induce the heat shock response to protect ⁇ -cells from the toxic effect of amylin exposure.
  • Monoclonal antibodies directed against the first 11 amino acids from the amino terminal extracellular domain of human FXYD2ya may be employed, in specific embodiments.
  • This is a specific ⁇ -cell biomarker that contains the FXYD domain of the regulatory subunit of the Na+-K+-ATPase.
  • the intrinsic properties of the pump are regulated by association with tissue-specific small single-span membrane proteins belonging to the FXYD family.
  • FXYD2ya is produced in ⁇ -cells, but not alpha or delta cells, making this a specific target for ⁇ -cells.
  • antibodies recognizing the first 11 amino acids from the amino terminal extracellular domain of FXYD2ya are conjugated to nanorods, thereby allowing their binding to ⁇ -cells. See Flamez et al. (2010).
  • a composition is delivered directly or indirectly to the pancreas, and the pancreas is heated directly, although in certain cases the pancreas is heated indirectly.
  • the heat is applied as a steady pulse, although in some cases the heat is applied as an intermittent pulse.
  • the level of heat applied will be such that HSP proteins and/or their expression are thereby enhanced but not to the level of killing the localized cells.
  • an increase in expression that is twofold or greater is encompassed in the invention, including three-fold, four-fold, five-fold, 10- fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, or greater.
  • the duration of heating is a specific range, for example no less than 5 seconds, for example but no longer than two hours, for example.
  • the type of heat that is applied to the pancreas may be of a particular kind.
  • near infrared heat is employed.
  • Gold nanorods that strongly absorb NIR light are utilized in certain embodiments as a strategy to locally heat and increase the levels of heat shock proteins within the pancreatic tissue. Increased levels of heat shock proteins in the pancreas therapeutically prevent amylin aggregation and consequently prevent amylin toxicity in pancreatic beta cells, in particular aspects of the invention.
  • antibodies are employed that target compositions of the invention to the pancreas of the diabetic individual.
  • the antibodies target the compositions of the invention to pancreatic B cells of the diabetic individual.
  • the antibodies bind to a target polypeptide on the surface of the pancreatic B cells, and in particular cases the target polypeptide is unique to the surface of pancreatic B cells.
  • the target polypeptide is an extracellular domain of a membrane protein of the pancreatic B cell.
  • the target polypeptide comprises Na-K- ATPase of the pancreatic B cell.
  • the antibody binds a region of the FXYD2 domain of the regulatory subunit of the Na-K-ATPase.
  • GenBank® NP_001120961.1 is provided in SEQ ID NO:9, and the FXYD2 nucleic acid sequence (GenBank® NM_001127489.1, and is
  • the antibody binds to a region of the FXYD2 domain, for example, within the N-terminal 1-20 amino acids of the FXYD2 domain, although in some cases it binds within the N-terminal 1-15 amino acids or N-terminal 1-11 amino acids of the FXYD2 domain, for example. According to Flamez et al. (2010), these regions are identified as beta cell-specific in pancreatic beta cells.
  • the term "antibody” is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
  • antibody is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • DABs single domain antibodies
  • Fv single chain Fv
  • scFv single chain Fv
  • the techniques for preparing and using various antibody-based constructs and fragments are well known in the art.
  • Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).
  • Polyclonal antibodies to the target polypeptide may be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the polypeptide (or respective fragment thereof) and an adjuvant.
  • sc subcutaneous
  • ip intraperitoneal
  • the animal boosted with the conjugate of the same target polypeptide, but conjugated to a different protein and/or through a different cross-linking reagent.
  • Conjugates also can be made in recombinant cell culture as protein fusions.
  • aggregating agents such as alum can be used to enhance the immune response.
  • Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally- occurring mutations that may be present in minor amounts.
  • the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.
  • the anti- target polypeptide monoclonal antibodies of the invention may be made using the hybridoma method first described by Kohler & Milstein, Nature 256:495 (1975), or may be made by recombinant DNA methods [Cabilly, et ah, U.S. Pat. No. 4,816,567], for example.
  • a mouse or other appropriate host animal such as hamster is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, pp.59- 103 (Academic Press, 1986)].
  • a suitable fusing agent such as polyethylene glycol
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA, for example.
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the target polypeptide.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson & Pollard, Anal. Biochem. 107:220 (1980), for example.
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods. Goding, Monoclonal Antibodies: Principles and Practice, pp.59- 104 (Academic Press, 1986). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison, et al., Proc. Nat. Acad. Sci. 81, 6851 (1984), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide.
  • “chimeric” or “hybrid” antibodies are prepared that have the binding specificity of an anti-target polypeptide monoclonal antibody herein.
  • non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for a target polypeptide and another antigen-combining site having specificity for a different antigen.
  • Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond.
  • suitable reagents for this purpose include iminothiolate and methyl- 4-mercaptobutyrimidate .
  • the antibodies of the invention may be labeled with a detectable moiety, in certain embodiments.
  • the detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, or 125 I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; biotin; radioactive isotopic labels, such as, e.g., 125 I, 32 P, 14 C, or 3 H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry 13: 1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982), for example.
  • the antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147- 158 (CRC Press, Inc., 1987), for example.
  • ком ⁇ онентs rely on the ability of a labeled standard (which may be a target polypeptide or an immunologically reactive portion thereof) to compete with the test sample analyte (target polypeptide) for binding with a limited amount of antibody.
  • the amount of target polypeptide in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies.
  • the antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected.
  • the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three part complex.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an antiimmunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay).
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature 321, 522-525 (1986); Riechmann et al, Nature 332, 323-327 (1988); Verhoeyen et al, Science 239, 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences.
  • Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e. the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • Human monoclonal antibodies can be made by the hybridoma method.
  • Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor, J. Immunol. 133, 3001 (1984), and Brodeur, et ah, Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987).
  • transgenic animals ⁇ e.g. mice
  • transgenic animals that are capable, upon immunization, of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production.
  • transgenic animals ⁇ e.g. mice
  • JH antibody heavy chain joining region
  • the phage display technology (McCafferty et al, Nature 348, 552-553 [1990]) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.
  • V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments on the surface of the phage particle.
  • the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties.
  • the phage mimics some of the properties of the B-cell.
  • Phage display can be performed in a variety of formats; for their review see, e.g. Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3, 564-571 (1993).
  • V-gene segments can be used for phage display.
  • Minibodies are sFv polypeptide chains which include oligomerization domains at their C-termini, separated from the sFv by a hinge region.
  • the oligomerization domain comprises self-associating alpha-helices, e.g., leucine zippers, that can be further stabilized by additional disulfide bonds.
  • oligomerization domain is designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein.
  • minibodies are produced using recombinant methods well known in the art. See, e.g., Pack et al. (1992) Biochem 31: 1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126.
  • Antibody-like binding peptidomimetics are also contemplated in the present invention. Liu et al. Cell Mol Biol (Noisy-le-grand). 2003 Mar;49(2):209-16 describe "antibody like binding peptidomimetics" (ABiPs), which are peptides that act as pared-down antibodies and have certain advantages of longer serum half-life as well as less cumbersome synthesis methods. G. Bispecific antibodies
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities is for a target polypeptide, the other one is for any other antigen, and preferably for another pancreatic B-cell-specific antigen.
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH 2 and CH 3 regions. It is preferred to have the first heavy chain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions.
  • CHI first heavy chain constant region
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in copending application Ser. No. 07/931,811 filed Aug. 17, 1992.
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (PCT application publication Nos. WO 91/00360 and WO 92/200373; EP 03089).
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • the present invention concerns a novel composition
  • lipids associated with at least one composition for providing to a pancreatic B cell.
  • the lipid is heatable.
  • a lipid is a substance that is characteristically insoluble in water and extractable with an organic solvent.
  • Lipids include, for example, the substances comprising the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which are well known to those of skill in the art which contain long chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.
  • a lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester- linked fatty acids and polymerizable lipids, and combinations thereof.
  • a neutral fat may comprise a glycerol and a fatty acid.
  • a typical glycerol is a three carbon alcohol.
  • a fatty acid generally is a molecule comprising a carbon chain with an acidic moeity (e.g., carboxylic acid) at an end of the chain.
  • the carbon chain may of a fatty acid may be of any length, however, it is preferred that the length of the carbon chain be of from about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, to about 30 or more carbon atoms, and any range derivable therein.
  • a preferred range is from about 14 to about 24 carbon atoms in the chain portion of the fatty acid, with about 16 to about 18 carbon atoms being particularly preferred in certain embodiments.
  • the fatty acid carbon chain may comprise an odd number of carbon atoms, however, an even number of carbon atoms in the chain may be preferred in certain embodiments.
  • a fatty acid comprising only single bonds in its carbon chain is called saturated, while a fatty acid comprising at least one double bond in its chain is called unsaturated.
  • Specific fatty acids include, but are not limited to, linoleic acid, oleic acid, palmitic acid, linolenic acid, stearic acid, lauric acid, myristic acid, arachidic acid, palmitoleic acid, arachidonic acid ricinoleic acid, tuberculosteric acid, lactobaciUic acid.
  • An acidic group of one or more fatty acids is covalently bonded to one or more hydroxyl groups of a glycerol.
  • a monoglyceride comprises a glycerol and one fatty acid
  • a diglyceride comprises a glycerol and two fatty acids
  • a triglyceride comprises a glycerol and three fatty acids.
  • a phospholipid generally comprises either glycerol or an sphingosine moiety, an ionic phosphate group to produce an amphipathic compound, and one or more fatty acids.
  • Types of phospholipids include, for example, phophoglycerides, wherein a phosphate group is linked to the first carbon of glycerol of a diglyceride, and sphingophospholipids (e.g., sphingomyelin), wherein a phosphate group is esterified to a sphingosine amino alcohol.
  • a sphingophospholipid is a sulfatide, which comprises an ionic sulfate group that makes the molecule amphipathic.
  • a phopholipid may, of course, comprise further chemical groups, such as for example, an alcohol attached to the phosphate group. Examples of such alcohol groups include serine, ethanolamine, choline, glycerol and inositol.
  • specific phosphoglycerides include a phosphatidyl serine, a phosphatidyl ethanolamine, a phosphatidyl choline, a phosphatidyl glycerol or a phosphotidyl inositol.
  • phospholipids include a phosphatidic acid or a diacetyl phosphate.
  • a phosphatidylcholine comprises a dioleoylphosphatidylcholine (a.k.a. cardiolipin), an egg phosphatidylcholine, a dipalmitoyl phosphalidycholine, a monomyristoyl phosphatidylcholine, a monopalmitoyl
  • phosphatidylcholine a monostearoyl phosphatidylcholine, a monooleoyl phosphatidylcholine, a dibutroyl phosphatidylcholine, a divaleroyl phosphatidylcholine, a dicaproyl
  • phosphatidylcholine a diheptanoyl phosphatidylcholine, a dicapryloyl phosphatidylcholine or a distearoyl phosphatidylcholine.
  • a glycolipid is related to a sphinogophospholipid, but comprises a carbohydrate group rather than a phosphate group attached to a primary hydroxyl group of the sphingosine.
  • a type of glycolipid called a cerebroside comprises one sugar group (e.g. , a glucose or galactose) attached to the primary hydroxyl group.
  • Another example of a glycolipid is a ganglioside (e.g. , a monosialoganglioside, a GMl), which comprises about 2, about 3, about 4, about 5, about 6, to about 7 or so sugar groups, that may be in a branched chain, attached to the primary hydroxyl group.
  • the glycolipid is a ceramide (e.g., lacto sylceramide) .
  • a steroid is a four-membered ring system derivative of a phenanthrene. Steroids often possess regulatory functions in cells, tissues and organisms, and include, for example, hormones and related compounds in the progestagen (e.g. , progesterone),
  • glucocoricoid e.g. , Cortisol
  • mineralocorticoid e.g. , aldosterone
  • androgen e.g. , testosterone
  • estrogen e.g. , estrone
  • Cholesterol is another example of a steroid, and generally serves structural rather than regulatory functions.
  • Vitamin D is another example of a sterol, and is involved in calcium absorption from the intestine.
  • a terpene is a lipid comprising one or more five carbon isoprene groups. Terpenes have various biological functions, and include, for example, vitamin A, coenyzme Q and carotenoids (e.g. , lycopene and ⁇ -carotene).
  • a lipid component of a composition is uncharged or primarily uncharged.
  • a lipid component of a composition comprises one or more neutral lipids.
  • a lipid component of a composition may be substantially free of anionic and cationic lipids, such as certain phospholipids (e.g. , phosphatidyl choline) and cholesterol.
  • a lipid component of an uncharged or primarily uncharged lipid composition comprises about 95%, about 96%, about 97%, about 98%, about 99% or 100% lipids without a charge, substantially uncharged lipid(s), and/or a lipid mixture with equal numbers of positive and negative charges.
  • a lipid composition may be charged.
  • charged phospholipids may be used for preparing a lipid composition according to the present invention and can carry a net positive charge or a net negative charge.
  • diacetyl phosphate can be employed to confer a negative charge on the lipid composition
  • stearylamine can be used to confer a positive charge on the lipid composition.
  • Lipids can be obtained from natural sources, commercial sources or chemically synthesized, as would be known to one of ordinary skill in the art.
  • phospholipids can be from natural sources, such as egg or soybean phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin and plant or bacterial phosphatidylethanolamine.
  • lipids suitable for use according to the present invention can be obtained from commercial sources. For example, dimyristyl
  • DMPC phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • stock solutions of lipids in chloroform or chloroform/methanol can be stored at about 20 C.
  • chloroform is used as the only solvent since it is more readily evaporated than methanol.
  • the composition for delivery to the pancreatic B cell may be associated with a lipid.
  • a composition associated with a lipid may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure.
  • composition of the present invention is not limited to any particular structure.
  • they may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in either size or shape.
  • they may be present in a bilayer structure, as micelles, or with a "collapsed" structure.
  • a bilayer structure as micelles, or with a "collapsed" structure.
  • lipofectamine (Gibco BRL) or Superfect (Qiagen) are also contemplated.
  • a lipid composition may comprise about 1%, about 2%, about 3%, about 4% about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about
  • a lipid composition may comprise about 10% to about 20% neutral lipids, and about 33% to about 34% of a cerebroside, and about 1% cholesterol.
  • a liposome may comprise about 4% to about 12% terpenes, wherein about 1% of the micelle is specifically lycopene, leaving about 3% to about 11% of the liposome as comprising other terpenes; and about 10%to about 35% phosphatidyl choline, and about 1% of a drug.
  • lipid compositions of the present invention may comprise any of the lipids, lipid types or other components in any combination or percentage range.
  • a lipid may be comprised in an emulsion.
  • a lipid emulsion is a substantially permanent heterogenous liquid mixture of two or more liquids that do not normally dissolve in each other, by mechanical agitation or by small amounts of additional substances known as emulsifiers. Methods for preparing lipid emulsions and adding additional components are well known in the art (e.g., Modern Pharmaceutics, 1990, incorporated herein by reference).
  • one or more lipids are added to ethanol or chloroform or any other suitable organic solvent and agitated by hand or mechanical techniques. The solvent is then evaporated from the mixture leaving a dried glaze of lipid. The lipids are resuspended in aqueous media, such as phosphate buffered saline, resulting in an emulsion.
  • aqueous media such as phosphate buffered saline
  • the mixture may be sonicated using conventional sonication techniques, further emulsified using microfluidization (using, for example, a Microfluidizer, Newton, Mass.), and/or extruded under high pressure (such as, for example, 600 psi) using an Extruder Device (Lipex Biomembranes, Vancouver, Canada).
  • microfluidization using, for example, a Microfluidizer, Newton, Mass.
  • high pressure such as, for example, 600 psi
  • Extruder Device Lipex Biomembranes, Vancouver, Canada
  • a lipid may be comprised in a micelle.
  • a micelle is a cluster or aggregate of lipid compounds, generally in the form of a lipid monolayer, and may be prepared using any micelle producing protocol known to those of skill in the art (e.g., Canfield et al., 1990; El- Gorab et al, 1973; Colloidal Surfactant, 1963; and Catalysis in Micellar and Macromolecular Systems, 1975, each incorporated herein by reference).
  • one or more lipids are typically made into a suspension in an organic solvent, the solvent is evaporated, the lipid is resuspended in an aqueous medium, sonicated and then centrifuged.
  • a lipid comprises a liposome.
  • a "liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes may be characterized as having vesicular structures with a bilayer membrane, generally comprising a phospholipid, and an inner medium that generally comprises an aqueous composition.
  • a multilamellar liposome has multiple lipid layers separated by aqueous medium. They form spontaneously when lipids comprising phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Lipophilic molecules or molecules with lipophilic regions may also dissolve in or associate with the lipid bilayer.
  • phospholipids from natural sources such as egg or soybean phosphatidylcholine, brain phosphatidic acid, brain or plant
  • phosphatidylinositol, heart cardiolipin and plant or bacterial phosphatidylethanolamine are preferably not used as the primary phosphatide, i.e., constituting 50% or more of the total phosphatide composition or a liposome, because of the instability and leakiness of the resulting liposomes.
  • a lipid and/or composition of the invention may be, for example, encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the composition, entrapped in a liposome, complexed with a liposome, etc.
  • a liposome used according to the present invention can be made by different methods, as would be known to one of ordinary skill in the art.
  • Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure.
  • a phospholipid (Avanti Polar Lipids, Alabaster, AL), such as for example the neutral phospholipid dioleoylphosphatidylcholine (DOPC), is dissolved in tert- butanol.
  • the lipid(s) is then mixed with the composition and/or other component(s).
  • Tween 20 is added to the lipid mixture such that Tween 20 is about 5% of the composition's weight.
  • tert-butanol is added to this mixture such that the volume of tert-butanol is at least 95%.
  • the mixture is vortexed, frozen in a dry ice/acetone bath and lyophilized overnight.
  • the lyophilized preparation is stored at -20°C and can be used up to three months. When required the lyophilized liposomes are reconstituted in 0.9% saline.
  • the average diameter of the particles obtained using Tween 20 for encapsulating the composition is about 0.7 to about 1.0 ⁇ in diameter.
  • a liposome can be prepared by mixing lipids in a solvent in a container, e.g., a glass, pear shaped flask.
  • a container e.g., a glass, pear shaped flask.
  • the container should have a volume ten times greater than the volume of the expected suspension of liposomes.
  • the solvent is removed at approximately 40°C under negative pressure.
  • the solvent normally is removed within about 5 min to 2 hours, depending on the desired volume of the liposomes.
  • the composition can be dried further in a desiccator under vacuum. The dried lipids generally are discarded after about 1 week because of a tendency to deteriorate with time.
  • Dried lipids can be hydrated at approximately 25 50 mM phospholipid in sterile, pyrogen free water by shaking until all the lipid film is resuspended.
  • the aqueous liposomes can be then separated into aliquots, each placed in a vial, lyophilized and sealed under vacuum.
  • liposomes can be prepared in accordance with other known laboratory procedures (e.g. , see Bangham et al., 1965; Gregoriadis, 1979; Deamer and Uster 1983, Szoka and Papahadjopoulos, 1978, each incorporated herein by reference in relevant part). These methods differ in their respective abilities to entrap aqueous material and their respective aqueous space to lipid ratios.
  • the dried lipids or lyophilized liposomes prepared as described above may be dehydrated and reconstituted in a solution of inhibitory peptide and diluted to an appropriate concentration with an suitable solvent, e.g. , DPBS.
  • DPBS a suitable solvent
  • Unencapsulated additional materials such as agents including but not limited to hormones, drugs, nucleic acid constructs and the like, are removed by centrifugation at 29,000 g and the liposomal pellets washed.
  • the washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g. , about 50 200 mM.
  • the amount of additional material or active agent encapsulated can be determined in accordance with standard methods.
  • the liposomes may be diluted to appropriate concentrations and stored at 4 C until use.
  • a pharmaceutical composition comprising the liposomes will usually include a sterile, pharmaceutically acceptable carrier or diluent, such as water or saline solution.
  • the size of a liposome varies depending on the method of synthesis.
  • Liposomes in the present invention can be a variety of sizes.
  • the liposomes are small, e.g., less than about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, or less than about 50 nm in external diameter.
  • any protocol described herein, or as would be known to one of ordinary skill in the art may be used.
  • a liposome suspended in an aqueous solution is generally in the shape of a spherical vesicle, having one or more concentric layers of lipid bilayer molecules.
  • Each layer consists of a parallel array of molecules represented by the formula XY, wherein X is a hydrophilic moiety and Y is a hydrophobic moiety.
  • the concentric layers are arranged such that the hydrophilic moieties tend to remain in contact with an aqueous phase and the hydrophobic regions tend to self associate.
  • the lipid molecules may form a bilayer, known as a lamella, of the arrangement XY YX.
  • Aggregates of lipids may form when the hydrophilic and hydrophobic parts of more than one lipid molecule become associated with each other.
  • the size and shape of these aggregates will depend upon many different variables, such as the nature of the solvent and the presence of other compounds in the solution.
  • lipid formulations often is accomplished by sonication or serial extrusion of liposomal mixtures after (I) reverse phase evaporation (II) dehydration- rehydration (III) detergent dialysis and (IV) thin film hydration.
  • a contemplated method for preparing liposomes in certain embodiments is heating sonicating, and sequential extrusion of the lipids through filters or membranes of decreasing pore size, thereby resulting in the formation of small, stable liposome structures.
  • This preparation produces liposomal/carrier/HSP of liposomes only of appropriate and uniform size, which are structurally stable and produce maximal activity.
  • Such techniques are well-known to those of skill in the art (see, for example Martin, 1990).
  • lipid structures can be used to encapsulate compounds that are toxic (e.g. , chemotherapeutics) or labile (e.g., nucleic acids) when in circulation.
  • toxic e.g. , chemotherapeutics
  • labile e.g., nucleic acids
  • the physical characteristics of liposomes depend on pH, ionic strength and/or the presence of divalent cations. Liposomes can show low permeability to ionic and/or polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state.
  • Liposomal encapsulation has resulted in a lower toxicity and a longer serum half-life for such compounds (Gabizon et al., 1990).
  • Liposomes interact with cells to deliver agents via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and/or neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic and/or electrostatic forces, and/or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and/or by transfer of liposomal lipids to cellular and/or subcellular membranes, and/or vice versa, without any association of the liposome contents. Varying the liposome formulation can alter which mechanism is operative, although more than one may operate at the same time.
  • lipid based gene transfer strategies to enhance conventional or establish novel therapies, in particular therapies for treating hyperproliferative diseases.
  • Advances in liposome formulations have improved the efficiency of gene transfer in vivo (Templeton et al., 1997) and it is contemplated that liposomes are prepared by these methods.
  • Alternate methods of preparing lipid-based formulations for nucleic acid delivery are described (W0 99/18933).
  • SDMC solvent dilution microcarrier
  • the SDMCs can be used to deliver lipopolysaccharides, polypeptides, nucleic acids and the like.
  • any other methods of liposome preparation can be used by the skilled artisan to obtain a desired liposome formulation in the present invention.
  • association of the composition with a liposome may improve biodistribution and other properties of the composition.
  • liposome mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987).
  • the feasibility of liposome mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980).
  • Successful liposome mediated gene transfer in rats after intravenous injection has also been accomplished (Nicolau et al., 1987).
  • a liposome composition of the invention may comprise additional materials for delivery to a tissue.
  • the lipid or liposome may be associated with an antibody that targets the liposome to a pancreatic B cell.
  • Targeted delivery is achieved by the addition of antibodies or other ligands without compromising the ability of these liposomes to deliver large amounts of the composition of the invention. It is contemplated that this will enable delivery to specific cells, tissues and organs.
  • the targeting specificity of the ligand-based delivery systems are based on the distribution of the ligand receptors on different cell types.
  • the targeting ligand may either be non-covalently or covalently associated with the lipid complex, and can be conjugated to the liposomes by a variety of methods.
  • bifunctional cross-linking reagents have been extensively used for a variety of purposes including preparation of affinity matrices,
  • Liposomes in particular, multilamellar vesicles (MLV) or unilamellar vesicles such as microemulsified liposomes (MEL) and large unilamellar liposomes (LUVET), each containing phosphatidylethanolamine (PE), have been prepared by established procedures.
  • MEL microemulsified liposomes
  • LVET large unilamellar liposomes
  • PE phosphatidylethanolamine
  • the inclusion of PE in the liposome provides an active functional residue, a primary amine, on the liposomal surface for cross-linking purposes.
  • Ligands such as epidermal growth factor (EGF) have been successfully linked with PE-liposomes. Ligands are bound covalently to discrete sites on the liposome surfaces.
  • EGF epidermal growth factor
  • cross-linking reagents include glutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE), and a water soluble carbodiimide, preferably l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).
  • GAD glutaraldehyde
  • OXR bifunctional oxirane
  • EGDE ethylene glycol diglycidyl ether
  • EDC water soluble carbodiimide
  • antibodies are associated with the lipid complex.
  • liposomes have been described further that specifically target cells of the mammalian central nervous system (U.S. Patent 5,786,214, incorporated herein by reference).
  • the liposomes are composed essentially of N glutarylphosphatidylethanolamine, cholesterol and oleic acid, wherein a monoclonal antibody specific for neuroglia is conjugated to the liposomes. It is contemplated that a monoclonal antibody or antibody fragment may be used to target delivery to specific cells, tissues, or organs in the animal, such as the pancreas.
  • the actual dosage amount of a lipid composition (e.g. , a liposome- pancreatic B cell composition) administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, idiopathy of the patient and on the route of administration. With these considerations in mind, the dosage of a lipid composition for a particular subject and/or course of treatment can readily be determined.
  • the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, rectally,topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally, intrapericardially, orally, topically, locally and/or using aerosol, injection, infusion, continuous infusion, localized perfusion bathing target cells directly or via a catheter and/or lavage.
  • compositions of the present invention comprise an effective amount of one or more compositions of the invention dissolved or dispersed in a
  • pharmaceutically acceptable carrier refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • pharmaceutical composition that contains at least one composition or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference).
  • preservatives e.g., antibacterial agents, antifungal agents
  • isotonic agents e.g., absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like
  • compositions may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's
  • the composition may be formulated into a composition in a free base, neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as formulated for parenteral
  • administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.
  • the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent.
  • the carrier should be assimilable and includes liquid, semisolid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate.
  • carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof.
  • composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g., methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.
  • the composition is combined or mixed thoroughly with a semi- solid or solid carrier.
  • the mixing can be carried out in any convenient manner such as grinding.
  • Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.
  • stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
  • the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include the composition and an aqueous solvent.
  • lipid will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term "lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance.
  • Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
  • neutral fats phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
  • lipids are also encompassed by the compositions and methods of the present invention.
  • One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle.
  • the composition may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art.
  • the dispersion may or may not result in the formation of liposomes.
  • the actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • compositions may comprise, for example, at least about 0.1% of an active compound.
  • the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500
  • microgram/kg/body weight about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
  • the composition is formulated to be administered via an alimentary route.
  • Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually.
  • these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et ah, 1997; Hwang et ah, 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety).
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.
  • a binder such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof
  • an excipient such as, for
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001.
  • the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells.
  • a syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally- administered formulation.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a
  • compositions may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
  • suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
  • traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.
  • suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
  • composition may be administered via a parenteral route.
  • parenteral includes routes that bypass the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308,
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • aqueous solutions for parenteral administration in an aqueous solution
  • the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
  • the active compound may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.
  • topical i.e., transdermal
  • mucosal administration intranasal, vaginal, etc.
  • inhalation inhalation
  • compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder.
  • Ointments include all oleaginous, adsorption, emulsion and water-solubly based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only.
  • Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram.
  • compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base.
  • Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture.
  • Transdermal administration of the present invention may also comprise the use of a "patch".
  • the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
  • the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety).
  • the delivery of drugs using intranasal microparticle resins Takenaga et ah, 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts.
  • transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).
  • aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant.
  • the typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent.
  • Suitable propellants include hydrocarbons and hydrocarbon ethers.
  • Suitable containers will vary according to the pressure requirements of the propellant.
  • Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.
  • the methods and compositions of the invention are administered to an individual who has taken or is taking Type II diabetes therapy, including therapy for one or more complications from diabetes.
  • the present invention generally concerns treatment for Type 2 diabetes, and the individual may or may not have been treated or is being treated for T2DM.
  • Type II diabetes may be asymptomatic or they may have one or more symptoms as follows: blurred vision; erectile dysfunction; fatigue; frequent or slow-healing infections; increased appetite, thirst, and/or urination.
  • Type 2 diabetes can be diagnosed by measuring fasting blood glucose level, hemoglobin Ale test, oral glucose tolerance test, and/or random (non-fasting) blood glucose level.
  • the methods and compositions of the invention are administered to an individual who has taken or is taking therapy for one or more complications from diabetes.
  • therapy may include any means to lower high blood glucose levels, including by one or more drugs, exercise, and/or diet, for example.
  • Medications to treat diabetes include alpha-glucosidase inhibitors; biguanides; exenatide, mitiglinide, pramlintide, sitagliptin, and saxagliptin; meglitinides; sulfonylureas; and/or thiazolidinediones.
  • Medications to prevent complications may also include an ACE inhibitor, statin drugs, or aspirin.
  • the diabetic individual may also be treated for cataracts; vascular damage; diabetic retinopathy; foot sores or ulcers; glaucoma; high blood pressure; high cholesterol; kidney disease or kidney failure; or macular edema, for example.
  • an individual at risk for developing Type II diabetes or at risk for developing additional complications from diabetes are treated with methods or compositions of the invention.
  • Such individuals at risk include those with an age greater than 45 years; HDL cholesterol of less than 35 mg/dL or triglyceride level of greater than 250 mg/dL; high blood pressure; history of gestational diabetes; polycystic ovarian syndrome; impaired glucose tolerance; and/or having a race/ethnicity including African Americans, Hispanic Americans, and Native Americans, for example.
  • the combination therapy that is in addition to the methods or compositions of the present invention may be delivered before, during, and/or after administration of the present invention.
  • compositions described herein may be comprised in a kit.
  • a composition of the invention may be comprised in a kit.
  • the kits will thus comprise, in suitable container means, one or more of an antibody, nanoparticle, HSP, chitosan, or lipid-based compound, such as a liposome.
  • the components of the kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted.
  • kits of the present invention also will typically contain a means for containing the composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained, for example.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to the pancreas, for example.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • kits of the invention may also comprise, and/or be packaged with, an instrument for assisting with the injection/administration and/or placement of the ultimate composition within the body of an individual, including to the pancreas.
  • an instrument may be a syringe, pipette, forceps, and/or any such medically approved delivery vehicle.
  • Transgenic C. elegans model expressing h-IAPP in vivo Due to the fact that animal diabetes model systems take approximately 18-24 months to develop diabetes, the inventors developed a human-IAPP (h-IAPP) C. elegans model to determine h-IAPP toxicity in a living organism. Animals which chronically express h-IAPP and mouse-IAPP (m-IAPP) as a control were first generated. These animals were tagged with YFP (yellow fluorescent protein) in pharynx, body wall muscles, neurons and intestines under the control of promoters lev-11, tnt- 4, aex-3 and ges-1, respectively.
  • YFP yellow fluorescent protein
  • Plasmids were co-injected into the pha-l(e2123); him- 5(el490); Ute-l(ce314) hermaphrodites at concentrations of 37.5ng ⁇ l each, along with SOng/ ⁇ of the pha-1 rescuing plasmid pBXl.
  • pha-1 is a temperature-sensitive embryonic lethal mutation, which allows worms to grow normally at 15°C; however, at 20°C the mutation is 100% embryonic lethal.
  • transgenic animals that carry pBXl are selected due to their ability to grow at 20°C.
  • Transgenes were generated by PCR amplification of h-IAPP and m-IAPP cDNA clones (Origene) that were subcloned into BamHl sites of expression vector pSX95.77YFP by In-Fusion PCR cloning system (Clontech, Mountain View, CA) using human-amylin forward (5'- CGACTCTAGAGGATCCATGGGCATCCTGAAGCTGCAAG-3'; SEQ ID NO: 11) and reverse (5 '-CCAATCCCGGGGATCC AAGGGGCAAGTAATTCAGTGG-3 '; SEQ ID NO: 12) primers for h-IAPP cloning and m-amylin forward (5'-
  • Gateway Vector Conversion Reading frame Cassette C.l (Life Technologies) to generate Gateway destination vectors pNGl and pNG2 that contained the h-IAPP and m-IAPP clones, respectively.
  • Entry clones pLR22, pLR25 and pLR35 contained the C. elegans promoter regions lev-11, tnt-4 and aex-3, respectively. All these entry clones contained Gateway ATTL sites, which allowed promoter sequences in pLR22, pLR25 and pLR35 to be recombined, using the LR and pNG2, to generate C. elegans transgenic tissue-specific plasmids.
  • h-IAPP plasmids Three h-IAPP plasmids: pPR3 (lev-11 promoter), pPR4 (tnt-4 promoter) and pPR5 (aex-3 promoter); and three m-IAPP plasmids: pPR8 (lev-11 promoter), pPR9 (tnt-4 promoter) and pPRlO (aex-3 promoter) were generated. Positive plasmids were identified by restriction enzyme pattern digestion.
  • the animal did not respond then the worm's head was gently tapped with a platinum pick. If there was no response, the worm was scored as dead and removed from the plate.
  • Self-fertilization was assessed by detecting the presence of larvae on the Petri dish. The period between day 1 and the last day of self -progeny production was referred to as the self-fertilization span. Larvae were synchronized by hypochlorite treatment and transgenic animals were identified using a fluorescence microscope. Seventy-two hours after treatment, larvae stage or adult stage were determined and compared with the control.
  • Beta-TC-6 cells were grown in HEPES -buffered Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% of heat-inactivated FBS (Gibco, Grand Island, NY), 100 IU/ml penicillin and 100 ⁇ g/ml streptomycin (Gibco). Cells were maintained in a humidified 5% CO2 atmosphere at 37°C.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS heat-inactivated FBS
  • streptomycin Gibco
  • Beta-TC-6 cells were plated on 96-well and 6- well tissue culture plates at a density of 6 x 104 or 1 x 106 cells per well, respectively, and allowed to attach overnight.
  • cells were transfected with h- IAPP cDNA clone (vector pCMV6-XL5, Origene, Rockville, MD) or m-IAPP cDNA clone (vector pCMV6-Kan/NEO, Origene).
  • Transfection complex were prepared with Opti-MEM (Gibco), and Lipofectamine 2000 (Life Technologies, Grand Island, NY), according to the manufacturer's instructions. Medium was changed after 6h and cells were incubated for 72h.
  • Hsp72 was co-transfected with h-IAPP in Beta-TC-6 cells.
  • Human Hsp72 cDNA clone (vector pEGFP, Addgene, Cambridge, MA) and h-IAPP cDNA clone (vector pCMV6-XL5, Origene) was used to prepare transfection complex as explained above.
  • cells plated on 96-well plates were used to evaluate cell viability by MTS assay and cells plated on 6-well plates were harvested for Western blot analysis to detect the protein levels of h-IAPP and Hsp72.
  • Empty vectors pCMV6 and pEGFP-C3 will serve as transfection controls.
  • Cell viability assay Cell viability was measured by MTS assay (Promega). MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt] according to the manufacturer's instructions. Briefly, 60 ⁇ 1 of MTS solution was added to each well and incubated for 4h at 37°C, 5% CO2. The absorbance was read at 490nm on a spectrophotometer Spectra Max Plus 384. Cell viability was expressed as the percentage of MTS reduction with respect to the controls.
  • Amylin treatment of mouse pancreatic ⁇ -cells and human pancreatic islets According to the results of Lorenzo et al. (1994), islet toxicity is first detected at concentrations of 5 ⁇ human amylin and almost complete islet cell death occurs after 24 hours of incubation with 20 ⁇ amylin in both rat and human islet cell cultures. For this reason one can conduct amylin toxicity assays with freshly prepared human amylin solution (in water), at concentrations of 5, 10, 20 and 40 ⁇ . Note that the solution should be prepared immediately before the assay and used within 5 min of being dissolved. According to the results reported by Janson et al.
  • FIG. 1 shows the toxicity of amylin at different concentrations in murine pancreatic beta cells determined by trypan blue exclusion assay.
  • Mouse insulinoma Beta-TC-6 cells ATCC, Manassas, VA
  • ATCC Manassas, VA
  • h-IAPP exogenous human islet amyloid polypeptide
  • h-IAPP ICso was demonstrated to be 10.43+1.9 ⁇ (FIG. 2).
  • heat shock treatment increases the levels of Hsp72 (as an exemplary HSP of the invention) in ⁇ -cells preventing the formation of amylin fibrils and ⁇ -cell death.
  • Heat shock is used as a prophylactic treatment in order to induce higher levels of Hsp72 before exposure to toxic amylin, in certain aspects.
  • intracellular HSPs help to deal with increased levels of exogenous amylin.
  • the mitigator effect of heat treatment in ⁇ -cells already exposed to toxic amylin is demonstrated.
  • exposure of ⁇ -cells to amylin followed by heat shock treatment attenuates the toxic response.
  • these cells have a better chance of survival due to reduced formation of amylin fibrils compared with controls not receiving heat shock.
  • the beneficial effect of heat shock treatment depends on the induction of Hsp72 expression by using quercetin, which is one of the most effective inhibitors of heat- induced Hsp72 expression (Wang et ah, 2009); quercetin blocks the prophylactic and mitigator effects of heat shock, in particular embodiments.
  • Peroxidase-conjugated goat anti-rabbit IgG was used as a secondary antibody (Jackson
  • Beta-TC-6 cells were plated on a 96- well plate at 6xl0 4 cells/well and cultured for 24h. On day 2, cells were heat shocked at 43°C for 30min and then returned to the incubator for 24h to allow for Hsp72 expression. On day 3, different concentrations of h-IAPP solution were applied to the supernatant of the heat shocked cells reaching final concentrations of 40 ⁇ , 20 ⁇ , ⁇ , 8 ⁇ , 5 ⁇ , 3 ⁇ , ⁇ , vehicle, or medium. After 24h of incubation cell viability was assessed. We demonstrated that heat shock treatment effectively mitigated the toxic effect of h-IAPP in ⁇ -cells (FIG. 4).
  • the foregoing results were next validated in a complex animal system, the C. elegans model.
  • the inventors developed a human-IAPP (h-IAPP) and mouse-IAPP (m-IAPP) C. elegans model to determine h-IAPP toxicity. This is because h-IAPP, but not m-IAPP induces aggregates in the C. elegans and results in its ultimate death.
  • h- IAPP transgenic model showed high fluorescence in coelomocytes (cells that have a similar function to the macrophages of vertebrates, but are in a fixed position), spermatheca, vulva muscles, body wall muscles, pharynx and anal depressor muscle; while the m-IAPP transgenic model did not show significant aggregate in coelomocytes, body wall muscles and pharynx, nor in vulva, spermatheca and anal depressor (FIG. 5). It was next demonstrated that heat shock (HS) treatment of h-IAPP C. elegans for 30 min at 33°C significantly reduced the number of visible h-IAPP toxic aggregates (FIG. 6).
  • HS heat shock
  • HEAT SHOCK TREATMENT CAN BE ACHIEVED UTILIZING GOLD NANORODS SPECIFICALLY TARGETED TO MOUSE PANCREATIC B-CELLS AND IRRADIATED WITH A NIR PULSED-LASER TO INDUCE LOCALIZED
  • targeted gold nanorods irradiated with NIR induce the synthesis of Hsp72 in ⁇ -cells in response to the thermal stress.
  • Gold nanorods (axial diameter lOnm, wavelength) conjugated with rabbit polyclonal antibodies directed against the first 11 amino acids from the amino terminal extracellular domain of mouse FXYD2ya
  • NIR irradiation is accomplished using an Integrated Fiber Array Packet, FAP-I System, with a wavelength of 808 nm (Coherent, Santa Clara, CA) at a power density of 4 W/cm (Gobin, 2007) and a spot size of 85.6 mm diameter in order to cover all wells in one column at a time for 5, 10 or 15 min through the culture medium.
  • Cell viability and Hsp72 levels are determined 24, 48 and 72 h after laser irradiation using the same assays as described above.
  • the optimal time is the one with the least number of dead cells and highest Hsp72 levels, in particular cases.
  • Photothermal therapy Protocols may be the same as in Example 1, but instead of heat shock the cells are treated with gold nanorods and NIR for a period of time determined in the previous section.
  • Results are calculated as means + SEM. Comparisons of cytotoxicity (reduction in the number of murine ⁇ -cells after heat shock treatments or nanorod irradiation treatment) relative to the control are analyzed by one-way analysis of variance (ANOVA) with Student-Newman-Keuls test for identifying differences among means. Comparison of levels of Hsp72 and amylin aggregate formation in cell medium is qualitative and is assessed with imaging processing software. Statview 5.0 software will be used. Controls for each of the experiments were described in individual experimental sections.
  • targeting gold nanorods to ⁇ -cells increases the efficiency of nanorod endocytosis.
  • nanorods suspended in the medium could make contact with the cells and uptake could occur by nonspecific endocytosis, washing the cell monolayer prior to irradiation will remove the nanorods that have just landed on the cells and are not specifically attached, in particular cases. Therefore, one can expect a significantly larger uptake of nanorods by ⁇ -cells and a concomitant increase in Hsp72 levels after irradiation compared to the naked gold nanorods.
  • antibodies may be humanized to decrease provocation of an immune response in humans.
  • T2DM treatment should target both insulin sensitivity and deterioration and loss of ⁇ -cell mass.
  • the use of heat to offset the detrimental effects of islet amyloid deposition and to prevent ⁇ -cell death is an innovative approach in the management of T2DM.
  • the creation of gold nanoparticles conjugated to polyclonal/monoclonal antibodies and delivered intravenously to a patient resulting in selective uptake in pancreatic ⁇ -cells and exposure to NIR is useful as an adjuvant therapy for T2DM. This would ultimately improve ⁇ -cell survival and insulin response to hyperglycemia. Optimization of the rod shape and size and the determination of the required concentration of rods for sufficient heating to induce a stress response without toxicity may be employed.
  • the duration, frequency and intensity of NIR irradiation may be determined experimentally or using computational models, in specific embodiments.
  • the use of multifunctional nanocarriers for cell imaging and NIR photothermal therapy is a novel approach that is under investigation for cancer treatment (Guo et al., 2010).
  • multifunctional nanocarriers based on chitosan (Guo et al., 2010) targeted to pancreatic ⁇ -cells using monoclonal antibodies and carrying gold nanorods to serve as a NIR thermotherapy device to induce the synthesis of HSPs, and optionally includes a fluorescent probe for real-time ⁇ -cell imaging, constitutes a useful strategy that not only helps to prevent ⁇ -cell death but also allows the evaluation of ⁇ -cell mass prior and during the treatment.
  • FIG. 6 is an example of a regimen of heat treatment of pancreatic B-cells.
  • Kaneda et al. "Increased expression of DNA cointroduced with nuclear protein in adult rat liver," Science, 243:375 378, 1989.
  • Kaneda et al. "Introduction and expression of the human insulin gene in adult rat liver," J Biol Chem., 264(21): 12126-12129, 1989.

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Abstract

La présente invention concerne des procédés et des compositions permettant d'augmenter les niveaux de protéines de choc thermique (HSP) dans les cellules bêta du pancréas afin de réduire l'agrégation de l'amyline chez des individus souffrant d'un diabète de type 2. Dans des cas spécifiques, des nanoparticules d'or sont administrées en direction des cellules bêta du pancréas, lesdites nanoparticules portant ou étant dépourvues d'une ou plusieurs HSP, et les cellules sont ensuite chauffées, par exemple avec de la lumière infrarouge proche. Dans des cas particuliers de ciblage, des anticorps dirigés contre un antigène spécifique des cellules bêta du pancréas sont également employés.
PCT/US2012/021246 2011-01-14 2012-01-13 Effet thérapeutique de protéines de choc thermique utilisées pour la prévention de l'agrégation de l'amyline dans le diabète sucré de type 2 WO2012097255A2 (fr)

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WO2017039074A1 (fr) * 2015-09-01 2017-03-09 한국과학기술원 Procédé destiné à l'administration efficace de gène dans des cellules à l'aide de l'effet photothermique de nanoparticules d'or
US10500217B2 (en) 2018-04-30 2019-12-10 Leslie Ray Matthews, M.D., Llc Vitamin D3, heat shock proteins, and glutathione for the treatment of chronic inflammation and chronic diseases
US20210238691A1 (en) * 2013-08-08 2021-08-05 Onyx Therapeutics, Inc. Immunoglobulin expression levels as biomarker for proteasome inhibitor response

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210238691A1 (en) * 2013-08-08 2021-08-05 Onyx Therapeutics, Inc. Immunoglobulin expression levels as biomarker for proteasome inhibitor response
WO2017039074A1 (fr) * 2015-09-01 2017-03-09 한국과학기술원 Procédé destiné à l'administration efficace de gène dans des cellules à l'aide de l'effet photothermique de nanoparticules d'or
US10500217B2 (en) 2018-04-30 2019-12-10 Leslie Ray Matthews, M.D., Llc Vitamin D3, heat shock proteins, and glutathione for the treatment of chronic inflammation and chronic diseases

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