WO2014039501A1 - Composés bolaamphiphiles, compositions et leurs utilisations - Google Patents

Composés bolaamphiphiles, compositions et leurs utilisations Download PDF

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Publication number
WO2014039501A1
WO2014039501A1 PCT/US2013/057956 US2013057956W WO2014039501A1 WO 2014039501 A1 WO2014039501 A1 WO 2014039501A1 US 2013057956 W US2013057956 W US 2013057956W WO 2014039501 A1 WO2014039501 A1 WO 2014039501A1
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Prior art keywords
pharmaceutical composition
compound
glh
bolaamphiphilic
composition according
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PCT/US2013/057956
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English (en)
Inventor
Charles Linder
Eliahu Heldman
Sarina Grinberg
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Lauren Sciences Llc
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Priority to CA2883784A priority Critical patent/CA2883784C/fr
Priority to EP13834981.6A priority patent/EP2892510A4/fr
Priority to AU2013312908A priority patent/AU2013312908A1/en
Application filed by Lauren Sciences Llc filed Critical Lauren Sciences Llc
Publication of WO2014039501A1 publication Critical patent/WO2014039501A1/fr
Priority to US14/328,419 priority patent/US20150110875A1/en
Priority to IL237541A priority patent/IL237541B/en
Priority to US15/099,956 priority patent/US20160367678A1/en
Priority to AU2018203992A priority patent/AU2018203992B2/en
Priority to US16/188,232 priority patent/US20190216895A1/en
Priority to IL267888A priority patent/IL267888B/en
Priority to AU2020203496A priority patent/AU2020203496B2/en
Priority to IL280316A priority patent/IL280316B/en
Priority to US17/351,373 priority patent/US20220072098A1/en
Priority to AU2022200494A priority patent/AU2022200494A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • 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
    • A61K47/54—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 organic compound
    • A61K47/545—Heterocyclic compounds
    • 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
    • A61K47/54—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 organic compound
    • A61K47/55—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 organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers

Definitions

  • nanovesicles comprising bolaamphiphilic compounds, and complexes thereof with neurotrophins (NTFs), such as glial cell derived growth factor (GDNF) or nerve growth factor (NGF), and pharmaceutical compositions thereof.
  • NTFs neurotrophins
  • GDNF glial cell derived growth factor
  • NTF nerve growth factor
  • the present disclosure is further directed to compounds, compositions, and method of the treatement of neurological diseases including, for illustrative potposes Parkinson's disease, Alzheimers and amyotrophic lateral sclerosis (ALS).
  • NTFs neurotrophins
  • GDNF glial cell derived growth factor
  • NTF nerve growth factor
  • the present disclosure is further directed to compounds, compositions, and method of the treatement of neurological diseases including, for illustrative memeposes Parkinson's disease, Alzheimers and amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • compositions provided herein are provided herein.
  • NTFs neurotrophins
  • GDNF glial cell derived growth factor
  • NGF nerve growth factor
  • GDNF or NGF do not permeate through the blood-brain barrier (BBB), thus they have to be delivered directly into the brain in order to exert its therapeutic action.
  • BBB blood-brain barrier
  • attempts to deliver GDNF directly into the brain had little benefit, most probably because its distribution within the brain was restricted to only 2-9% of the area receiving the GDNF [2].
  • convection-enhanced delivery of GDNF resulted in a great deal of variability in its distribution within the injected site [3]. The variability in GDNF distribution, and its limited diffusion throughout the brain, is most probably due to its binding to the extracellular matrix [4].
  • a delivery system which is capable of distributing GDNF uniformly within the brain, and not concentrating it at a small site (that might cause toxicity), should increase the probability that all affected neurons are exposed to GDNF's therapeutic activity and, thus, increase GDNF's efficacy in the treatment of PD.
  • the brain capillary endothelial cells (BCECs) that form the BBB play important role in brain physiology by maintaining selective permeability and preventing passage of various compounds from the blood into the brain.
  • One consequence of the highly effective barrier properties of the BBB is the limited penetration of therapeutic agents into the brain, which makes treatment of many brain diseases extremely challenging.
  • a delivery system that uses the intense capillary network that supplies blood to the brain should deliver GDNF or NGF to a wide area within the brain, provided that the delivery system is capable of crossing the BBB and releasing the NTF there. Targeting to specific sites within the brain is alos greatly facilitated by an efficient penetration through the BBB into the brain after systemic administration.
  • CF carboxyfluorescein
  • a fluorescent marker a fluorescent marker
  • CF carboxyfluorescein
  • single headed amphiphiles of opposite charge in cationic vesicles formed by mixing single-tailed cationic and anionic surfactants
  • a certain portion of the CF is passively encapsulated within the core of the formed vesicles.
  • the present disclosure employing bolaamphilies includes embodiments in which a portion of the active agent may be complexed to the head groups of the bolaamphiphiles and another fraction of the active agents are encapsulated within the core of the vesicles.
  • the major portion of the active agent is encapsulated by complexation with the head groups.
  • WO 02/05501 1 and WO 03/047499 both of the same applicant of the present disclosure, disclose amphiphilic derivatives composed of at least one fatty acid chain derived from natural vegetable oils such as vernonia oil, lesquerella oil and castor oil, in which functional groups such as epoxy, hydroxy and double bonds were modified into polar and ionic headgroups.
  • WO 10/128504 reports a series of amphiphiles and bolamphiphiles (amphiphiles with two head groups) useful for targeted drug delivery of insulin, insulin analogs, TNF, GDNF, DNA, NA (including siRNA), enkephalin class of analgesics, and others.
  • bolaamphiphiles that interact with and encapsulate a variety of small and large molecules including peptides, proteins and plasmid DNAs delivering them across biological membranes.
  • These bolaamphiphiles are a unique class of compounds that have two hydrophilic headgroups placed at each ends of a hydrophobic domain.
  • Bolaamphiphiles can form vesicles that consist of monolayer membrane that surrounds an aqueous core.
  • Vesicles made from natural bolaamphiphiles, such as those extracted from archaebacteria (archaesomes) are very stable and, therefore, might be employed for targeted drug delivery.
  • bolaamphiphiles from archaebacteria are heterogeneous and cannot be easily extracted or chemically synthesized.
  • NTF nerve growth factor
  • compositions comprising of a bolaamphiphile complex.
  • novel nano-sized vesicles comprising of bolaamphiphilic compounds .
  • novel nano-sized vesicles comprising of bolaamphiphilic compounds which are capable of encapsulating NTF, GDNF or NGF.
  • novel nano-sized bola vesicles that encapsulate
  • GDNF or NGF are capable of delivering the encapsulated material into the brain.
  • novel nano-sized bola vesicles that encapsulate
  • GDNF or NGF are capable of delivering the encapsulated material to the brain, specifically to dopaminergic neurons.
  • novel nano-sized bola vesicles that encapsulate GDNF or NGF and are capable of delivering the encapsulated material into brain regions affected in neurological disorders.
  • the neurological disorder is Parkinson's disease (PD) or Alzheimer's disease (AD).
  • novel bolaamphiphile complexes comprising bolaamphiphilic compounds and a compound active against PD.
  • the compound active against AD is GDNF.
  • novel bolaamphiphile complexes comprising bolaamphiphilic compounds and a compound active against AD.
  • the compound active against PD is NGF.
  • novel formulations of GDNF or NGF with bolaamphiphilic compounds or with bolaamhphile vesicles are provided herein.
  • the method comprises the step of administering to the animal or human a pharmaceutical composition comprising of a bolaamphiphile complex; and wherein the bolaamphiphile complex comprises a bolaamphiphilic compound and GDNF.
  • the complex comprises bolaamphiphilic compound and NGF.
  • the bolaamphiphilic compound consists of two hydrophilic headgroups linked through a long hydrophobic chain.
  • the hydrophilic headgroup is an amino containing group.
  • the hydrophilic headgroup is a tertiary or quaternary amino containing group.
  • the bolaamphiphilic compound is a compound according to formula I:
  • each HG 1 and HG 2 is independently a hydrophilic head group
  • L 1 is alkylene, alkenyl, heteroalkylene, or heteroalkenyl linker; unsubstituted or substituted with C1-C20 alkyl, hydroxyl, or oxo.
  • the pharmaceutically acceptable salt is a quaternary ammonium salt.
  • the bolaamphiphilic compound of formula I is a compound according to formula II, III, IV, V, or VI:
  • each HG 1 and HG 2 is independently a hydrophilic head group
  • each Z 1 and Z 2 is independently -C(R 3 ) 2 -, -N(R 3 )- or -0-;
  • each R la , R lb , R 3 , and R 4 is independently H or Ci-C 8 alkyl
  • each R 2a and R 2b is independently H , Ci-C 8 alkyl, OH, alkoxy, or O-HG 1 or O-HG 2 ; each n8, n9, ni l, and nl2 is independently an integer from 1-20;
  • nlO is an integer from 2-20;
  • each dotted bond is independently a single or a double bond.
  • each HG 1 and HG 2 is independently selected from:
  • X is -NR 5a R 5b , or -N + R 5a R Sb R 5c ; each R 5a , and R 5b is independently H or substituted or unsubstituted C1-C2 0 alkyl or R 5a and R 5b may join together to form an N containing substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclyl;
  • each R 5c is independently substituted or unsubstituted C1-C2 0 alkyl; each R 8 is independently
  • ml is 0 or 1
  • each nl3, nl4, and nl5 is independently an integer from 1-20.
  • this disclosure provides novel monlayer nanovescicles.
  • the nanovesicles comprise bolaamphiphilic compounds with head groups faciliatating penetration of the bood brain barrier.
  • the nanovesicles comprise bolaamphiphilic compounds with head groups that facilitate targeting to dopaminergic neurons in the brain.
  • the vesicles formed from the bolaamphiphiles contain additives that help to stabilize the vesicles, by stabilizing the vesicle's membranes, such as but not limited to cholesterol derivcatives such as cholesteryl hemisuccinate and cholesterol itself and combinations such as cholesteryl hemisuccinate and cholesterol.
  • the vesicles comprise the bolaamphiphiles, vesicle membrane stabilizing additives, stearyl amine, and GDNF and GF.
  • the vesicles in addition to these components have another addiitves which decorates the outer vesicle memrbanes with groups or pendants that enhance penetration though biological barriers such as the BBB and groups for targeting.
  • a non limiting example of such additives may be alkyl conjugates of chitosan or bolaamphiphiles where one of the head groups is chitiosan.
  • the present disclosure provides nanovesicles that comprise bolaamphiphilic compounds with chitosan head groups. [0029] In certain embodiments, the present disclosure provides nanovesicles that comprise bolaamphiphilic compounds with head groups that can function as ligands for the dopamine transporter.
  • the present disclosure provides nanovesicles that comprise bolaamphiphilic compounds with head groups that can function as ligands for the dopamine transporter as well as with bolaamphiphilic compounds with that comprise chitosan head groups.
  • the present disclosure provides monlayer nanovesicles comprising the bolaamphiphilic compound designated herein as GLH-55a, the bolaamphiphilic compound designated herein as GLH-57, as well as encapsulated GDNF.
  • the present disclosure provides a method of treatment of a neurotrophic disease comprising administration of an effective amount of monlayer nanovesicles of the disclosure comprising an encapsulated active agent.
  • the neutrophic disese is Parkinson's disease
  • the administered monlayer nanovesicles comprise the bolaamphiphilic compound designated herein as GLH-55a, the bolaamphiphilic compound designated herein as GLH-57, as well as encapsulated GDNF.
  • the present disclosure further provides compositions and methods for controlling the rate of release of vesicle-encapsulated materials by varying the length of alkyl chains adjacent to hydrolysable head groups of bolaamphiphilic vesicles.
  • the head groups are acetylcholine head groups.
  • Figure 1 TEM micrograph of vesicles from GLH-20 (A) and their size distribution determined by DLS (B).
  • Figure 2 Head group hydrolysis by AChE (A) of GLH-19 (blue) and GLH-20 (red) and release of CF from GLH-19 vesicles (B) and GLH-20 vesicles (C).
  • Figure 3 CF accumulation in brain after i.v. injection of encapsulated and non- encapsulated CF. Only GLH-20 vesicles allow accumulation of CF in the brain (A). CS improves GLH-20 vesicles' penetration into the brain (B).
  • FIG. 4 Analgesia after i.v. injection of enkephalin non-encapsulated and encapsulated in vesicles.
  • Analgesia (compared with morphine, which was used as a positive control) is obtained only when enkephalin is encapsulated in GLH-20 vesicles (A), the head groups of which are hydro lyzed by ChE.
  • the vesicles do not disrupt the BBB since non- encapsulated enkephalin co- injected with empty vesicles (extravesicular enkephalin) did not cause analgesia (B).
  • **Significantly different from free leu-enkephalin (t-test, PO.01).
  • Figure 5 Fluorescence in mouse cerebral cortex after i.v. injection of albumin-FITC (non-encapsulated) (A) encapsulated in GLH-20 vesicles (B).
  • Figure 6 Mass spectra of GLH-20(A) and GLH- 19 (B).
  • Figure 7 FT-IR spectra of original (CS) (spectrum a) and LMWCS (spectrum b).
  • Figure 10 X H NMR of compound ⁇ -CFT 5.
  • Figure 1 1 X H-NMR (A) and 13 C-NMR (B) spectra of the demethylated ⁇ -CFT fluoronortropane 7.
  • Figure 12 X H-NMR spectrum of GLH-57 (panel (A)), enlargement of the section 2.6- 3.5 ppm (panel (B)).
  • FIG. 13 CryoTEM micrographs of vesicles made from the basic bolas. Vesicles were prepared by film hydration followed by probe sonication from a formulation containing 10 mg/ml bola, 2.1 mg/ml cholesteryl hemisuccinate and 1.6 mg/ml cholesterol.
  • Figure 14 CryoTEM micrographs of vesicles made from a mixture of GLH-19 and GLH-20. Vesicles were prepared by film hydration followed by sonication from a formulation containing GLH-19 and GLH-20 at a ratio of 2: 1, respectively (total of 10 mg/ml bolas), cholesterol (1.6 mg/ml) and cholesteryl hemisuccinate (2.1 mg/ml).
  • Figure 17 Representative data from DLS measurements of size distribution by intensity for vesicles made from GLH-19 (Panel A); GLH-20 (Panel B); and a mixture of GLH- 19 and GLH-20 at a ratio of 2: 1 (Panel C).
  • Vesicles were prepared by film hydration followed by sonication from 10 mg/ml bolas, 2.1 mg/ml cholesteiyl hemisuccinate and 1.6 mg/ml cholesterol. Each sample was measured by the DLS 3 times, and each profile shows the three measurements overlaid.
  • Figure 18 Size distribution of GLH-20 vesicles, with and without encapsulated trypsinogen. Vesicles were prepared by film hydration followed by sonication from 10 mg/ml GLH-20, 2.1 mg/ml cholesteryl hemisuccinate and 1.6 mg/ml cholesterol, in presence and absence of trypsinogen. Size distribution was measured by DLS.
  • FIG. 19 Stability of GLH-20 vesicles in storage. Encapsulation of CF was determined after diluting the vesicles to reduce the extravesicular CF concentration, then, the vesicles were disrupted by Triton X100 and the fluorescence of released CF was measured at various times as indicated. Encapsulation was normalized using encapsulation at time 0 as 100%
  • Figure 20 Stability of bolaamphiphilic vesicles in whole serum. Vesicles were prepared from GLH-19 or GLH-20 or from mixtures of both bolas using two ratios as shown. Vesicles were added to the serum in a ratio of 1 : 10 (vesicles:serum). Percent CF encapsulation was determined by fluorescence measurements as described in FIG. 14. Encapsulation was normalized using encapsulation at time 0 as 100%
  • FIG. 21 Release of CF from bolavesicles in response to AChE.
  • Vesicles were prepared from either GLH-20 alone (plus the standard additives) (Panel A), or a mixture of GLH- 19 and GLH-20 (plus the standard additives) (Panel B) and both loaded with CF.
  • the vesicles were placed in a cuvette, and fluorescence was measured as a function of time until stable reading was achieved. Then, 2 units of AChE was added to each vesicle preparation, and the fluorescence measurement continued. The release of the encapsulated CF causes increase in the fluorescence. About 7 minutes after the addition of the AChE, triton X100 was added (to a final concentration of 0.15%), to fully disrupt the vesicles and to release the remaining CF for the determination of the total CF that was encapsulated.
  • Figure 22 Elution profile of a vesicle formulation that contained encapsulated (peak 1) and free trypsinogen (peak 2). The vesicles were applied on Sephadex G50 column and eluted with PBS.
  • Figure 23 Quantification of encapsulated trypsinogen using the data obtained from the experiment described in FIG. 17.
  • Figure 24 Encapsulation of trypsinogen following vesicle preparation by film hydration and sonication or by extrusion.
  • Upper graph shows the overlap of the elution profiles obtained running each vesicle preparation on the Sephadex G50 column.
  • the lower graphs show the quantification of encapsulation for sonicated vesicles (Panel A) and extruded vesicles (Panel B).
  • Figure 25 Encapsulation of AlexaFluor®-488-labeled trypsinogen in bolaamphiphilc vesicles. Vesicles were made by film hydration followed by sonication from a mixture of 10 mg/ml GLH-19 and GLH-20 (2: 1) with 2.1 mg/ml choesteryl hemisuccinate and 1.6 mg/ml cholesterol. Trypsinogen was labeled with AlexaFluor®-488, as described in the method section, and was included in the formulation at a concentration of 0.2 mg/ml.
  • Figure 26 Encapsulation efficiencies of trypsinogen and GDNF.
  • Vesicles were prepared by film hydration followed by sonication from a mixture of GLH-19 and GLH-20 at a concentration of 10 mg/ml with 1.6 mg/ml cholesterol and 2.1 mg/ml cholesteryl hemisuccinate.
  • the formulations contained 50 ⁇ g/ml trypsinogen (Panel A), 100 ⁇ g/ml trypsinogen (Panel B) and 12.5 ⁇ g/ml GDNF (Panel C). All proteins were labeled with AlexaFluorD-488. After encapsulation, the vesicles were eluted from a Sephadex G50 column by PBS and the fluorescence of each fraction was determined.
  • Figure 27 The effect of the encapsulation process on GDNF integrity and activity.
  • Figure 28 Uptake of CF-loaded vesicles into cells in culture. Vesicles were made from 10 mg/ml GLH-19:GLH-20 (2: 1) without (uncoated vesicles) and with 0.8 mg/ml GLH-57, a bola that contains DAT ligand as the head group (DAT -vesicles). Cells were incubated for 1 h with the vesicles, and tested by flow cytometry. A shift to the right of the peak indicates fluorescent cells due to uptake of the vesicles.
  • DAT -vesicles a bola that contains DAT ligand as the head group
  • Figure 29 Accumulation of CF in the brain following i.v. administration.
  • Vesicles were made by film hydration followed by sonication from a 10 mg/ml mixture of GLH-19 and GLH-20 (2: 1), 1 mg/ml CS-fatty acid (vernolate) conjugate, 2.1 mg/ml cholesteryl hemisuccinate and 1.6 mg/ml cholesterol in absence (empty vesicles) and in presence of 0.2/ml CF (CF-loaded vesicles).
  • Mice were pretreated with 0.5 mg/kg (i.m.) pyridostigmine and 15 min afterward the mice were injected i.v.
  • Figure 30 CF concentration in the brain after delivering it encapsulated in vesicles with CS surface groups.
  • Vesicles were prepared as described in FIG. 24, except that in one case, 1 mg/ml GLH-55a was used in the vesicle formulation to provide CS surface groups (vesicles with CS-bola), and in the other case, 1 mg/ml CS-fatty acid conjugate was used. Conditions of this experiment were similar to those presented in FIG. 24.
  • Figure 3 1 Distribution of CF in the brain after injecting CF-loaded vesicles with and without surface DAT ligand.
  • Vesicles were prepared by film hydration followed by sonication from a 10 mg/ml mixture of GLH-19 and GLH-20 (2: 1), 1 mg/ml GLH-55a (a bola with CS head group), 2.1 mg/ml cholesteryl hemisuccinate, 1.6 mg/ml cholesterol, 0.2 mg/ml CF and without (vesicle CS bola) or with GLH-57 (vesicles DAT CS bola).
  • mice were pretreated with 0.5 mg/kg (i.m.) pyridostigmine (to inhibit peripheral ChE) and 15 min afterward the vesicles were injected i.v. After 30 min the mice were sacrificed, perfused with 10 ml PBS and the brain removed and dissected into cortex, striatum and cerebellum. The tissues were weighed, homogenized and deproteinated by trichloroacetic acid, centrifuged and fluorescence was determined in the homogenates. The amount of the CF in each brain region was calculated from a calibration curve of CF, taking into consideration the weight of the tissue and the dilution done during the homogenization. Each bar represent an average value obtained from 5 mice +/- SEM.
  • Figure 32 Representative histofluorescence slides showing AlxaFlour-488-labeld trypsinogen in brain (Panels A-C); liver (Panels D-F) and kidney (Panels G-I) of mice that were injected with the labeled protein encapsulated in CS-coated vesicles or with the free protein.
  • Panels A, D and G are micrographs taken from control untreated mice.
  • Panels B, E and H are micrographs taken from mice injected with 200 ⁇ g of free trypsinogen labeled with
  • AlexaFluor®-488 and Panels C, F and I are micrographs taken from mice that were injected with 200 ⁇ g of encapsulated trypsinogen labeled with AlexaFluor®-488.
  • Figure 33 Distribution of trypsinogen labeled with AlexaFluor®-488 in brain, kidney and liver after the injection (i.v.) of the labeled protein in its free form or encapsulated in vesicles. For the quantification, data obtained in the experiment described in FIG. 27 were used. Each bar represent an average value of 5 mice +/- SEM
  • Figure 34 Representative brain sections stained for GDNF-biotin with avidine- AlexaFluor®-488. Mice were pretreated with 0.5 mg/kg (i.m.) pyridostigmine, then injected i.v. with vesicles coated with CS groups and DAT ligand with encapsulated GDNF-biotin. After 30 min, animals were sacrificed, perfused with 10 ml PBS, brains removed and striata, cortex and cerebella were dissected out, frozen and cryosectioned. Brain sections from these mice were stained with DAPI (blue) and avidine-AlexaFluor®-488 (green) and observed using confocal microscopy at a magnification of 10X.
  • DAPI blue
  • avidine-AlexaFluor®-488 green
  • Figure 35 Distribution of exogenous GDNF-biotin in the brain after delivering the protein encapsulated in bolavesicles. These micrographs of high magnification, (60X) were taken from brain sections obtained from the mice used in the experiment described in FIG. 29. The nuclei of the cells appear in blue, due to DAPI staining, and the GDNF-biotin appears in green, due to the binding of the avidine-AlexaFluorl SS.
  • Figure 36 Chemical shifts of the chloromethylene (-CHjCl) and alkoxymethylene (C(0)-0-CH 2 -) groups of comound 4.
  • Figure 37 Comparison of the NMR spectrum in CDCI 3 of the dichloroacetate intermediate 4 and the bolaamphiphile 5.
  • Figure 38 TEM micrographs of particles formed from bolaamphiphile GLH-20 (Panel A) and bolaamphiphile GLH-32 (Panel B). Vesicles were prepared by film-hydration- extrusion (FHE) using 200 nm and 100 nm membranes, consecutively
  • Figure 39 TEM micrographs of vesicles made from bolaamphiphile GLH-20 (left) and bolaamphiphile GLH-32 (right) formulated with CHOL and CHEMS at a molar ratio of 2: 1 : 1. Vesicles were prepared by FHE using 200 nm and 100 nm membranes, consecutively [0074]
  • Figure 40 Vesicle stability determine by changes in vesicle size (Panel A) and changes in percent encapsulation (Panel B) using vesicles made from GLH-20 and GLH-32 with CHOL and CHEMS at a ratio of 2: 1 : 1.
  • Figure 41 Hydrolysis of the ACh head group of bioamphiphiles GLH-20 and GLH-32 by AChE. Hydrolysis was measured by determining the pH change after addition of AChE to the incubation medium and was converted to change in the proton concentration.
  • Figure 42 Lineweaver-Burk plots of ATC hydrolysis by AChE in presence of several concentrations of GLH-20 and GLH-32
  • Figure 43 The effect of AChE on the release of CF from vesicles made from GLH-20 and GLH-32.
  • the released CF was monitored by measuring the fluorescence before and after the addition of X units of AChE dissolved in X ⁇ PBS. The experiment was terminated by the addition of Triton X- 100 to disrupt the vesicles and release all the encapsulated CF.
  • Figure 44 Percent release of encapsulated CF at different time after exposing bolaamphiphilic vesicles to AChE. Percent release was calculated from the amount of CF that was released at a particular time point versus the total amout of encapsulated CF, which was determined after lysing the vesicles with Triton XI 00.
  • Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers.
  • the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer.
  • Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et ah,
  • Ci_6 alkyl is intended to encompass, Ci, C 2 , C3, C 4 , C5, Ce, C ⁇ , Ci- 5, Ci-4, Ci-3, Ci-2, C2-6, C 2 -5, C4-6, C4-5, and C5-6 alkyl.
  • Alkyl refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms (“Ci_2o alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“Ci_i2 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“Ci-io alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“Ci_9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“Ci_8 alkyl”). In some embodiments,
  • an alkyl group has 1 to 7 carbon atoms (“Ci_7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“Ci_6 alkyl”, also referred to herein as "lower alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“Ci_5 alkyl”). In some
  • an alkyl group has 1 to 4 carbon atoms ("C1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“Ci_3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“Ci_ 2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“Ci alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C 2 -6 alkyl”). Examples of Ci_6 alkyl groups include methyl (Ci), ethyl (C 2 ), n-propyl (C ?
  • alkyl groups include n-heptyl (C 7 ), n-octyl (Cs) and the like.
  • each instance of an alkyl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") with one or more substituents; e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • the alkyl group is unsubstituted Ci_io alkyl (e.g., -CH 3 ).
  • the alkyl group is substituted Ci-10 alkyl.
  • Alkylene refers to a substituted or unsubstituted alkyl group, as defined above, wherein two hydrogens are removed to provide a divalent radical.
  • exemplary divalent alkylene groups include, but are not limited to, methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), the propylene isomers (e.g., -CH 2 CH 2 CH 2 - and -CH(CH 3 )CH 2 -) and the like.
  • alkenyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds ("C 2 - 20 alkenyl”).
  • an alkenyl group has 2 to 10 carbon atoms ("C 2 _io alkenyl”).
  • an alkenyl group has 2 to 9 carbon atoms ("C 2 _9 alkenyl”).
  • an alkenyl group has 2 to 8 carbon atoms (“C2-8 alkenyl”).
  • an alkenyl group has 2 to 7 carbon atoms (“C 2 _ 7 alkenyl”).
  • an alkenyl group has 2 to 6 carbon atoms ("C 2 _6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C 2 _5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms ("C 2 ⁇ alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C 2 _3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms ("C 2 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl).
  • Examples of C 2 ⁇ alkenyl groups include ethenyl (C 2 ), 1-propenyl (C 3 ), 2- propenyl (C 3 ), 1-butenyl (C 4 ), 2-butenyl (C 4 ), butadienyl (C 4 ), and the like.
  • Examples of C 2 _6 alkenyl groups include the aforementioned C 2 _4 alkenyl groups as well as pentenyl (C 5 ), pentadienyl (C 5 ), hexenyl (Ce), and the like. Additional examples of alkenyl include heptenyl (C 7 ), octenyl (Cs), octatrienyl (Cs), and the like.
  • each instance of an alkenyl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkenyl") or substituted (a "substituted alkenyl") with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • the alkenyl group is unsubstituted C2-1 0 alkenyl.
  • the alkenyl group is substituted C 2 _ 1 0 alkenyl.
  • alkenylene refers a substituted or unsubstituted alkenyl group, as defined above, wherein two hydrogens are removed to provide a divalent radical.
  • Alkynyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon triple bonds, and optionally one or more double bonds ("C2-2 0 alkynyl”).
  • an alkynyl group has 2 to 10 carbon atoms ("C2-1 0 alkynyl”).
  • an alkynyl group has 2 to 9 carbon atoms ("C2-9 alkynyl”).
  • an alkynyl group has 2 to 8 carbon atoms (“C 2 - 8 alkynyl”).
  • an alkynyl group has 2 to 7 carbon atoms ("C2-7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms ("C2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C 2 _s alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms ("C2-4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms ("C 2 alkynyl”).
  • the one or more carbon-carbon triple bonds can be internal (such as in 2- butynyl) or terminal (such as in 1-butynyl).
  • alkynyl groups include, without limitation, ethynyl (C 2 ), 1-propynyl (C ? ), 2-propynyl (C 3 ), 1-butynyl (C 4 ), 2-butynyl (C 4 ), and the like.
  • C2-6 alkenyl groups include the aforementioned C 2 ⁇ i alkynyl groups as well as pentynyl (C5), hexynyl (Ce), and the like.
  • alkynyl examples include heptynyl (C 7 ), octynyl (Cs), and the like.
  • each instance of an alkynyl group is independently optionally substituted, i.e. , unsubstituted (an "unsubstituted alkynyl") or substituted (a "substituted alkynyl") with one or more substituents; e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • the alkynyl group is unsubstituted C 2 _ 10 alkynyl.
  • the alkynyl group is substituted C2-10 alkynyl.
  • Alkynylene refers a substituted or unsubstituted alkynyl group, as defined above, wherein two hydrogens are removed to provide a divalent radical.
  • exemplary divalent alkynylene groups include, but are not limited to, ethynylene, propynylene, and the like.
  • '"Aryl refers to a radical of a monocyclic or poly cyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 ⁇ electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system ("C6-i 4 aryl").
  • an aiyl group has six ring carbon atoms ("Ce aryl”; e.g., phenyl).
  • an aryl group has ten ring carbon atoms ("C 10 aryl”; e.g., naphthyl such as 1- naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms ("Ci 4 aryl”; e.g., anthracyl).
  • Aryl also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.
  • Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthiylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, and trinaphthalene.
  • aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl.
  • each instance of an aryl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more substituents.
  • the aryl group is unsubstituted C6-i 4 aryl.
  • the aryl group is substituted C6-i 4 aryl.
  • one of R and R may be hydrogen and at least one of R and R is each independently selected from Ci-Cs alkyl, Ci-C$ haloalkyl, 4-10 membered heterocyclyl, alkanoyl, Ci-Cs alkoxy, heteroaryloxy, alkylamino, arylamino, heteroarylamino, NR 58 COR 59 , NR 58 SOR 59 R 58 S0 2 R 59 , COOalkyl, COOaryl, CONR S8 R 59 , CONR 58 OR 59 , NR 58 R 59 ,
  • S0 2 NR 58 R 59 S-alkyl, SOalkyl, S0 2 alkyl, Saryl, SOaryl, S0 2 aryl; or R 56 and R 57 may be joined to form a cyclic ring (saturated or unsaturated) from 5 to 8 atoms, optionally containing one or more heteroatoms selected from the group N, O, or S.
  • R 60 and R 61 are independently hydrogen, Ci-Cs alkyl, C1-C4 haloalkyl, C3-C10 cycloalkyl, 4-10 membered heterocyclyl, C6-C10 aryl, substituted C6-C1 0 aryl, 5-10 membered heteroaryl, or substituted 5-10 membered heteroaryl.
  • fused aryl refers to an aryl having two of its ring carbon in common with a second aryl ring or with an aliphatic ring.
  • Aralkyl is a subset of alkyl and aryl, as defined herein, and refers to an optionally substituted alkyl group substituted by an optionally substituted aryl group.
  • Heteroaryl refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 ⁇ electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur ("5-10 membered heteroaryl").
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • Heteroaiyl bicyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heteroaryl includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system.
  • Heteroaryl also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system.
  • Bicyclic heteroaiyl groups wherein one ring does not contain a heteroatom e.g., indolyl, quinolinyl, carbazolyl, and the like
  • the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).
  • a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl").
  • a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heteroaryl").
  • a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heteroaryl").
  • the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.
  • each instance of a heteroaryl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted heteroaryl") or substituted (a "substituted heteroaryl") with one or more substituents.
  • the heteroaryl group is unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is substituted 5-14 membered heteroaryl.
  • Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl.
  • Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl.
  • Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.
  • Exemplaiy 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl.
  • Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl.
  • Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl.
  • Exemplaiy 6- membered heteroaiyl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively.
  • Exemplary 7-membered heteroaiyl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl.
  • Exemplary 5,6- bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl,
  • benzotriazolyl benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.
  • Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.
  • Examples of re resentative heteroaryls include the following:
  • each Y is selected from carbonyl, N, NR , O, and S; and R is independently hydrogen, Ci-Cs alkyl, C3-C1 0 cycloalkyl, 4-10 membered heterocyclyl, C6-C1 0 aryl, and 5-10 membered heteroaryl.
  • R is independently hydrogen, Ci-Cs alkyl, C3-C1 0 cycloalkyl, 4-10 membered heterocyclyl, C6-C1 0 aryl, and 5-10 membered heteroaryl.
  • each W is selected from C(R ) 2 , NR , O, and S; and each Y is selected from carbonyl, NR 66 , O and S; and R 66 is independently hydrogen, Ci-C 8 alkyl, C3-C10 cycloalkyl, 4-10 membered heterocyclyl, C6-C10 aryl, and 5-10 membered heteroaryl.
  • Heteroaralkyl is a subset of alkyl and heteroaryl, as defined herein, and refers to an optionally substituted alkyl group substituted by an optionally substituted heteroaryl group.
  • Carbocyclyl or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms ('3 ⁇ 4_ ⁇ carbocyclyl”) and zero heteroatoms in the non-aromatic ring system.
  • a carbocyclyl group has 3 to 8 ring carbon atoms ("C3_s carbocyclyl”).
  • a carbocyclyl group has 3 to 6 ring carbon atoms ("C 3 _ 6 carbocyclyl”).
  • a carbocyclyl group has 3 to 6 ring carbon atoms ("C 3 _6 carbocyclyl”).
  • a carbocyclyl group has 5 to 10 ring carbon atoms ("Cs-io carbocyclyl").
  • Exemplary C3-6 carbocyclyl groups include, without limitation, cyclopropyl (C 3 ), cyclopropenyl (C 3 ), cyclobutyl (C 4 ), cyclobutenyl (C 4 ), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (Ce), cyclohexenyl (Ce), cyclohexadienyl (Ce), and the like.
  • Exemplary C 3 _ 8 carbocyclyl groups include, without limitation, the aforementioned C3-6 carbocyclyl groups as well as cycloheptyl (C 7 ), cycloheptenyl (C 7 ), cycloheptadienyl (C 7 ), cycloheptatrienyl (C 7 ), cyclooctyl (Cs), cyclooctenyl (Cs), bicyclo[2.2.1 ]heptanyl (C 7 ), bicyclo[2.2.2]octanyl (Cs), and the like.
  • Exemplary C 3 _io carbocyclyl groups include, without limitation, the aforementioned C 3 _g carbocyclyl groups as well as cyclononyl (C 9 ), cyclononenyl (C 9 ), cyclodecyl (Cio), cyclodecenyl (Cio), octahydro-lH-indenyl (C9), decahydronaphthalenyl (Cio), spiro[4.5]decanyl (Cio), and the like.
  • the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) and can be saturated or can be partially unsaturated.
  • “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system.
  • each instance of a carbocyclyl group is independently optionally substituted, i.e. , unsubstituted (an "unsubstituted carbocyclyl") or substituted (a "substituted carbocyclyl") with one or more substituents.
  • the carbocyclyl group is unsubstituted C3-10 carbocyclyl.
  • the carbocyclyl group is a substituted C3_io carbocyclyl.
  • “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 10 ring carbon atoms ("C3_io cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms ("C 3 -8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3_6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms ("C5_6 cycloalkyl").
  • a cycloalkyl group has 5 to 10 ring carbon atoms ("Cs-io cycloalkyl").
  • C5-6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5).
  • C , ⁇ cycloalkyl groups include the aforementioned C5-6 cycloalkyl groups as well as cyclopropyl (C 3 ) and cyclobutyl (C 4 ).
  • C3_8 cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (Cs). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an "unsubstituted cycloalkyl") or substituted (a
  • substituted cycloalkyl with one or more substituents.
  • the cycloalkyl group is unsubstituted C 3 -10 cycloalkyl.
  • the cycloalkyl group is substituted C3_io cycloalkyl.
  • Heterocyclyl or “heterocyclic” refers to a radical of a 3- to 10-membered non- aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3-10 membered heterocyclyl").
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • a heterocyclyl group can either be monocyclic ("monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system ("bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated.
  • Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heterocyclyl also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.
  • each instance of heterocyclyl is independently optionally substituted, i.e.
  • the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3-10 membered heterocyclyl.
  • a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("5-10 membered heterocyclyl").
  • a heterocyclyl group is a 5-8 membered non- aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heterocyclyl").
  • a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heterocyclyl").
  • the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.
  • Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl.
  • Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl.
  • Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione.
  • Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one.
  • Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl.
  • Exemplary 6- membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl.
  • Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl.
  • Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl.
  • Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl.
  • Exemplary 5-membered heterocyclyl groups fused to a C 6 aryl ring include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like.
  • Exemplary 6-membered heterocyclyl groups fused to an aiyl ring include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.
  • each W is selected from CR , C(R ) 2 , NR , O, and S; and each Y is selected from NR 67 , O, and S; and R 67 is independently hydrogen, d-C 8 alkyl, C3-C10 cycloalkyl, 4- 10 membered heterocyclyl, C6-C10 aryl, 5-10 membered heteroaryl.
  • heterocyclyl rings may be optionally substituted with one or more substituents selected from the group consisting of the group consisting of acyl, acylamino, acyloxy, alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl (carbamoyl or amido), aminocarbonylamino, aminosulfonyl, sulfonylamino, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, halogen, hydroxy, keto, nitro, thiol, -S-alkyl, -S-aryl, -S(0)-alkyl,-S(0)-aryl, -S(0) 2 -alkyl, and -S(0) 2 - aryl.
  • Substituting groups include carbonyl or thiocarbonyl which provide, for example, lactam and urea derivatives.
  • Hetero when used to describe a compound or a group present on a compound means that one or more carbon atoms in the compound or group have been replaced by a nitrogen, oxygen, or sulfur heteroatom. Hetero may be applied to any of the hydrocarbyl groups described above such as alkyl, e.g., heteroalkyl, cycloalkyl, e.g., heterocyclyl, aryl, e.g,. heteroaryl, cycloalkenyl, e.g,. cycloheteroalkenyl, and the like having from 1 to 5, and particularly from 1 to 3 heteroatoms.
  • alkyl e.g., heteroalkyl, cycloalkyl, e.g., heterocyclyl, aryl, e.g,. heteroaryl, cycloalkenyl, e.g,. cycloheteroalkenyl, and the like having from 1 to 5, and particularly from 1 to 3 heteroatoms.
  • Acyl refers to a radical -C(0)R 20 , where R 20 is hydrogen, substituted or unsubstitued alkyl, substituted or unsubstitued alkenyl, substituted or unsubstitued alkynyl, substituted or unsubstitued carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstitued heteroaryl, as defined herein.
  • “Alkanoyl” is an acyl group wherein R 20 is a group other than hydrogen.
  • t is an integer from 0 to 4.
  • R 21 is Ci-Cs alkyl, substituted with halo or hydroxy; or C3-C1 0 cycloalkyl, 4-10 membered heterocyclyl, C6-C1 0 aryl, arylalkyl, 5-10 membered heteroaryl or heteroarylalkyl, each of which is substituted with unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy.
  • Acylamino refers to a radical -NR 22 C(0)R 23 , where each instance of R 22 and R23 is independently hydrogen, substituted or unsubstitued alkyl, substituted or unsubstitued alkenyl, substituted or unsubstitued alkynyl, substituted or unsubstitued carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstitued heteroaryl,, as defined herein, or R 22 is an amino protecting group.
  • acylamino groups include, but are not limited to, formylamino, acetylamino, cyclohexylcarbonylamino, cyclohexylmethyl-carbonylamino, benzoylamino and benzylcarbonylamino.
  • acylamino groups are -NR 24 C(0)-Ci-C 8 alkyl, -NR 24 C(O)-(CH 2 ),(C 6 -Ci 0 aryl), - NR 24 C(O)-(CH 2 ) t (5-10 membered heteroaryl), -NR 24 C(O)-(CH 2 ) t (C 3 -Ci 0 cycloalkyl), and - NR 24 C(O)-(CH 2 ) t (4- 10 membered heterocyclyl), wherein t is an integer from 0 to 4, and each R 24 independently represents H or Ci-Cs alkyl.
  • R 25 is H, Ci-Cs alkyl, substituted with halo or hydroxy; C3-C1 0 cycloalkyl, 4-10 membered heterocyclyl, C6-C1 0 aryl, arylalkyl, 5-10 membered heteroaryl or heteroarylalkyl, each of which is substituted with unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy; and R 26 is H, Ci-Cs alkyl, substituted with halo or hydroxy;
  • C3-C1 0 cycloalkyl 4- 10 membered heterocyclyl, C6-C1 0 aryl, arylalkyl, 5- 10 membered heteroaryl or heteroa r yl a lkyl, each of which is substi t ut e d with unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxyl; provided that at least one of R 25 and R 26 is other than H.
  • Acyloxy refers to a radical -OC(0)R 27 , where R 27 is hydrogen, substituted or unsubstitued alkyl, substituted or unsubstitued alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, as defined herein.
  • Representative examples include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl and benzylcarbonyl.
  • R is Ci-C 8 alkyl, substituted with halo or hydroxy; C3-Q0 cycloalkyl, 4-10 membered heterocyclyl, C6-C10 aryl, arylalkyl, 5-10 membered heteroaryl or heteroarylalkyl, each of which is substituted with unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy.
  • Alkoxy refers to the group -OR 29 where R 29 is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • Particular alkoxy groups are methoxy, ethoxy, n- propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1,2- dimethylbutoxy.
  • Particular alkoxy groups are lower alkoxy, i.e. with between 1 and 6 carbon atoms. Further particular alkoxy groups have between 1 and 4 carbon atoms.
  • R 29 is a group that has 1 or more substituents, for instance, from 1 to 5 substituents, and particularly from 1 to 3 substituents, in particular 1 substituent, selected from the group consisting of amino, substituted amino, C6-C10 aryl, aryloxy, carboxyl, cyano, C3-C1 0 cycloalkyl, 4-10 membered heterocyclyl, halogen, 5-10 membered heteroaryl, hydroxyl, nitro, thioalkoxy, thioaryloxy, thiol, alkyl-S(O)-, aryl-S(O)-, alkyl-S(0)2- and aryl- S(0)2-.
  • substituents for instance, from 1 to 5 substituents, and particularly from 1 to 3 substituents, in particular 1 substituent, selected from the group consisting of amino, substituted amino, C6-C10 aryl, aryloxy, carboxyl, cyano, C3-C1 0 cycloal
  • Exemplary 'substituted alkoxy' groups include, but are not limited to, -0-(CH 2 ) t (C6-Cio aryl), -O-(CH 2 ) t (5-10 membered heteroaryl), -O-(CH 2 ) t (C 3 -Ci 0 cycloalkyl), and -O-(CH 2 ) t (4-10 membered heterocyclyl), wherein t is an integer from 0 to 4 and any aryl, heteroaryl, cycloalkyl or heterocyclyl groups present, may themselves be substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C 1-C4 haloalkyl, unsubstituted C 1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy.
  • Particular exemplary 'substituted alkoxy' groups are -OCF 3 , -OCH 2 CF 3 , -OCH 2 Ph, -OCH 2 -cyclopropyl, -OCH 2 CH 2 OH, and -OCH 2 CH 2 NMe 2 .
  • Amino refers to the radical -NH 2 .
  • Substituted amino refers to an amino group of the formula -N(R" 8 ) 2 wherein R 38 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstitued alkenyl, substituted or unsubstitued alkynyl, substituted or unsubstitued carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstitued heteroaryl, or an amino protecting group, wherein at least one of R 38 is not a hydrogen.
  • each R 38 is independently selected from: hydrogen, Ci-Cs alkyl, C3-C8 alkenyl, C3-C8 alkynyl, C6-C10 aryl, 5-10 membered heteroaryl, 4-10 membered heterocyclyl, or C3-C1 0 cycloalkyl; or Ci-Cs alkyl, substituted with halo or hydroxy; C3-C8 alkenyl, substituted with halo or hydroxy; C3-C8 alkynyl, substituted with halo or hydroxy, or -(CH 2 ) t (C6-Cio aryl), -(CH 2 ) t (5-10 membered heteroaryl), - (CH 2 ) t (C 3 -C !
  • cycloalkyl or -(CH 2 ) t (4-10 membered heterocyclyl), wherein t is an integer between 0 and 8, each of which is substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy; or both R 38 groups are joined to form an alkylene group.
  • Exemplary 'substituted amino' groups are -NR 39 -Ci-C 8 alkyl, -NR 39 -(CH 2 )t(C 6 -Cio aryl), -NR 9 -(CH 2 ) t (5-10 membered heteroaryl), -NR 39 -(CH 2 ) t (C 3 -Cio cycloalkyl), and -NR 39 - (CH 2 ) t (4-10 membered heterocyclyl), wherein t is an integer from 0 to 4, for instance 1 or 2, each R 39 independently represents H or Q-Cs alkyl and any alkyl groups present, may themselves be substituted by halo, substituted or unsubstituted amino, or hydroxy; and any aryl, heteroaryl, cycloalkyl, or heterocyclyl groups present, may themselves be substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4
  • substituted amino includes the groups alkylamino, substituted alkylamino, alkylarylamino, substituted alkylarylamino, arylamino, substituted arylamino, dialkylamino, and substituted dialkylamino as defined below.
  • Substituted amino encompasses both monosubstituted amino and disubstituted amino groups.
  • Carbamoyl or “amido” refers to the radical -C(0)NH 2 .
  • Substituted carbamoyl or “substituted amido” refers to the radical -C(0)N(R 62 ) 2 wherein each R 62 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstitued alkenyl, substituted or unsubstitued alkynyl, substituted or unsubstitued carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstitued heteroaryl, or an amino protecting group, wherein at least one of R 62 is not a hydrogen.
  • R 62 is selected from H, Ci-Cs alkyl, C3-C1 0 cycloalkyl, 4- 10 membered heterocyclyl, C6-C1 0 aryl, aralkyl, 5-10 membered heteroaryl, and heteroaralkyl; or Ci-Cs alkyl substituted with halo or hydroxy; or C3-C1 0 cycloalkyl, 4-10 membered heterocyclyl, C6-C1 0 aryl, aralkyl, 5-10 membered heteroaryl, or heteroaralkyl, each of which is substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy; provided that at
  • Exemplary 'substituted carbamoyl' groups include, but are not limited to, -C(O) NR 64 - Ci-C 8 alkyl, -C(O)NR 64 -(CH 2 ) t (C 6 -Ci 0 aryl), -C(O)N 64 -(CH 2 ) t (5-10 membered heteroaryl), - C(0)NR 64 -(CH 2 ) t (C 3 -Cio cycloalkyl), and -C(O)NR 64 -(CH 2 ) t (4-10 membered heterocyclyl), wherein t is an integer from 0 to 4, each R 64 independently represents H or Ci-Cs alkyl and any aryl, heteroaryl, cycloalkyl or heterocyclyl groups present, may themselves be substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstit
  • Carboxy' refers to the radical -C(0)OH.
  • Cyano refers to the radical -CN.
  • Halo or "halogen” refers to fluoro (F), chloro (CI), bromo (Br), and iodo (I).
  • the halo group is either fluoro or chloro. In further embodiments, the halo group is iodo.
  • Haldroxy refers to the radical -OH.
  • Niro refers to the radical -N0 2 .
  • Cycloalkylalkyl refers to an alkyl radical in which the alkyl group is substituted with a cycloalkyl group.
  • Typical cycloalkylalkyl groups include, but are not limited to,
  • cyclopropylmethyl cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, cyclooctylmethyl, cyclopropylethyl, cyclobutylethyl, cyclopentylethyl, cyclohexylethyl, cycloheptylethyl, and cyclooctylethyl, and the like.
  • Heterocyclylalkyl refers to an alkyl radical in which the alkyl group is substituted with a heterocyclyl group.
  • Typical heterocyclylalkyl groups include, but are not limited to, pyrrolidinylmethyl, piperidinylmethyl, piperazinylmethyl, morpholinylmethyl, pyrrolidinylethyl, piperidinylethyl, piperazinylethyl, morpholinylethyl, and the like.
  • Cycloalkenyl refers to substituted or unsubstituted carbocyclyl group having from 3 to 10 carbon atoms and having a single cyclic ring or multiple condensed rings, including fused and bridged ring systems and having at least one and particularly from 1 to 2 sites of olefinic unsaturation.
  • Such cycloalkenyl groups include, by way of example, single ring structures such as cyclohexenyl, cyclopentenyl, cyclopropenyl, and the like.
  • Fused cycloalkenyl refers to a cycloalkenyl having two of its ring carbon atoms in common with a second aliphatic or aromatic ring and having its olefinic unsaturation located to impart aromaticity to the cycloalkenyl ring.
  • Ethylene refers to substituted or unsubstituted -(C-C)-.
  • Nonrogen-containing heterocyclyl means a 4- to 7- membered non-aromatic cyclic group containing at least one nitrogen atom, for example, but without limitation, morpholine, piperidine (e.g. 2-piperidinyl, 3-piperidinyl and 4-piperidinyl), pyrrolidine (e.g. 2- pyrrolidinyl and 3-pyrrolidinyl), azetidine, pyrrolidone, imidazoline, imidazolidinone, 2- pyrazoline, pyrazolidine, piperazine, and N-alkyl piperazines such as N-methyl piperazine.
  • piperidine e.g. 2-piperidinyl, 3-piperidinyl and 4-piperidinyl
  • pyrrolidine e.g. 2- pyrrolidinyl and 3-pyrrolidinyl
  • azetidine pyrrolidone
  • imidazoline imidazolidinone
  • 2- pyrazoline pyr
  • Particular examples include azetidine, piperidone and piperazone.
  • Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaiyl groups, as defined herein, are optionally substituted (e.g., "substituted” or "unsubstituted” alkyl,
  • substituted means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
  • a "substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position.
  • substituted is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein that results in the formation of a stable compound.
  • the present invention contemplates any and all such combinations in order to arrive at a stable compound.
  • heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
  • each instance of R aa is, independently, selected from CL_LO alkyl, Ci_io perhaloalkyl, C 2 _io alkenyl, C 2 _i 0 alkynyl, C 3 -10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two R aa groups are joined to form a 3-14 membered heterocyclyl or 5- 14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1 , 2, 3, 4, or 5 R dd groups;
  • each instance of R cc is, independently, selected from hydrogen, Ci_io alkyl, Ci_io perhaloalkyl, C 2 -io alkenyl, C2-10 alkynyl, C3_io carbocyclyl, 3-14 membered heterocyclyl, C 14 aryl, and 5-14 membered heteroaryl, or two R cc groups are joined to form a 3-14 membered heterocyclyl or 5- 14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1 , 2, 3, 4, or 5 R dd groups;
  • each instance of R ff is, independently, selected from hydrogen, Ci_ 6 alkyl, Ci_ 6 perhaloalkyl, C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, C6-10 aryl and 5-10 membered heteroaryl, or two R ff groups are joined to form a 3-14 membered heterocyclyl or 5- 14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ss groups; and each instance of R ss is, independently, halogen, -CN, -N0 2 , -N 3 , -S0 2 H, -SO3H, -OH, -OCi_ 6 alkyl, -ON(d_ 6 alkyl) 2 , -N
  • a "counterion” or “anionic counterion” is a negatively charged group associated with a cationic quaternary amino group in order to maintain electronic neutrality.
  • exemplary counterions include halide ions (e.g., F ⁇ CI “ , Br “ , ⁇ ), N0 3 ⁇ , C10 4 , OFT, H 2 P0 4 HS0 4 sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene- 1 -sulfonic acid-5-sulfonate, ethan-1 -sulfonic acid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate, ethanoate, propanoate,
  • Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quarternary nitrogen atoms.
  • the substituent present on a nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group).
  • Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • Nitrogen protecting groups such as carbamate groups include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9- (2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7— di— i— butyl-[9-(10,10-dioxo-10, 10,10, 10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4- methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2- trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), l-(l-adamantyl)-l- methyl
  • cyclopropylmethyl carbamate 7-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1, l-dimethyl-3-(N,N- dimethylcarboxamido)propyl carbamate, 1, 1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p '-methoxyphenylazo)benzyl carbamate, 1- methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1 -methyl- 1 -cyclopropylmethyl carbamate, l-methyl-l-(3,5-dimethoxypheny
  • Nitrogen protecting groups such as sulfonamide groups include, but are not limited to, -toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4- methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl- 4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyW-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6- dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6- sulfonamide (Pmc), methan
  • nitrogen protecting groups include, but are not limited to, phenothiazinyl-(lO)- acyl derivative, N - -toluenesulfonylaminoacyl derivative, N'-phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2- one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5- dimethylpyrrole, N-l , l ,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5- substituted l,3-dimethyl-l ,3,5-triazacyclohexan-2-one, 5-substituted l,3-dibenzyl-l,3,5- triazacyclohexan-2
  • diphenylphosphinamide Dpp
  • dimethylthiophosphinamide Mpt
  • diphenylthiophosphinamide Ppt
  • dialkyl phosphoramidates dibenzyl phosphoramidate, diphenyl phosphoramidate
  • benzenesulfenamide o-nitrobenzenesulfenamide (Nps)
  • 2,4-dinitrobenzenesulfenamide pentachlorobenzenesulfenamide, 2-nitro ⁇ l-methoxybenzenesulfenamide
  • the substituent present on an oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group).
  • Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), ?-butylthiomethyl,
  • benzisothiazolyl S,S-dioxido trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t- butyldimethylsilyl (TBDMS), ?-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), ?-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, -chlorophenoxy
  • the substituent present on an sulfur atom is an sulfur protecting group (also referred to as a thiol protecting group).
  • Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • “Pharmaceutically acceptable” means approved or approvable by a regulatory agency of the Federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans.
  • “Pharmaceutically acceptable salt” refers to a salt of a compound of the invention that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.
  • such salts are non-toxic may be inorganic or organic acid addition salts and base addition salts.
  • such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pymvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2 -hydroxy ethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenes
  • Salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the compound contains a basic functionality, salts of non toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.
  • pharmaceutically acceptable cation refers to an acceptable cationic counter-ion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like (see, e.g., Berge, et ah, J. Pharm. Sci. 66(1): 1-79 (Jan.”77) .
  • “Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered.
  • “Pharmaceutically acceptable metabolically cleavable group” refers to a group which is cleaved in vivo to yield the parent molecule of the structural Formula indicated herein.
  • Examples of metabolically cleavable groups include -COR, -COOR,-CONRR and -CH 2 OR radicals, where R is selected independently at each occurrence from alkyl, trialkylsilyl, carbocyclic aryl or carbocyclic aryl substituted with one or more of alkyl, halogen, hydroxy or alkoxy.
  • Specific examples of representative metabolically cleavable groups include acetyl, methoxycarbonyl, benzoyl, methoxymethyl and trimethylsilyl groups.
  • Prodrugs refers to compounds, including derivatives of the compounds of the invention,which have cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention that are pharmaceutically active in vivo. Such examples include, but are not limited to, choline ester derivatives and the like, N- alkylmorpholine esters and the like. Other derivatives of the compounds of this invention have activity in both their acid and acid derivative forms, but in the acid sensitive form often offers advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, FL, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985).
  • Prodrugs include acid derivatives well know to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides.
  • Simple aliphatic or aromatic esters, amides and anhydrides derived from acidic groups pendant on the compounds of this invention are particular prodrugs.
  • double ester type prodrugs such as (acyloxy)alkyl esters or
  • ((alkoxycarbonyl)oxy)alkylesters Particularly the Ci to Cg alkyl, C2-C 8 alkenyl, C2-C 8 alkynyl, aryl, C7-C12 substituted aryl, and C7-C12 arylalkyl esters of the compounds of the invention.
  • Solvate refers to forms of the compound that are associated with a solvent or water (also referred to as "hydrate”), usually by a solvolysis reaction. This physical association includes hydrogen bonding.
  • solvents include water, ethanol, acetic acid and the like.
  • the compounds of the invention may be prepared e.g. in crystalline form and may be solvated or hydrated.
  • Suitable solvates include pharmaceutically acceptable solvates, such as hydrates, and further include both stoichiometric solvates and non-stoichiometric solvates. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid.
  • “Solvate” encompasses both solution-phase and isolable solvates. Representative solvates include hydrates, ethanolates and methanolates.
  • a "subject" to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g, infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or a non- human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs.
  • the subject is a human.
  • the subject is a non-human animal.
  • the terms "human", “patient” and “subject” are used interchangeably herein.
  • “Therapeutically effective amount” means the amount of a compound that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease.
  • the “therapeutically effective amount” can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated.
  • Preventing refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject not yet exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.
  • prophylaxis is related to "prevention", and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease.
  • prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.
  • Treating” or “treatment” of any disease or disorder refers, in certain embodiments, to ameliorating the disease or disorder (i.e., arresting the disease or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof).
  • “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the subject.
  • “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.
  • “treating” or “treatment” relates to slowing the progression of the disease.
  • isotopic variant refers to a compound that contains unnatural proportions of isotopes at one or more of the atoms that constitute such compound.
  • an “isotopic variant” of a compound can contain one or more non-radioactive isotopes, such as for example, deuterium ( 2 H or D), carbon-13 ( 13 C), nitrogen-15 ( 15 N), or the like.
  • the following atoms, where present, may vaiy, so that for example, any hydrogen may be 2 H/D, any carbon may be L, C, or any nitrogen may be 15 N, and that the presence and placement of such atoms may be determined within the skill of the art.
  • the invention may include the preparation of isotopic variants with radioisotopes, in the instance for example, where the resulting compounds may be used for drug and/or substrate tissue distribution studies.
  • the radioactive isotopes tritium, i.e., 3 H, and carbon-14, i.e., 14 C, are particularly useful for this purpose in view of their ease of incoiporation and ready means of detection.
  • compounds may be prepared that are substituted with positron emitting isotopes, such as n C, 18 F, 15 0 and 13 N, and would be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. All isotopic variants of the compounds provided herein, radioactive or not, are intended to be encompassed within the scope of the invention.
  • enantiomers Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”.
  • An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e. , as (+) or (-)-isomers respectively).
  • a chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a "racemic mixture".
  • Tautomers refer to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of ⁇ electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Another example of tautomerism is the aci- and nitro- forms of phenylnitromethane, which are likewise formed by treatment with acid or base.
  • Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.
  • a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess).
  • an "S” form of the compound is substantially free from the "R” form of the compound and is, thus, in enantiomeric excess of the "R” form.
  • enantiomerically pure or “pure enantiomer” denotes that the compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 98.5% by weight, more than 99% by weight, more than 99.2% by weight, more than 99.5% by weight, more than 99.6% by weight, more than 99.7% by weight, more than 99.8% by weight or more than 99.9% by weight, of the enantiomer.
  • the weights are based upon total weight of all enantiomers or stereoisomers of the compound.
  • the term “enantiomerically pure R- compound” refers to at least about 80% by weight R-compound and at most about 20% by weight S-compound, at least about 90% by weight R-compound and at most about 10% by weight S-compound, at least about 95% by weight R-compound and at most about 5% by weight S-compound, at least about 99% by weight R-compound and at most about 1% by weight S- compound, at least about 99.9% by weight R-compound or at most about 0.1% by weight S- compound.
  • the weights are based upon total weight of compound.
  • the term “enantiomerically pure S- compound” or “S-compound” refers to at least about 80% by weight S-compound and at most about 20% by weight R-compound, at least about 90% by weight S-compound and at most about 10% by weight R-compound, at least about 95% by weight S-compound and at most about 5% by weight R-compound, at least about 99% by weight S-compound and at most about 1% by weight R-compound or at least about 99.9% by weight S-compound and at most about 0.1% by weight R-compound.
  • the weights are based upon total weight of compound.
  • an enantiomerically pure compound or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof can be present with other active or inactive ingredients.
  • a pharmaceutical composition comprising enantiomerically pure R-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure R-compound.
  • the enantiomerically pure R- compound in such compositions can, for example, comprise, at least about 95% by weight R- compound and at most about 5% by weight S-compound, by total weight of the compound.
  • a pharmaceutical composition comprising enantiomerically pure S-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure S-compound.
  • the enantiomerically pure S-compound in such compositions can, for example, comprise, at least about 95% by weight S-compound and at most about 5% by weight R-compound, by total weight of the compound.
  • the active ingredient can be formulated with little or no excipient or carrier.
  • the compounds of this invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)- stereoisomers or as mixtures thereof.
  • heterocyclic ring may have one to four heteroatoms so long as the heteroaromatic ring is chemically feasible and stable.
  • compositions comprising of a bolaamphiphile complex.
  • novel nano-sized vesicles comprising of bol aamphiphi!ie compounds .
  • novel nano-sized vesicles comprising of bolaamphiphilic compounds which are capable of encapsulating NTF, GDNF or NGF.
  • the vesicles formed from the bolaamphiphiles to encapsulating NTF, GDNF or NGF contain additives that help to stabilize the vesicles, by stabilizing the vesicle's membranes, such as but not limited to cholesterol derivatives such as cholesteryl hemisuccinate and cholesterol itself and combinations such as cholesteryl hemisuccinate and cholesterol.
  • the vesicles in addition to these components have another addiitves which decorates the outer vesicle memrbanes with groups or pendants that enhance penetration though biological barriers such as the BBB, or groups for targeting to specific sites such as dopaminergic neurons.
  • the bolaamphiphile head groups that comprise the vesicles membranes can interact with the neuro active agents such as GDNF or NDF to be delivered in to the brain and brain sites ionic interactions to enhance the % encapsulation via complexation and well as passive encapsulation within the vesicles core.
  • the formuation may contain other additives within the veieles membranes to futhjer enhance the degree of encapsulation of neuro active agents such as GDNF or NDF.
  • the pH in which the vesicle formation and encapsulation of the neuro active agent such as GDNF or NDF is such as to maximize the electrostatic or inonic interactions between the said agents and the sai bolaamphiphiles and or additives to maximize the % encapsulation.
  • novel nano-sized bola vesicles described above that encapsulate GDNF or NGF and are capable of delivering the encapsulated material into the brain.
  • novel nano-sized bola vesicles that encapsulate GDNF or NGF and are capable of delivering the encapsulated material to the brain, specifically to dopaminergic neurons.
  • novel nano-sized bola vesicles that encapsulate GDNF or NGF and are capable of delivering the encapsulated material into brain regions affected in neurological disorders.
  • the neurological disorder is Parkinson's disease (PD) or Alzheimer's disease (AD).
  • novel bolaamphiphile complexes comprising bolaamphiphilic compounds and a compound active against PD.
  • the compound active against PD is GDNF.
  • novel bolaamphiphile complexes comprising bolaamphiphilic compounds and a compound active against AD.
  • the compound active against AD is NGF.
  • novel formulations of GDNF or NGF with bolaamphiphilic compounds or with bolaamhphile vesicles are provided herein.
  • methods of delivering GDNF or NGF agents into animal or human brain comprises the step of administering to the animal or human a pharmaceutical composition comprising of a bolaamphiphile complex; and wherein the bolaamphiphile complex comprises a bolaamphiphilic compound and GDNF.
  • the complex comprises bolaamphiphilic compound and NGF.
  • the bolaamphiphilic compound consists of two hydrophilic headgroups linked through a long hydrophobic chain.
  • the hydrophilic headgroup is an amino containing group.
  • the hydrophilic headgroup is a tertiary or quaternary amino containing group.
  • the bolaamphiphilic compound is a compound according to formula I:
  • each HG 1 and HG 2 is independently a hydrophilic head group
  • L 1 is alkylene, alkenyl, heteroalkylene, or heteroalkenyl linker; unsubstituted or substituted with C1-C2 0 alkyl, hydroxyl, or oxo.
  • the pharmaceutically acceptable salt is a quaternary ammonium salt.
  • L 1 is heteroalkylene, or heteroalkenyl linker comprising C, N, and O atoms; unsubstituted or substituted with C1-C2 0 alkyl, hydroxyl, or oxo.
  • each L 2 and L 3 is C4-C2 0 alkenyl linker; unsubstituted or substituted with C i-Cs alkyl or hydroxy;
  • n4 is independently an integer from 4-20.
  • L 1 is -0-(CH 2 ) nl -0-C(0)-(CH 2 ) n2 -C(0)-0-(CH 2 ) n3 -0-.
  • each Z 1 and Z 2 is independently -C(R 3 ) 2 -, -N(R 3 )- or -0-;
  • each R la , R lb , R 3 , and R 4 is independently H or Ci-Cg alkyl
  • each R 2a and R 2b is independently H , Ci-C 8 alkyl, OH, or alkoxy;
  • each n8, n9, ni l, and nl2 is independently an integer from 1-20; nlO is an integer from 2-20; and
  • each dotted bond is independently a single or a double bond.
  • each methylene carbon is unsubstituted or substituted with C1-C4 alkyl; and each nl, n2, and n3 is independently an integer from 4-20.
  • the bolaamphiphilic compound is a compound according to formula II, III, IV, V, or VI:
  • each HG 1 and HG 2 is independently a hydrophilic head group
  • each Z 1 and Z 2 is independently -C(R 3 ) 2 -, -N(R 3 )- or -0-;
  • each R la , R lb , R 3 , and R 4 is independently H or d-C 8 alkyl
  • each R 2a and R 2b is independently H , Ci-C 8 alkyl, OH, alkoxy, or O-HG 1 or O-HG 2 ; each 118, n9, nl 1, and nl2 is independently an integer from 1-20;
  • nlO is an integer from 2-20;
  • each dotted bond is independently a single or a double bond.
  • each n9 and nl 1 is independently an integer from 2-12. In another embodiment, n9 and nl 1 is independently an integer from 4-8. In a particular embodiment, each n9 and nl 1 is 7 or 11.
  • each n8 and nl2 is independently 1, 2, 3, or 4. In a particular embodiment, each n8 and nl2 is 1.
  • each R 2a and R 2b is independently H, OH, or alkoxy.
  • each R 2a and R 2b is independently H, OH, or OMe.
  • each R 2a and R 2b is independently-O-HG 1 or O-HG 2 .
  • each R 2a and R 2b is OH.
  • each R la and R lb is independently H, Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, sec-Bu, n- pentyl, isopentyl, n-hexyl, n-heptyl, or n-octyl.
  • each R la and R lb is independently n-pentyl.
  • each dotted bond is a single bond. In another embodiment, each dotted bond is a double bond.
  • nlO is an integer from 2-16. In another embodiment, nlO is an integer from 2-12. In a particular embodiment, nlO is 2, 4, 6, 8, 10, 12, or 16.
  • R 4 is H, Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, sec-Bu, n-pentyl, or isopentyl. In another embodiment, R 4 is Me, or Et. In a particular embodiment, R 4 is Me. [00199] In one embodiment, with respect to the bolaamphiphilic compound of formula II, III, IV, V, or VI, each Z 1 and Z 2 is independently C(R 3 )2-, or -N(R 3 )-.
  • each Z 1 and Z 2 is independently C(R 3 ) 2 -, or -N(R 3 )-; and each R 3 is independently H, Me, Et, n-Pr, i- Pr, n-Bu, i-Bu, sec-Bu, n-pentyl, or isopentyl.
  • R" is H.
  • each Z 1 and Z 2 is -0-.
  • each HG 1 and HG 2 is independently selected from:
  • X is -NR 5a R 5b , or -N + R 5a R 5b R 5c ; each R 5a , and R 5b is independently H or substituted or unsubstituted C1-C2 0 alkyl or R 5a and R 5b may join together to form an N containing substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclyl;
  • each R Sc is independently substituted or unsubstituted C1-C20 alkyl; each R 8 is independently
  • ml is 0 or 1
  • each nl3, nl4, and nl5 is independently an integer from 1-20.
  • HG 1 and HG 2 are as defined above, and each ml is 0.
  • HG 1 and HG 2 are as defined above, and each ml is 1.
  • HG 1 and HG 2 are as defined above, and each nl3 is 1 or 2.
  • HG 1 and HG 2 are as defined above, and each nl4 and nl5 is independently 1, 2, 3, 4, or 5. In another embodiment, each nl4 and nl5 is independently 2 or 3.
  • the bolaamphiphilic compound is a compound according to formula Vila, Vllb, VIIc, or Vlld:
  • each X is -NR 5a R 5b , or -N + R 5a R 5b R 5c ; each R 5a , and R 5b is independently H or substituted or unsubstituted Ci-C 2 o alkyl or R 5a and R 5b may join together to form an N containing substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclyl;
  • each R 5c is independently substituted or unsubstituted Ci-C 2 o alkyl;
  • nlO is an integer from 2-20;
  • each dotted bond is independently a single or a double bond.
  • the bolaamphiphilic compound is a compound according to formula Villa, Vlllb, VIIIc, or VHId:
  • each X is -NR 5a R 5b , or -N + R 5a R 5b R 5c ; each R 5a , and R 5b is independently H or substituted or unsubstituted Ci-C 2 o alkyl or R 5a and R 5b may join together to form an N containing substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclyl;
  • each R 5c is independently substituted or unsubstituted Ci-C 2 o alkyl;
  • nlO is an integer from 2-20;
  • each dotted bond is independently a single or a double bond.
  • the bolaamphiphilic compound is a compound according to formula IXa, IXb, or IXc:
  • IXc or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or a combination thereof;
  • each X is -NR 5a R 5b , or -N + R 5a R 5b R 5c ; each R 5a , and R 5b is independently H or substituted or unsubstituted Ci-C 2 o alkyl or R 5a and R 5b may join together to form an N containing substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclyl;
  • each R 5c is independently substituted or unsubstituted Ci-C 2 o alkyl;
  • nlO is an integer from 2-20;
  • each dotted bond is independently a single or a double bond.
  • the bolaamphiphilic compound is a compound according to formula Xa, Xb, or Xc:
  • Xc or a pharmaceutically acceptable salt, solvate, hydrate, prodmg, stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or a combination thereof;
  • each X is -NR 5a R 5b , or -N + R 5a R 5b R 5c ; each R 5a , and R 5b is independently H or substituted or unsubstituted C1-C2 0 alkyl or R 5a and R 5b may join together to form an N containing substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclyl;
  • each R 5c is independently substituted or unsubstituted C1-C2 0 alkyl;
  • nlO is an integer from 2-20;
  • each dotted bond is independently a single or a double bond.
  • each dotted bond is a single bond. In another embodiment, each dotted bond is a double bond.
  • nlO is an integer from 2-16.
  • nlO is an integer from 2-12.
  • nlO is 2, 4, 6, 8, 10, 12, or 16.
  • each R 5a , R 5b , and R Sc is independently substituted or unsubstituted C1-C20 alkyl.
  • each R 5a , R 5b , and R 5c is independently unsubstituted d- C 20 alkyl.
  • R 5a , R 5b , and R 5c is C1-C20 alkyl substituted with - OC(0)R 6 ; and R 6 is C 1 -C20 alkyl.
  • R 5a , R 5b , and R 5c are independently C1-C20 alkyl substituted with -OC(0)R 6 ; and R 6 is C 1 -C 20 alkyl.
  • R 6 is Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, sec-Bu, n-pentyl, isopentyl, n-hexyl, n-heptyl, or n-octyl.
  • R 6 is Me.
  • R 5a , R 5b , and R 5c is C1-C20 alkyl substituted with amino, alkylamino or dialkylamino.
  • R 5a , R 5b , and R 5c are independently C1-C20 alkyl substituted with amino, alkylamino or dialkylamino.
  • X is -NMe 2 or -N + Me 3 .
  • X is -N(Me)-CH 2 CH 2 -OAc or -N + (Me) 2 -CH 2 CH 2 -OAc.
  • X is a chitosanyl group; and the chitosanyl group is a poly-(D)glucosaminyl group with MW of 3800 to 20,000 Daltons, and is attached to the core via N.
  • each pi and p2 is independently an integer from 1-400; and each R 7a is H or acyl.
  • X is a mannose group.
  • X is a maleimide group.
  • the bolaamphiphilic compound is a pharmaceutically acceptable salt.
  • the bolaamphiphilic compound is in a form of a quaternary salt.
  • the bolaamphiphilic compound is in a form of a quaternary salt with pharmaceutically acceptable alkyl halide or alkyl tosylate.
  • the bolaamphiphilic compound is any one of the bolaambphilic compounds listed in Table 1.
  • the bolavesicle comporises one or more bolaamphilic compounds described herein.
  • GDNF brain-targeted drug delivery using the bolavesicles incorporated with GDNF.
  • GDNF GDNF
  • nano-particles comprising one or more bolaamphiphilic compounds and GDNF.
  • the bolaamphiphilic compounds and GDNF are encapsulated within the nano-particle.
  • compositions comprising a nano-sized particle comprising one or more bolaamphiphilic compounds and GDNF; and a pharmaceutically acceptable carrier.
  • provided herein are methods for incorporating NGF in the bolavesicles.
  • the bolavesicle comporises one or more bolaamphilic compounds described herein.
  • methods for brain-targeted drug delivery using the bolavesicles incorporated with NGF are provided herein.
  • nano-particles comprising one or more bolaamphiphilic compounds and NGF.
  • the bolaamphiphilic compounds and NGF are encapsulated within the nano-particle.
  • compositions comprising a nano-sized particle comprising one or more bolaamphiphilic compounds and NGF; and a pharmaceutically acceptable carrier.
  • the bolaamphiphilic compound is other than Comound ID GLH- 16, GLH-19, GLH-20, GLH-26, GLH-29, or GLH-41.
  • the bolaamphiphilic compound is other than Comound ID GLH-6, GLH-8, GLH-12, GLH-13, GLH-13a, or GLH-49 to GLH-54 (all can be used as intermediates for bolaamphiphiles).
  • composition of novel bolaamphiphilic compounds wherein the bolaamphiphilic compound is selected from the bolaambphilic compounds listed in Table 1.
  • the bolaamphiphilic compound is other than Comound ID GLH-16, GLH- 19, GLH-20, GLH-26, GLH-29, or GLH-41.
  • the compound is other than compound with ID GLH-3, GLH-4, GLH-5, or GLH-21.
  • bolaamphiphilic compound is selected from the bolaambphilic compounds listed in Table 1 , and the compound is compound with ID GLH-7, GLH-9, GLH- 10, GLH- 1 1 , GLH-14, GLH- 15, GLH- 17, GLH- 18, GLH-22, GLH-23, GLH-24, GLH-25, GLH-27, GLH-28, GLH-30 to GLH-48, GLH-55, GLH-56, or GLH-57.
  • the bolaamphiphilic compound of formula I, II, III, IV, V, VI, Vlla-VIIc, Villa- VIIIc, IXa-IXc and Xa-Xc is Comound ID GLH-19, or GLH-20.
  • the bolaamphiphilic compound of formula I, II, III, IV, V, VI, Vlla-VIIc, Villa- VIIIc, IXa-IXc and Xa-Xc the bolaamphiphilic compound is Comound ID GLH-16, GLH-26, GLH-29, or GLH-41.
  • the syntheses can involve initial construction of, for example, vernonia oil or direct functionalization of natural derivatives by organic synthesis manipulations such as, but not limiting to, epoxide ring opening.
  • organic synthesis manipulations such as, but not limiting to, epoxide ring opening.
  • the epoxy group is opened by the addition of reagents such as carboxylic acids or organic or inorganic nucleophiles.
  • Such ring opening results in a mixture of two products in which the new group is introduced at either of the two carbon atoms of the epoxide moiety.
  • This provides beta substituted alcohols in which the substitution position most remote from the CO group of the main aliphatic chain of the vernonia oil derivative is arbitrarily assigned as position 1, while the neighboring substituted carbon position is designated position 2.
  • the Derivatives and Precursors shown herein may indicate structures with the hydroxy group always at position 2 but the Derivatives and Precursors wherein the hydroxy is at position 1 are also encompassed by the invention.
  • a radical of the formula— CH(OH)— CH(R)— refers to the substitution of—OH at either the carbon closer to the CO group, designated position 2 or to the carbon at position 1.
  • vesicles are prepared using the mixture of unfractionated positional isomers.
  • the bola used in vesicle preparation can actually be a mixture of three different positional isomers.
  • the three different derivatives are isolated. Accordingly, the vesicles disclosed herein can be made from a mixture of the three isomers of each derivative or, in other embodiments, the individual isomers can be isolated and used for preparation of vesicles.
  • Symmetrical bolaamphiphiles can form relatively stable self aggregate vesicle structures by the use of additives such as cholesterol and cholesterol derivatives (e.g., cholesterol hemisuccinate, cholesterol oleyl ether, anionic and cationic derivatives of cholesterol and the like), or other additives including single headed amphiphiles with one, two or multiple aliphatic chains such as phospholipids, zwitterionic, acidic, or cationic lipids.
  • zwitterionic lipids are phosphatidylcholines, phosphatidylethanol amines and sphingomyelins.
  • Examples of acidic amphiphilic lipids are phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, and phosphatidic acids.
  • Examples of cationic amphipathic lipids are diacyl trimethylammonium propanes, diacyl dimethylammonium propanes, and stearylamines cationic amphiphiles such as spermine cholesterol carbamates, and the like, in optimum concentrations which fill in the larger spaces on the outer surfaces, and/or add additional hydrophilicity to the particles.
  • Such additives may be added to the reaction mixture during formation of nanoparticles to enhance stability of the nanoparticles by filling in the void volumes of in the upper surface of the vesicle membrane.
  • Stability of nano vesicles according to the present disclosure can be demonstrated by dynamic light scattering (DLS) and transmission electron microscopy (TEM).
  • DLS dynamic light scattering
  • TEM transmission electron microscopy
  • suspensions of the vesicles can be left to stand for 1, 5, 10, and 30 days to assess the stability of the nanoparticle solution/suspension and then analyzed by DLS and TEM.
  • the vesicles disclosed herein may encapsulate within their core the active agent, which in particular embodiments is selected from peptides, proteins, nucleotides and or non- polymeric agents.
  • the active agent is also associated via one or more non-covalent interactions to the vesicular membrane on the outer surface and/or the inner surface, optionally as pendant decorating the outer or inner surface, and may further be incorporated into the membrane surrounding the core.
  • biologically active peptides, proteins, nucleotides or non-polymeric agents that have a net electric charge may associate ionically with oppositely charged headgroups on the vesicle surface and/or form salt complexes therewith.
  • additives which may be used in particular aspects of these embodiments, additives which may be used in particular aspects of these embodiments, additives which may be used in particular.
  • bolaamphiphiles or single headed amphiphiles comprise one or more branching alkyl chains bearing polar or ionic pendants, wherein the aliphatic portions act as anchors into the vesicle's membrane and the pendants (e.g., chitosan derivatives or polyamines or certain peptides) decorate the surface of the vesicle to enhance penetration through various biological barriers such as the intestinal tract and the BBB, and in some instances are also selectively hydrolyzed at a given site or within a given organ.
  • the concentration of these additives is readily adjusted according to experimental determination.
  • the oral formulations of the present disclosure comprise agents that enhance penetration through the membranes of the GI tract and enable passage of intact nanoparticles containing the drug.
  • agents may be any of the additives mentioned above and, in particular aspects of these embodiment, include chitosan and derivatives thereof, serving as vehicle surface ligands, as decorations or pendants on the vesicles, or the agents may be excipients added to the formulation.
  • the nanoparticles and vesicles disclosed herein may comprise the fluorescent marker carboxyfluorescein (CF) encapsulated therein while in particular aspects, the nanoparticle and vesicles of the present disclosure may be decorated with one or more of PEG, e.g. PEG2000-vernonia derivatives as pendants.
  • PEG e.g. PEG2000-vernonia derivatives
  • two kinds of PEG - vernonia derivatives can be used: PEG-ether derivatives, wherein PEG is bound via an ether bond to the oxygen of the opened epoxy ring of, e.g., vernolic acid and PEG-ester derivatives, wherein PEG is bound via an ester bond to the carboxylic group of, e.g., vernolic acid.
  • vesicles made from synthetic amphiphiles, as well as liposomes, made from synthetic or natural phospholipids, substantially (or totally) isolate the therapeutic agent from the environment allowing each vesicle or liposome to deliver many molecules of the therapeutic agent.
  • the surface properties of the vesicle or liposome can be modified for biological stability, enhanced penetration through biological barriers and targeting, independent of the physico-chemical properties of the encapsulated drug.
  • the headgroup is selected from: (i) choline or thiocholine, O-alkyl, N-alkyl or ester derivatives thereof; (ii) non-aromatic amino acids with functional side chains such as glutamic acid, aspartic acid, lysine or cysteine, or an aromatic amino acid such as tyrosine, tryptophan, phenylalanine and derivatives thereof such as levodopa (3,4-dihydroxy-phenylalanine) and p-aminophenylalanine; (iii) a peptide or a peptide derivative that is specifically cleaved by an enzyme at a diseased site selected from enkephalin, N-acetyl- ala-ala, a peptide that constitutes a domain recognized by beta and gamma secretases, and a peptide that is recognized by stromelysins; (iv) saccharides such as glucose,
  • nano-sized particle and vesicles disclosed herein further comprise at least one additive for one or more of targeting purposes, enhancing permeability and increasing the stability the vesicle or particle.
  • additives may selected from from: (i) a single headed amphiphilic derivative comprising one, two or multiple aliphatic chains, preferably two aliphatic chains linked to a midsection/spacer region such as— H— (CH 2 ) 2 ⁇ N ⁇ (CH 2 ) 2 ⁇ N ⁇ , or ⁇ 0 ⁇ (CH 2 ) 2 ⁇ N ⁇ (CH 2 ) 2 ⁇ 0 ⁇ , and a sole headgroup, which may be a selectively cleavable headgroup or one containing a polar or ionic selectively cleavable group or moiety, attached to the N atom in the middle of said midsection.
  • the additive can be selected from among cholesterol and cholesterol derivatives such as cholesteryl hemmisuccinate; phospholipids, zwitterionic, acidic, or cationic lipids; chitosan and chitosan derivatives, such as vernolic acid-chitosan conjugate, quaternized chitosan, chitosan- polyethylene glycol (PEG) conjugates, chitosan-polypropylene glycol (PPG) conjugates, chitosan N-conjugated with different amino acids, carboxyalkylated chitosan, sulfonyl chitosan, carbohydrate-branched N-(carboxymethylidene) chitosan and N-(carboxymethyl) chitosan;
  • cholesterol and cholesterol derivatives such as cholesteryl hemmisuccinate
  • polyamines such as protamine, polylysine or polyarginine
  • ligands of specific receptors at a target site of a biological environment such as nicotine, cytisine, lobeline, 1 -glutamic acid MK801, morphine, enkephalins, benzodiazepines such as diazepam (valium) and librium, dopamine agonists, dopamine antagonists tricyclic antidepressants, muscarinic agonists, muscarinic antagonists, cannabinoids and arachidonyl ethanol amide
  • polycationic polymers such as polyethylene amine
  • peptides that enhance transport through the BBB such as OX 26, transferrins, polybrene, histone, cationic dendrimer, synthetic peptides and polymyxin B nonapeptide (PMBN)
  • monosaccharides such as glucose, mannose, ascorbic acid and derivatives thereof
  • the aforementioned head groups on the additives designed for one or more of targeting memeposes and enhancing permeability may also be a head group, preferably on an asymmetric bolaamphiphile wherein the other head group is another moiety, or the head group on both sides of a symmetrical bolaamphiphile.
  • nano-sized particle and vesicles discloser herein may comprises at least one biologically active agent is selected from: (i) a natural or synthetic peptide or protein such as analgesics peptides from the enkephalin class, insulin, insulin analogs, oxytocin, calcitonin, tyrotropin releasing hormone, follicle stimulating hormone, luteinizing hormone, vasopressin and vasopressin analogs, catalase, interleukin-II, interferon, colony stimulating factor, tumor necrosis factor (TNF), melanocyte-stimulating hormone, superoxide dismutase, glial cell derived neurotrophic factor (GDNF) or the Gly-Leu-Phe (GLF) families; (ii) nucleosides and polynucleotides selected from DNA or RNA molecules such as small interfering RNA (siRNA) or a DNA plasmid; (iii) anti
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of Formula I or a complex thereof.
  • compositions When employed as pharmaceuticals, the compounds provided herein are typically administered in the form of a pharmaceutical composition.
  • Such compositions can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
  • the carrier is a parenteral carrier, oral or topical carrier.
  • the present invention also relates to a compound or pharmaceutical composition of compound according to Formula I; or a pharmaceutically acceptable salt or solvate thereof for use as a pharmaceutical or a medicament.
  • the compounds provided herein are administered in a therapeutically effective amount.
  • the amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
  • compositions provided herein can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal.
  • routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal.
  • the compounds provided herein are preferably formulated as either injectable or oral compositions or as salves, as lotions or as patches all for transdermal administration.
  • compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.
  • the compound is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.
  • Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like.
  • Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • Injectable compositions are typically based upon injectable sterile saline or phosphate- buffered saline or other injectable carriers known in the art.
  • the active compound in such compositions is typically a minor component, often being from about 0.05 to 10% by weight with the remainder being the injectable carrier and the like.
  • Transdermal compositions are typically formulated as a topical ointment or cream containing the active ingredient(s), generally in an amount ranging from about 0.01 to about 20% by weight, preferably from about 0.1 to about 20% by weight, preferably from about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight.
  • the active ingredients When formulated as a ointment, the active ingredients will typically be combined with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with, for example an oil-in-water cream base.
  • Such transdermal formulations are well-known in the art and generally include additional ingredients to enhance the dermal penetration of stability of the active ingredients or the formulation. All such known transdermal formulations and ingredients are included within the scope provided herein.
  • transdermal administration can be accomplished using a patch either of the reservoir or porous membrane type, or of a solid matrix variety.
  • the compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems.
  • sustained release materials can be found in Remington 's Pharmaceutical Sciences.
  • the present invention also relates to the pharmaceutically acceptable formulations of compounds of Formula I.
  • the formulation comprises water.
  • the formulation comprises a cyclodextrin derivative.
  • the formulation comprises hexapropyl-P-cyclodextrin.
  • the formulation comprises hexapropyl-P-cyclodextrin (10-50% in water).
  • the present invention also relates to the pharmaceutically acceptable acid addition salts of compounds of Formula I.
  • the acids which are used to prepare the pharmaceutically acceptable salts are those which form non-toxic acid addition salts, i.e. salts containing pharmacologically acceptable aniovs such as the hydrochloride, hydroiodide, hydrobromide, nitrate, sulfate, bisulfate, phosphate, acetate, lactate, citrate, tartrate, succinate, maleate, fumarate, benzoate, para-toluenesulfonate, and the like.
  • a compound of the invention may be dissolved or suspended in a buffered sterile saline injectable aqueous medium to a concentration of approximately 5 mg/mL.
  • the nano-sized stable vesicles [5,6,7,8,9] can be used to deliver GDNF to the brain.
  • These nano-sized vesicles are made of novel bolaamphiphiles (bolas).
  • the vesicles that these novel bolas form were shown to aggregate into vesicles or nano particleds that cross the BBB and deliver small molecules, peptides and proteins to the brain.
  • Bolas are promising building block candidates for vesicles used as a drug delivery system targeted to the brain, since they can form vesicles with monolayer membranes, which are more stable than liposomes with bilayer membranes, due to the high energy barrier for lipid exchange that characterizes bolas [10].
  • the high stability of such vesicles allows them to circulate in the blood stream until they reach the brain, and then penetrate the BBB in their intact form.
  • the monolayer membrane is thinner than a bilayer membrane, thus providing higher inner volume for encapsulation as compared to vesicles of the same size made of an encapsulating bilayer membrane [11].
  • a controlled release mechanism is more likely to be achieved with vesicles made of bolas that form monolayer membranes, as compared to classical liposomes made of bilayer encapsulating membranes, since monolayer membranes are known to rapidly change their morphology from vesicles to fibers and sheets upon small changes in their surface groups [10].
  • a controlled release mechanism should allow release of the encapsulated material only after the vesicles penetrate into the brain, thus preventing leakage in non relevant tissues. Indeed, the vesicles made from bolas do cross the BBB, transport encapsulated small molecules, peptides and proteins into the brain and release them primarily there.
  • This novel delivery system can be an effective delivery system for GDNF, and has the potential to be used in the treatment of PD, since it can distribute the NTF within a wide brain area and, thus, can positively affect degenerating neurons throughout the brain.
  • the resulting bola-GDNF delivery systems or formulations may be capable of delivering other neurotrophic factors for the treatment of several neurodegenerative diseases, particularly PD.
  • various PD active drug molecueles such as GDNF
  • GDNF can be encapsulated in the bolaamphiphilic vesicles and then delivered to the brain in sufficient concentrations for therapeutic use.
  • NGF may contain additives that help to stabilize the vesicles, by stabilizing the vesicle's membranes, such as but not limited to cholesterol derivatives such as cholesteryl hemisuccinate and cholesterol itself and combinations such as cholesteryl hemisuccinate and cholesterol.
  • the bola vesicles in addition to these components have another addiitves which decorates the outer vesicle memrbanes with groups or pendants that enhance penetration though biological barriers such as the BBB, or groups for targeting to specific sites such as dopaminergic neurons.
  • the bolaamphiphile head groups that comprise the vesicles membranes can interact with the neuro active agents such as GDNF or NDF to be delivered in to the brain and brain sites ionic interactions to enhance the % encapsulation via complexation and well as passive encapsulation within the vesicles core.
  • the formuation may contain other additives within the veicles membranes to futhjer enhance the degree of encapsulation of neuro active agents such as GDNF or NDF.
  • the pH in which the vesicle formation and encapsulation of the neuro active agent such as GDNF or NDF is such as to maximize the electrostatic or inonic interactions between the said agents and the sai bolaamphiphiles and or additives to maximize the % encapsulation.
  • the compounds provided herein may be isolated and purified by known standard procedures. Such procedures include (but are not limited to) recrystallization, column chromatography or HPLC. The following schemes are presented with details as to the preparation of representative substituted biarylamides that have been listed herein.
  • the compounds provided herein may be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis.
  • the bolaamphiphile compounds may be used as racemic mixtures or mixtures of geometric isomors such as cis or trans, or as mixtures of geometric isomers unless otherwise specified as being enantiomerically pure compounds.
  • enantiomerically pure compounds thay may be provided herein may be prepared according to any techniques known to those of skill in the art. For instance, they may be prepared by chiral or asymmetric synthesis from a suitable optically pure precursor or obtained from a racemate by any conventional technique, for example, by chromatographic resolution using a chiral column, TLC or by the preparation of diastereoisomers, separation thereof and regeneration of the desired enantiomer. See, e.g., "Enantiomers, Racemates and Resolutions," by J. Jacques, A. Collet, and S.H. Wilen, (Wiley-Interscience, New York, 1981); S.H. Wilen, A. Collet, and J.
  • an enantiomerically pure compound of formula (1) may be obtained by reaction of the racemate with a suitable optically active acid or base.
  • suitable acids or bases include those described in Bighley et ah, 1995, Salt Forms of Drugs and Adsorption, in Encyclopedia of Pharmaceutical Technology, vol. 13, Swarbrick & Boylan, eds., Marcel Dekker, New York; ten Hoeve & H. Wynberg, 1985, Journal of Organic Chemistry 50:4508-4514; Dale & Mosher, 1973, J Am. Chem. Soc. 95:512; and CRC Handbook of Optical Resolution via Diastereomeric Salt Formation, the contents of which are hereby incorporated by reference in their entireties.
  • Enantiomerically pure compounds can also be recovered either from the crystallized diastereomer or from the mother liquor, depending on the solubility properties of the particular acid resolving agent employed and the particular acid enantiomer used.
  • the identity and optical purity of the particular compound so recovered can be determined by polarimetry or other analytical methods known in the art.
  • the diasteroisomers can then be separated, for example, by chromatography or fractional crystallization, and the desired enantiomer regenerated by treatment with an appropriate base or acid.
  • the other enantiomer may be obtained from the racemate in a similar manner or worked up from the liquors of the first separation.
  • enantiomerically pure compound can be separated from racemic compound by chiral chromatography.
  • Various chiral columns and eluents for use in the separation of the enantiomers are available and suitable conditions for the separation can be empirically determined by methods known to one of skill in the art.
  • Exemplary chiral columns available for use in the separation of the enantiomers provided herein include, but are not limited to CHIRALCEL® OB, CHIRALCEL® OB-H, CHIRALCEL® OD, CHIRALCEL® OD-H, CHIRALCEL® OF, CHIRALCEL® OG, CHIRALCEL® OJ and CHIRALCEL® OK.
  • BBB blood brain barrier BCECs
  • GUVs giant unilamellar vesicles
  • boloamphiphles or bolaamphiphilic compounds of the invention can be synthesized following the procedures described previously (see below).
  • the carboxylic group of methyl vernolate or vernolic acid was interacted with aliphatic diols to obtain bisvernolesters.
  • the epoxy group of the vernolate moiety located on C12 and CI 3 of the aliphatic chain of vernolic acid, was used to introduce two ACh headgroups on the two vicinal carbons obtained after the opening of the oxirane ring.
  • the ACh head group was attached to the vernolate skeleton through the nitrogen atom of the choline moiety.
  • the bolaamphiphile was prepared in a two-stage synthesis: First, opening of the epoxy ring with a haloacetic acid and, second, quaternization with the N,N- dimethylamino ethyl acetate.
  • the bolaamphiphile was prepared in a three-stage synthesis, including opening of the epoxy ring with glutaric acid, then esterification of the free carboxylic group with N,N-dimethyl amino ethanol and the final product was obtained by quaternization of the head group, using methyl iodide followed by exchange of the iodide ion by chloride using an ion exchange resin.
  • Each bolaamphiphile was characterized by mass spectrometry, NMR and IR spectroscopy. The purity of the two bolaamphiphiles was >97% as determined by HPLC.
  • Iron(III) acetylacetonate Fe(acac)3
  • diphenyl ether 1,2-hexadecanediol
  • oleic acid oleylamine
  • carboxyfluorescein CF
  • TMA trimethylammoniumphenyl-6-phenyl-l ,3,5-hexatriene
  • the synthesis bolaamphiphilic compounds of this invention can be carried out in accordance with the methods described previously (Chemistry and Physics of Lipids 2008, 153, 85-97; Journal of Liposome Research 2010, 20, 147-59; WO2002/05501 1 ; WO2003/047499; or WO2010/128504) and using the appropriate reagents, starting materials, and purification methods known to those skilled in the art.
  • Table 1 lists the representative bolaamphiphilic compounds of the invention.
  • vesicle formation and their optimization [00302] The vesicles shown to be effective in delivering enkephalin and albumin to the CNS were made from the bola GLH-20, or a mixture of GLH-19 and GLH-20 [Table 1]. Both of these bolas contain acetyl choline (ACh) head groups [8], but only GLH-20 is hydrolyzed by choline esterases (ChE). The mixture of these two bolas enables extended release of the encapsulated material. Stability and release rates can be used as the criteria to get the optimal ratio between GLH-19 and GLH-20. Stability and release rates can be studied using fluorescent measurements of encapsulated CF as described by us previously [7, 8].
  • ACh acetyl choline
  • ChE choline esterases
  • Each of the vesicle formulations can be examined for vesicle size (by dynamic light scattering), morphology (by cryo-transmission electron microscopy), zeta potential (by Zeta Potential Analyzer) and stability (by fluorescence measurements of encapsulated CF at various incubation times before and after exposing the vesicles to AChE and then to a detergent) [5,7,8].
  • Optimal formulations can be selected based on stability and ability to release encapsulated, material by AChE.
  • Vesicle stability can be tested first in PBS and, then, if stable, in whole serum at 37°C in the presence and absence of pyridostigmine— an AChE inhibitor.
  • the encapsulated GDNF can be run on acrylamide gel electrophoresis (after release from vesicles by a detergent) to confirm that it maintained its integrity during the encapsulation process.
  • the activity of the GDNF affected by the encapsulation process can be tested by measuring the ability of the encapsulated material to induce tyrosine hydroxylase (TH) gene expression in comparison to free GDNF.
  • SK-N-MC cells stably transfected with expression constructs of c-ret and with a luciferase reporter gene driven by 2 kb of the rat TH gene promoter region can be used. In the presence of GDNF, luciferase activity is expected to increase [15].
  • Specimens studied by cryo-TEM were prepared. Sample solutions (4 ⁇ ) are deposited on a glow discharged, 300 mesh, lacey carbon copper grids (Ted Pella, Redding, CA, USA). The excess liquid is blotted and the specimen was vitrified in a Leica EM GP vitrification system in which the temperature and relative humidity are controlled. The samples are examined at -180 °C using a FEI Tecnai 12 G2 TWIN TEM equipped with a Gatan 626 cold stage, and the images are recorded (Gatan model 794 charge-coupled device camera) at 120 kV in low-dose mode.
  • Figure 1 shows TEM micrograph of vesicles from GLH-20 (A) and their size distribution determined by DLS (B).
  • Lipid/polydiacetylene (PDA) vesicles (PDA/DMPC 3:2, mole ratio) are prepared by dissolving the lipid components in chloroform/ ethanol and drying together in vacuo. Vesicles are subsequently prepared in DDW by probe-sonication of the aqueous mixture at 70°C for 3 min. The vesicle samples are then cooled at room temperature for an hour and kept at 4°C overnight. The vesicles are then polymerized using irradiation at 254 nm for 10-20 s, with the resulting emulsions exhibiting an intense blue appearance.
  • PDA fluorescence is measured in 96-well microplates (Greiner Bio-One GmbH, Frickenhausen, Germany) on a Fluoroscan Ascent fluorescence plate reader (Thermo Vantaa, Finland). All measurements are performed at room temperature at 485 nm excitation and 555 nm emission using LP filters with normal slits.
  • Fi is the fluorescence emission of the lipid/PDA vesicles after addition of the tested membrane-active compounds
  • F 0 is the fluorescence of the control sample (without addition of the compounds)
  • Fioo is the fluorescence of a sample heated to produce the highest fluorescence emission of the red PDA phase minus the fluorescence of the control sample.
  • the b.End3 cells were cultured in DMEM medium
  • the cells are maintained in an incubator at 37°C in a humidified atmosphere with 5% CO 2 .
  • b.End3 cells are grown on 24-well plates or on coverslips (for FACS and fluorescence microscopy analysis, respectively). The medium is replaced with culture medium without semm and CF solution, or tested bolavesicles (equivalent to 0.5 ⁇ g/mL CF), or equivalent volume of the medium are added to the cells and incubated for 5 hr at 4°C or at 37°C.
  • the Figure 2 shows head group hydrolysis by AChE (A) of GLH-19 (blue) and GLH-20 (red) and release of CF from GLH-19 vesicles (B) and GLH- 20 vesicles (C).
  • AChE causes the release of encapsulated material from GLH-20 vesicles, but not from GLH-19 vesicles (Fig.2).
  • the vesicles are capable of delivering small molecules, such as carboxyfluorescein (CF), into a mouse brain, but the fluorescent dye accumulates only if it is delivered in vesicles that release their encapsulated CF in presence of AChE, namely, GLH-20 vesicles (Fig. 3A).
  • the ACh head groups also provide the vesicles with cationic surfaces, which promote penetration through the BBB [Lu et al, 2005] and transport of the encapsulated material into the brain. Toxicity studies showed that the dose which induced the first toxic signs was 10-20 times higher than the doses needed to obtain analgesia by encapsulated analgesic peptides.
  • vesicles can be made to encapsulate other molecules, such as agents agains neurodegerative diseases such as GDNF and NGF, and other agents agains other diseases such as anti-retroviral drugs, and deliver them into the brain without harming the BBB.
  • agents agains neurodegerative diseases such as GDNF and NGF
  • other agents agains other diseases such as anti-retroviral drugs
  • GDNF glial cell line-derived neurotrophic factor
  • Delivering GDNF to brain regions affected in PD may be beneficial in slowing down the progression of PD and may even promote neurorestoration, thus improving the status of the PD patient
  • SNpc Substantia Nigra pars compacta
  • STR striatum
  • GDNF does not permeate through the blood-brain barrier (BBB) and, to demonstrate efficacy, it has to be delivered directly into the brain [Slevin JT, Gash DM, Smith CD, Gerhardt GA, Kryscio R, Chebrolu H, Walton A, Wagner R, Young AB. Unilateral intraputamenal glial cell line-derived neurotrophic factor in patients with Parkinson disease: response to 1 year of treatment and 1 year of withdrawal. J Neurosurg. 2007; 106(4):614-620].
  • BBB blood-brain barrier
  • GDNF glial- derived neurotrophic factor
  • a delivery system capable of transporting GDNF to a wide area within the brain and targeting it to brain regions which are affected in PD should increase the probability that all affected neurons are exposed to therapeutic concentrations of the neurotrophin and, thus, increase its efficacy in the treatment of PD.
  • nano-sized stable vesicles are made of novel bolas that are the building block materials for nanoparticles used as a drug delivery system that can self-assemble into vesicles with monolayer membranes. These nanoparticles are more stable than liposomes made of bilayer membranes, due to the higher energy barrier for lipid exchange that characterizes monolayer membranes made from bolas [Fuhrhop JH, Wang T. Bolaamphiphiles, Chem Rev. 2004; 104:2901-2937].
  • vesicles The high stability of such vesicles allows them to circulate in the blood stream until they reach the brain, and then penetrate the blood brain barrier (BBB) to deliver their cargo into the brain.
  • BBB blood brain barrier
  • the monolayer membrane is thinner than the bilayer membrane, which is an important parmater in nano-sized vesicles, since is provides a higher inner volume for encapsulating drugs and biologically active compounds, as compared to liposomes of the same size that are made of a bilayer membrane.
  • the vesicles described herein may also be characterized as providing a controlled-release mechanism that enables the release of the cargo preferentially in the brain.
  • the experiments herein describe, for delivery of GDNF to brain regions that are affected in PD, provide vesicles two important components: a) a bola with a chitosan (CS) head group for increasing BBB permeability of the vesicles, and b) a bola with a dopamine transporter (DAT) ligand for targeting the vesicles to dopaminergic cells in the brain.
  • CS chitosan
  • DAT dopamine transporter
  • GDNF Human GDNF
  • GDNF-biotin Human GDNF
  • Cocaine that was used for the synthesis of the DAT ligand was obtained under license from the Chief Pharmacist of the Regional Health Office, Southern Region, Ministry of Health.
  • AlexaFluor -488 Protein Labeling Kit (A 10235) and AlexaFluor®-488 Microscale Protein Labeling Kit (A30006) were bought from Invitrogen. Other standard chemicals were all purchased from commercial sources.
  • Vernolic acid has an epoxy group that provides a reactive moiety to which functional groups are conjugated.
  • Vernolate-Chitosan that was used for comparison to the bola with the chitosan head groups, as described below, was synthesized by attaching a low molecular weight chitosan to N- hydroxy succineimide vernolate.
  • Elemental analysis was outsourced to a commercial laboratory.
  • FT-IR analysis was carried out on a Nicolet spectrometer.
  • X H and 1 C NMR (500 MHz) spectra were recorded on a Brucker WP-500 SY spectrometers, respectively, in CDCI 3 with TMS as the internal standard or de DMSO solutions.
  • HPLC analysis was carried out on a C18RP column with an evaporative light scattering detector (evaporation temperature 46 °C; mobile phase methanohwater (9: 1, v/v); flow rate 0.5 ml/min).
  • MS analysis was carried out on a Waters Micromass Q-TOF Premier Mass spectrometer (Waters-Micromass, Milford, MA, USA).
  • the basic components of the vesicles were the bolas GLH-19 and GLH-20.
  • the vesicle formulation contained the additives cholesterol and cholesteryl hemisuccinate and as indicated in the text, some formulations included also CS-vernolate conjugate or GLH-55a (a bola with CS head group) and/or GLH-57 (a bola with DAT ligand head groups).
  • CS-vernolate conjugate or GLH-55a a bola with CS head group
  • GLH-57 a bola with DAT ligand head groups.
  • the ratio between GLH-19 and GLH-20 was 2: 1. This ratio was found to give stable vesicles that release their content in a controlled manner (see Results).
  • the different formulations used in this project were: (a) GLH-19+GLH-20 (2: l)/cholesterol/cholesteryl-hemisuccinate (10/1.6/2.1); (b) GLH-19+GLH- 20/cholesterol/cholesteryl-hemisuccinate/CS-conjugate (10/1.6/2.1/1); (c) GLH-19+GLH- 20/cholesterol/cholesteryl hemisuccinate/GLH-55a (CS-bola) (10/1.6/2.1/1); (d) GLH-19+GLH- 20/cholesterol/cholesteryl hemisuccinate/GLH-57 (bola DAT) (10/1.6/2.1/0.8); (e) GLH- 19+GLH-20/cholesterol/cholesteryl hemisuccinate/CS conjugate or GLH-55a/GLH-57
  • Vesicles were prepared from the formulation described above by known methods: (a) Film Hydration followed by extrusion; or (b) Film hydration followed by sonication. Vesicle formation was conducted at room temperature (i.e. 25°C), which is above the transition point of the bolaamphiphilic compounds used in the present study. When the vesicles were prepared by extrusion, the bolas and the additives were dissolved in an organic solvent (usually chloroform). The solution was then placed in a vial and dried under stream of nitrogen. The film that was formed in the vial was then placed under vacuum overnight to remove residual solvent.
  • an organic solvent usually chloroform
  • the thin film was hydrated by adding an aqueous solution containing the material to be encapsulated in the appropriate buffer solution. Then the solution was vortexed and extruded using a LipexTM extruder (Northern Lipids Inc.) via 0.2 and then 0.1 ⁇ Polycarbonate membranes until the solution became transparent (approx. 8- 10 times for each membrane).
  • the polycarbonate membranes were manufactured by GE Water & Process Technologies, and purchased from Tamar Laboratory Supplies Ltd., Israel.
  • the vesicles were characterized with respect to morphology (by cryo-transmission electron microscopy - cryoTEM), size and size distribution (by dynamic light scattering - DLS), surface charge (by Zeta potential analyzer) and stability (by fluorescent measurements).
  • Cryo-Transmission Electron Microscope (Cryo-TEM): Samples of vesicles (about 5 - 10 ⁇ ) were deposited on 300-mesh holey carbon cupper grids (Ted Pella, Inc. Redding, CA). A drop of 5 ⁇ was applied to the grid and blotted with a filter paper to form a thin liquid film of the solution. Grids were rapidly plunged into a liquid ethane bath cooled with liquid nitrogen and maintained at a temperature of approximately -170 °C using a cryo-holder.
  • Zeta-potential measurements Particle size and zeta potential were measured by using zeta potential and Particle size analyzer, ZetaPlus, (Brookhaven Instruments Corporation Ltd, NY, USA), in the range of 10-1000 nm, in the Chemistry Department of BGU. Vesicle solutions were diluted 1 : 10 in appropriate buffers and loaded into a 4 ml cuvette for light scattering measurements. The measurements were conducted at an angle of 90°, at 10 repeated
  • Vesicle stability To determine vesicle stability, samples of carboxyfluorescein (CF)- loaded vesicles (see below method for loading vesicles with CF and determination of percent encapsulation) were incubated in PBS and percent encapsulation was determined at different times. For the measurement of vesicle decapsulation by acetylcholine esterase (AChE), the fluorescence of a sample of intact CF-loaded vesicles was monitored in a quartz cuvette under constant stirring for a few minutes, until a stable fluorescence reading was obtained, and then, AChE (2 ⁇ containing 2 units) was added and the fluorescence measurement continued for additional 5 min. At this point Triton XI 00 (0.15% final concentration) was added to break the remaining vesicles and to obtain the total fluorescence of the encapsulated CF.
  • AChE acetylcholine esterase
  • vesicle samples were incubated in PBS and the percent encapsulation was determined at different times. Stability was also determined by changes in the vesicle size (by DLS, as was described in previous sections) at various time points after vesicle preparation.
  • Encapsulation was achieved by including the material to be encapsulated in the hydration buffer during the hydration stage of the vesicle preparation (see above). After the vesicles were formed and the material in the hydrating buffer was encapsulated, non encapsulated material was removed over a Sephadex G-50 column (for details see below). Encapsulation efficiency was determined initially with CF as described in Popov et al [10, 13].
  • the encapsulation capacity of CF was assessed by measuring the fluorescence intensity (at excitation wavelength of 492 nm and emission wavelength of 517 nm) of CF-loaded vesicular preparation before and after disrupting the vesicular structure by Triton XI 00 at a final concentration of 0.15%.
  • the released CF is dequenched and emits a fluorescent signal, which is quantified by comparing to a calibration curve.
  • RB is the initial fluorescence reading and RAf is the fluorescence reading after the addition of Triton X-100.
  • Percent encapsulation was determined by dividing the area under the curve of the vesicle fractions by the total area under the curve, which was the sum of the area under the curve of the vesicle preparation and the area under the curve of the free protein. With the use of a calibration curve the concentrations in each were determined.
  • biotinylated GDNF GDNF-biotin
  • the following procedure was used for 100 ⁇ g GDNF-biotin:
  • the biotinylated GDNF is dissolved in 1 ml distilled water.
  • Empty vesicles are prepared by film hydration followed by sonication, using formulation e, which is described in the section on vesicle preparation above.
  • the GDNF-biotin solution is then added to 1 ml vesicle suspension and the solution is sonicated on ice to form vesicles made of 5 mg/ml of the basic bolaamphiphile with about 70 ⁇ g of encapsulated GDNF- biotin (the encapsulation efficiency is about 70%).
  • GDNF would be used at sub milligram range and its spectroscopic absorption could not be accurately measured at these concentrations, therefore, fluorescent tagging was investigated as a means of increasing the sensitivity of the determination of low quantities of the encapsulated protein.
  • AlexaFluor®-488 which emits a strong and stable fluorescent signal.
  • A30006 microscale Protein Labeling Kit
  • the vesicles were purified by size exclusion chromatography on Sephadex G50 columns.
  • the eluting buffer for the routine vesicle purification was 16 mM NaCl in phosphate buffer pH 7.3 but other eluting buffers were used as described in the Results Section.
  • the Flow rate used for the elution was 15 ml/hr.
  • Column dimensions were 20cm X 0.7cm (length and diameter, respectively).
  • the volume of each fraction collected from the column was 0.5 ml (equal to 1-2% of the total column volume). Optical density or fluorescence of each fraction was measured to determine the concentration of the eluted material.
  • the lysates were boiled, sonicated and centrifuged and then loaded onto 10% acrylamide gel for SDS-PAGE. After the electrophoresis, the samples were immune-blotted using antibodies against phospho- MAPK44/42 and against phospho-AKT.
  • Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to examine whether the encapsulation process affects the integrity of the encapsulated GDNF.
  • the test samples were applied on a 5%-/ 15% SDS-polyacrylamide gel and electrophoresis was performed using a running buffer of 14.4 g glycine and 3 g Tris base per Liter with 1% SDS. The gels were then stained by Coomasie blue using the Bio-Safe Coomassie staining protocol and then destained for 30 min in water.
  • DAT dopamine transporter
  • Three cell lines were used to test the ability of the surface DAT ligand to target the vesicles to the dopamine transporter: (a) HeLa cells - human cervical cancer cells that do not express the dopamine transporter and were used as control cells. HeLa cells were grown in DMEM medium supplemented with 5% fetal bovine serum, 2 mM L-Glutamine, 100 IU/mL penicillin and 100 ⁇ g/mL streptomycin at 37°C under humidified atmosphere with 5% CO 2 .
  • PC-12 cells were grown in RPMI-1640 medium, supplemented with heat- inactivated horse serum to a final concentration of 10%, fetal bovine serum to a final concentration of 5%, 2 mM L-Glutamine, 100 IU/mL penicillin and 100 ⁇ g mL streptomycin at 37°C under humidified atmosphere with 5% CO 2 .
  • SH-SY5Y human neuroblastoma cells that were reported to express the dopamine transporter [20].
  • SH-SY5Y cells were grown in DMEM medium supplemented with 5% fetal bovine serum, 2 mM L-Glutamine, 100 IU/mL penicillin and 100 ⁇ g/mL streptomycin at 37°C under humidified atmosphere with 5% C0 2 .
  • Vesicles are taken up by these cells after the vesicles adhere to the cell surface. Vesicles that contain DAT ligand on their surface will bind to cells that express the dopamine transporter. The binding of the vesicles to the cells facilitate the uptake and therefore, the extent of the internalization of the vesicles into the cells may be used as an index for targeting.
  • CF carboxyfluorescein
  • the cells were contacted with these CF-loaded vesicles.
  • cells were plated in 24 well plates at a density of 200,000 cells/well and after 24 hours, the the medium was replaced with a culture medium without serum and incubated in this medium for 30 min.
  • Encapsulated or non-encapsulated CF were then added to the cells (free 0.1 ⁇ g CF or the same amount of CF encapsulated in 5 ⁇ g vesicles) and the cells were incubated for 3-5 hours. At the end of the incubation, the cells were washed with PBS and detached from the bottom of the well by trypson-EDTA. The cell suspension was analyzed by flow cytometry (FACS).The fluorescence intensity of the treated cells was done by the Flow o software.
  • mice Eight-week-old male ICR or 10-week-old male C57BL/6 mice, weighing between 25-30g, were maintained on standard mice chow and tap water ad lib. The mice were kept in 12 hours light/dark cycles at a temperature of 25 ⁇ 3°C. All the animal experiments were performed according to the protocol approved by the Animal Care and Use Committee of BGU, according to an approved protocol (# IL-24-04-2008).
  • mice were pretreated 15 min before the injection of the test material by pyridostigmine (o.5 mg/kg, i.m.) to inhibit peripheral ChE and thus, reduce vesicle decapsulation in the blood circulation before they enter the brain.
  • the test material was injected i.v. into the tail vein in a volume of 100-200 ⁇ 1 per mouse.
  • the test material (either free CF or encapsulated CF) was injected into the tail vein of mice in a volume of up to 200 ⁇ .
  • the quantity of encapsulated CF was always determined prior to the administration and similar amounts of either encapsulated or free CF were injected.
  • mice were anesthetized by Xylazine-Ketamine and blood was withdrawn through cardiac puncture, the mouse was perfused with 10 ml PBS and tissues were dissected out. The specimens were homogenized in PBS, deproteinated by 5% (final
  • TCA tricholoro acetic acid
  • mice were anesthetized by Xylazine-Ketamine mixture and blood was withdrawn through cardiac puncture, the mouse was perfused with 10 ml PBS and tissues were dissected out. The dissected tissues were attached to labeled paper stripes, frozen in isopentane cooled over liquid nitrogen, and stored at -80°C. The selected tissues were cryosectioned and the fluorescence of the sections was analyzed using confocal fluorescent microscopy. All images were acquired using the same imaging settings and were not corrected or modified.
  • Fluorescence of slices from different organs was quantified by imaging software after subtracting background fluorescence.
  • the average fluorescence obtained from the control mice, which were injected with PBS instead of the fluorescent material was subtracted from the fluorescence values obtained from the same tissue taken from animals that received Trypsinogen-AlexaFluor®-488.
  • At least 4 slices from each organ of each mouse were used for the quantitative analysis and each group contained 4-5 mice.
  • DAPI solution 20 ⁇ was placed on each section and incubated at room temperature for 10 min. The sections were then washed twice with PBS and wiped gently around the tissue with a paper towel. At this stage, the sections were left to dry in the air. After the staining procedure, the sections were mounted on slides using Mowiol-based mounting solution and fluorescence of the images (3-4 images per each section) were taken, using the CF and the DAPI channels of the confocal microscope.
  • Scheme 1 was reacted with aliphatic diols to obtain the bisvernolester 2_(Scheme 1, below).
  • This bisvernolester is the skeleton for both bolas GLH- 19 and GLH-20.
  • the epoxy group of the vernolate moiety located on C 12-C13 of the aliphatic chain of vernolic acid, was used to introduce two ACh headgroups on each side of the alkyl chain, via one of the two vicinal carbons obtained after opening of the oxirane ring.
  • the ACh head group was attached to the vernolate skeleton through the nitrogen atom of the choline moiety.
  • CS chitosan
  • LMWCS low molecular weight chitosan
  • asymmetric bola skeleton asymmetric bola skeleton
  • binding the head groups to the skeleton The starting material for the preparation of LMWCS is commercial chitosan, which is of high- molecular weight, and is insoluble in water and organic solvents.
  • the LMWCS which has improved water solubility, could be obtained by a depolymerization reaction of the commercial high molecular weight chitosan, using hydrogen peroxide (3 ⁇ 4(3 ⁇ 4), which is a strong oxidant that produces free radicals, which can attack the ⁇ -D- (l-4)-glycosidic bond and depolymerizes chitosan.
  • Oxidative depolymerization of chitosan by heterogeneous treatment of commercial high-molecular chitosan (MW ⁇ 50 kDa) with hydrogen peroxide was accomplished by a dropwise addition of 30% hydrogen peroxide solution to chitosan dispersed in water at 60° C for 6 h.
  • the filtrate after separation of insoluble fragments, was evaporated and precipitated by ethanol to obtain LMWCS, providing, e.g., 2 g of the LMWCS using the method described above.
  • the FT-IR spectrum of the LMWCS [00372] The FT-IR spectrum of the LMWCS [00373] The FT-IR spectrum of the LMWCS (FIG. 2) exhibited most of the characteristic absorption peaks of the original chitosan with some differences. The vibrational band at 1 154 cm “1 , which corresponds to the ether bond between the pyranose rings, was weakened, indicating rupture of the ⁇ -glucosidic bonds in the molecular chain of chitosan. The band at 1596 cm "1 in LMWCS becomes stronger than that of the original chitosan, suggesting that the content of amino groups and correspondingly, the degree of deacetylation (DD) changed.
  • DD degree of deacetylation
  • LMWCS The decrease in the FL (pH-potentiometric titration of amino groups) content in LMWCS may be explained by the presence of "other groups". These "other groups” may be titrated together with the amino groups, for example, carboxylic groups.
  • the carboxylic groups of the LMWCS were determined by a direct titration with sodium hydroxide. In fact, LMWCS contained 1.05 - 1.15 mmol carboxylic groups per gram of chitosan (Table 2), below:
  • Table 2 also shows that the depolymerization of the commercial chitosan led to a decrease in nitrogen, carbon and hydrogen contents, suggesting an increase of oxygen content.
  • the mass ratio N/C decreased, confirming the loss of nitrogen as the result of the
  • the composition of the LMWCS chitosan was also analyzed by MALDI-TOF mass spectrometry.
  • the analysis of the mixture of oligomers obtained by the depolymerization of the original chitosan was performed in a positive-ion mode.
  • Table 3 shows that the depolymerization of the original chitosan led to the formation of oligomers with a degree of polymerization (DP) between 3 and 8.
  • the peaks correspond to oligomers carrying fragments of deacetylated (GLcN) and acetylated (GLc NAc) chitosan.
  • Deacetylated chitosan contains a repeat unit of C 6 H11O4N, with a MW of 161 Da and the acetylated chitosan contains a repeat unit of C 8 H 13 0 5 N, with a MW of 203 Da.
  • Table 3 Deacetylated chitosan
  • the ACh head group on the bola-CS is smaller than the CS head group and is similar to the ACh head groups of the symmetrical bolaamphiphile GLH-20.
  • the ACh head group of the bola-CS is expected to be situated on the inner membrane surface of the vesicle, together with one of the ACh head groups of GLH-20 and GLH-19 (which also has an ACh head group, but attached in a different way to the hydrophobic skeleton).
  • the CS head group will thus become an outer surface moiety and will be free to interact with the endothelial cells of the BBB, thus enhancing BBB permeability of the vesicles.
  • the synthesis of this asymmetric bola-CS is a multi stage process that is depicted in Scheme 4, below.
  • Stage 1 For the asymmetric bola GLH-55a, the synthesis began with the formation of monochloroacetate of decanediol 3 through the esterification of 1, 10-decanediol 1 with chloroacetic acid 2 at a molar ratio of 5: 1 respectively (Scheme 4). The reaction was carried out in toluene by azeotropic distillation in the presence of Amberlyst 15 as a heterogeneous acidic catalyst that can be easily removed by filtration at the end of the reaction, avoiding the tedious work needed to neutralize the soluble acidic catalyst.
  • Stage 2 The second step of the synthesis (Scheme 4, above) includes the elongation of the hydrophobic chain.
  • the intermediate 5 was obtained by reacting the monochloroacetate of decanediol 3 with a dicarboxylic acid 4 (1, 10-decanedicarboxylic acid) at a molar ratio of 1 :5, respectively.
  • the reaction mixture was refluxed in toluene with constant removal of water by azeotropic distillation and was catalyzed by Amberlyst 15.
  • the crude product was purified over a silica gel flash chromatography.
  • the chloroacetate intermediates were then characterized by X H and 13 C NMR spectroscopy (Table 4, below).
  • Stage 3 The next step was the preparation of an active ester of the carboxylic acid with N-hydroxysuccinimide (NHS).
  • the active N-hydroxy- succinimide of the ester of the carboxylic acid was synthesized by reacting intermediate 5 (Scheme 4) and N- hydroxy - succinimide in the presence of a coupling agent (dicyclohexylcarbo-diimide DCC) at room temperature by the method of [25].
  • a coupling agent dicyclohexylcarbo-diimide DCC
  • Stage 4 In order to attach the acetylcholine head group, the chloro derivative obtained in the previous stage was reacted with intermediate 7, that will serve as the alkylating agent with a small excess of the tertiary amine, N,N-dimethylaminoethyl acetate. The reaction was carried out in acetone as the solvent at the reflux temperature for about 8 hours. The progress of the reaction was followed by TLC and HPLC. The reaction mixture was washed several times with diethyl ether to remove the excess of the unreacted amine. The degree of quaternization of the amphiphile intermediate 9 was about 95-98%, as determined by argentometric titration.
  • ESI -MS electrospray ionization mass spectrometry
  • Stage 5 This stage constitutes the conjugation of the low molecular weight chitosan (LMWCS) (prepared as described above) with the bolaskeleton 9 (Scheme 4) containing already the cationic head group at one end, and the N-hydroxysuccinimide ester at the other end.
  • the conjugation was performed by adding the solution of intermediate 9 in DMSO to the solution of LMWCS and triethylamine in DMSO.
  • the molar ratio LMWCS to the activated ester 9 was 10: 1.
  • the reaction mixture was stirred for 72 h at RT.
  • the solution was lyophilized.
  • the yellow powder obtained was triturated several times with ether and ethanol, to remove the unreacted intermediate 9, filtered and dried.
  • the obtained product, GLH-55a is the
  • bolaamphiphile having the chitosan head group on one side and the acetyl choline head group on the other side of the bolaskeleton, was soluble in DMSO and water.
  • Table 5 and Table 6 present the chemical shifts of the final bola GLH-55a. As can be seen, the chemical shifts of the original LMWCS could also be found in the modified CS (marked with a star).
  • the new sig nals at 52.56 ppm [N + (CH 3 ) 2 ], 58.22 ppm [N + -CH 2 -CH 2 ] 62.62 ppm [N + -CH 2 -CH 2 ], 61.40 ppm [CO-CH 2 -N + ] indicate the formation of conjugation product.
  • This new polymeric unit which contains the bola with the acetyl choline as one head group and the chitosan as the second head group, has a MW of 736 Da.
  • This has been used for imaging in humans as a marker of the nigrostriatal pathway to assess the severity of Parkinson's disease (PD) [27].
  • This ligand was selected, in part, for its high affinity to dopaminergic cells, as an illustrative surface group for targeting the vesicles to dopaminergic neurons in the Substantia Nigra.
  • Stage 1 encmpasses the three steps ((a) - (c)) described below.
  • Stage 2 encompassed the following reaction:
  • This stage involved the Michael addition of the aromatic Grignard reagent, (p- fluorophenyl) magnesium bromide, to the anhydroecgonine methyl ester 4.
  • the methyl ester 4 in anhydrous ether was added drop wise to a mixture of the Grignard reagent in anhydrous ether at -30°C under a stream of nitrogen.
  • the method of quenching can determine the relative distribution of the a- and ⁇ - carbomethoxy isomers. Since the a-isomer is biologically inactive, there was a need to optimize the yield of the ⁇ -carbomethoxy compound.
  • Stage 3 encompassed the following reaction:
  • This reaction involves a demethylation reaction providing derivative 7 to be attached to the bolaskeleton.
  • the N-demethylation of the N-methyltropane analog 5 was carried out by using a-chloroethyl chloroformate (ACE-Cl) [32,33].
  • ACE-Cl a-chloroethyl chloroformate
  • the reaction of chloroformates with tertiary aliphatic amines provides a convenient method for promoting dealkylation.
  • Compound 5 was reacted with ACE-Cl to provide the ⁇ -chloroethyl carbamate intermediate 6.
  • the hydrolysis was then carried out with methanol to obtain the crude compound 7 in a 68.4% yield with a purity of 83% (GC).
  • GC 68.4% yield with a purity of 83%
  • the mixture still contained 1 1.8% ⁇ -CFT and 4.2% byproducts.
  • Purification by flash chromatography gave fluoro nortropane 7 in about
  • the protons at position 38 have a characteristic peak at 5.5ppm in both I and II configurations.
  • the proton at position 39 of isomer II shifted from 5.35ppm to 5.50 ppm compared to isomer I and appears together with the protons at position 38.
  • 3H appeared at 5.5ppm (2 protons for isomers I and II on 38 atom and 1 proton from isomer I on the atom 39) and 1H on the 39 atom from isomer II appeared at 5.35ppm.
  • Trypsinogen wasas a model protein for GDNF in initial encapsulation studies, since (a) trypsinogen is considerably less expensive and is available in large quantities for initial exploratory studies, (b) both proteins have similar molecular weights and isoelectric points (molecular weights of 22KDa and 18KDa for trypsinogen [35] and glycosylated GDNF [36], respectively, and (c) have similar isoelectric points of 9.25-9.36 and 9.26 for trypsinogen [37] and GDNF [36], respectively. Thus encapsulation, which is mostly affected by molecular weight and isoelectric point, is expected to be almost identical for both proteins.
  • Vesicles were also prepared from a mixture of GLH- 19 and GLH-20.
  • the rationale behind such vesicles was an attempt to obtain vesicles with higher stability, lower toxicity (preliminary studies indicate that vesicles from GLH-19 have lower toxicity than vesicles made from GLH-20), and which release their encapsulated content upon exposure to choline esterases (ChE).
  • ChE choline esterases
  • GLH-19 vesicles do not release their encapsulated content when exposed to ChE, whereas vesicles made from GLH-20 release their encapsulated content in presence of ChE, yet GLH-19 form more stable vesicles with lower toxicity than GLH-20.
  • the formulations for preparing vesicles further contain small quantities (1 mg/ml) of a bolaamphiphile with a CS head group on one side of the hydrophobic skeleton and an ACh head group on the other side of the hydrophobic skeleton (GLH-55a), and also 0.8 mg /ml of a bolaamphiphile with the DAT ligand on each side of the hydrophobic skeleton (GLH-57).
  • GLH-55a and GLH-57 do not affect vesicle morphology and other vesicle characteristics.
  • FIG. 15 A the spherical shape and size of the vesicles was maintained after incorporating into the formulation GLH-55a (FIG. 15 A), GLH-57 (FIG. 15 B) or both GLH-55a and GLH-57 (FIG. 15 C); thus, vesicles that contained GLH-55a and GLH-57 were similar to vesicles made from a mixture of GLH-19 and GLH-20 without the CS and the DAT ligand decoration.
  • Vesicle size and size distribution determine by dynamic light scattering
  • FIG. 17 Representative dynamic light scattering profiles are provided in FIG. 17, i.e., for empty vesicles made from GLH-19 (FIG. 17 A), GLH-20 (FIG. 17 B), and a mixture of GLH-19 and
  • Table 7 Vesicle size and zeta potential values measured by DLS
  • Vesicles were prepared by film hydration followed by sonication from 10 mg/ml bolas, 2.1 mg/ml cholesteryl hemisuccinate and 1.6 mg/ml cholesterol. Each measurement was done on at least 3 different vesicle preparations and the values are means ⁇ SEM.
  • the average diameter of the empty vesicles ranged between 100-123 nm.
  • the empty vesicles are positively charged (cationic vesicles), with GLH- 20 vesicles having higher zeta potential than GLH-19 vesicles.
  • Vesicles made from a mixture of GLH-19 and GLH-20 show zeta potential in between those of GLH-19 vesicles and GLH-20 vesicles.
  • Encapsulation of trypsinogen at a concentration of 4 mg/ml, consistently increased the vesicle size and reduced somewhat the zeta potential in all the vesicle preparations, indicating that some protein binds to the vesicle surface and neutralizing the positively charged groups.
  • encapsulation of GDNF did not change the vesicle size or zeta potential, probably because the concentration of the GDNF was much smaller than the trypsinogen concentration (the GDNF concentration was 40 ⁇ g/ml).
  • Vesicle stability was determined by measuring the amount of encapsulated fluorescent dye (carboxyfluorescein (CF)) as a function of time, during either storage or incubation in whole serum.
  • CF encapsulated fluorescent dye
  • vesicle stability in whole serum is complicated by the presence of ChE activity. It had been demonstrated that vesicles from GLH-20 release their encapsulated material when exposed to ChE, whereas vesicles made from GLH-19 were not sensitive to ChE. Vesicles made from a mixture of GLH-19 and GLH-20 are believed (without wishing to be held to that belief) to be potentially more effective for the purpose of delivering GDNF to the brain since the GLH-19 component should reduce toxicity.
  • the half life of vesicles made from a mixture of GLH-19 and GLH-20 is about 4-6 hours (depending on the ratio between GLH-19 and GLH-20), compared with a half life of 2.5 hours for vesicles made from GLH-20 only.
  • vesicles made from GLH-20 decapsulate and release their content in presence of ChE. Since the vesicles are designed to release their content in the brain by the influence of the brain ChE, it was important to confirm that the relatively small amount of GLH-20 in the vesicles made from a mixture of GLH-19 and GLH-20 at a ratio of 2: 1, is sufficient to cause decapsulation when exposed to ChE. Accordingly, vesicles were prepared from GLH-20 and from a mixture of GLH-19 and GLH-20, and loaded with CF and exposed to ChE, the release of the fluorescent marker was measured as a function of time after exposing them to the enzyme. The results are shown in FIG. 20.
  • both vesicle preparations started to release their content after the addition of AChE.
  • release from the vesicles made from GLH-20 was somewhat faster than the release from the vesicles made from a mixture of GLH-19 and GLH-20.
  • the vesicles made from GLH-20 released 42% of their content whereas at this time point the vesicles made from the mixture of GLH- 19 and GLH-20 released 33% of the total CF that was encapsulated.
  • vesicles made from a mixture of GLH- 19 and GLH-20 would be predicted to release their content in the brain in response to brain ChE and therefore, these vesicles can be used to deliver compounds to the brain and release their cargo in the brain.
  • the concentraion of trypsinogen can be measured by UV absorbance at 280 nm. Since the vesicles are prepared in media that contain trypsinogen, encapsulated protein had to be separated from non-encapsulated protein to determine encapsulation efficiency. For example, encapsulated trypsinogen could be separated from free protein by size exclusion chromatography on a Sephadex G50 column. As can be seen from FIG.
  • the formulations that were used for the vesicle preparations contained 10 mg/ml bolas (the ratio between the bolas was always 2 parts of GLH- 19 and 1 part of GLH-20), and the ratios between the bolas and the cholesterol (CHOL) and cholesteryl hemisuccinate (CHEMS) were varided as indicated in the Table 8.
  • Vesicles were prepared in the media as indicated in Table 8. All media contained 4 mg/ml trypsinogen.
  • Vesicles were prepared by film hydration followed by sonication in presence of various amounts of AlexaFluor®-488-labeled trypsinogen and various concentrations of the bolas, as indicated. Values are percent encapsulation, calculated by using the amount of the labeled trypsinigen that was present during vesicle preparation as 100%.
  • vesicles were prepared by film hydration followed by sonication from a mixture of GLH- 19 and GLH-20 at a concentration of 10 mg/ml with 1.6 mg/ml cholesterol and 2.1 mg/ml cholesteryl hemisuccinate.
  • the formulations contained 50 ⁇ g/ml trypsinogen (A), 100 ⁇ g/ml trypsinogen (B) and 12.5 ⁇ g/ml GDNF (C). All proteins were labeled with AlexaFluor ⁇ 3 ⁇ 4-488.
  • the vesicles were eluted from a Sephadex G50 column by PBS and the fluorescence of each fraction was determined. The results are shown in FIG. 26.
  • FIG. 27 depicts the effect of the encapsulation process on GDNF integrity and activity.
  • A Analysis of GDNF on PAGE, where lane 1 is empty vesicles; lane 2 is GDNF encapsulated by the method of film hydration followed by sonication; lane 3 is encapsulated GDNF which was incubated before the PAGE at 40°C for one hour; and lane 4 is free GDNF.
  • AKT activity regulates cell survival and this activity is particularly relevant to neuroprotection conferred by GDNF and to PD therapy.
  • AKT and MAPK are activated when they are phosphorylated and GDNF induces phosphorylation of these enzymes in SH-SY5Y neuroblastoma cells. If GDNF activity is impaired, it will not phosphorylate AKT and MAPK to pAKT and pMAPK, respectively.
  • pAKT and pMAPK can be detected by specific antibodies for the phosphorylated forms of the enzymes on a Western blot.
  • GDNF caused the same degree of phosphorylation in its free form as in its encapsulated state, or after it was added to empty vesicles.
  • Vesicles GLH-57 were formed by additiona of bolas with DAT ligand head groups to the vesicle formulation of GLH 19/GLH 20 CHEMS/CHOL. When this bola is included in the formulation, the resulting vesicles are decorated on their surface with the DAT ligand, intended to target cells that express the dopamine transporter, namely, dopaminergic cells.
  • the vesicles were added to three types of cells: a) PC 12 cells that highly express DAT [19]; b) SH-SY5Y neuroblastoma cells that are known to express DAT [20]; and c) HeLa cells that do not express DAT.
  • Vesicles were loaded with CF, and each cell type contacted with added the fluorescently labeled vesicles uptake of the fluorescent dye into the cell measured by flow cytometry. Vesicles were made from 10 mg/ml GLH-19:GLH-20 (2: 1) without (uncoated vesicles) and with 0.8 mg/ml GLH-57, a bola that contains DAT ligand as the head group (DAT- vesicles). Cells were incubated for 1 h with the vesicles, and tested by flow cytometry. A shift to the right of the peak indicates fluorescent cells due to uptake of the vesicles.
  • This experiment measure accumulation of CF in the brain following i.v. administration.
  • Vesicles were made by film hydration followed by sonication from a 10 mg/ml mixture of GLH- 19 and GLH-20 (2: 1), 1 mg/ml CS-fatty acid (vernolate) conjugate, 2.1 mg/ml cholesteryl hemisuccinate and 1.6 mg/ml cholesterol in absence (empty vesicles) and in presence of 0.2/ml CF (CF-loaded vesicles).
  • Mice were pretreated with 0.5 mg/kg (i.m.) pyridostigmine and 15 min afterward the mice were injected i.v.
  • vesicles were prepared as described in FIG. 29, except that in one case 1 mg/ml GLH-55a was used in the vesicle formulation to provide CS surface groups (vesicles with CS-bola), and in the other case, 1 mg/ml CS-fatty acid conjugate was used.
  • the amount of the CF in the brain was increased by about 100% when the CF was encapsulated in vesicles in which the CS surface group was an integral part of the membrane (by using the bola-CS - GLH-55a). These results indicate that bola-CS is better than fatty acid-CS for enhancing the permeability of the vesicles via the BBB.
  • the vesicles described herein transport their encapsulated content through the BBB into the brain.
  • This experiment was intended to demonstrate that the vesicles that are coated with DAT ligand will be targeted to brain regions that contain dopaminergic neurons.
  • Vescicles were loaded with CF and injected into the tail vein of mice and 30 min after the injection, the mice were sacrificed, the brain removed and dissected into three brain regions: (1) the cortex; (2) the striatum; and (3) the cerebellum. Each of these brain regions was homogenized, deproteinized by trichloroacetic acid, and fluorescence intensity was measured in the supernatant that was obtained after centrifugation.
  • vesicles were prepared by film hydration followed by sonication from a 10 mg/ml mixture of GLH-19 and GLH-20 (2: 1), 1 mg/ml GLH-55a (a bola with CS head group), 2.1 mg/ml cholesteryl hemisuccinate, 1.6 mg/ml cholesterol, 0.2 mg/ml CF and without (vesicle CS bola) or with GLH-57 (vesicles DAT CS bola).
  • Mice were pretreated with 0.5 mg/kg (i.m.) pyridostigmine (to inhibit peripheral ChE) and 15 min afterward the vesicles were injected i.v.
  • mice were sacrificed, perfused with 10 ml PBS and the brain removed and dissected into cortex, striatum and cerebellum.
  • the tissues were weighed, homogenized and deproteinated by trichloroacetic acid, centrifuged and fluorescence was determined in the homogenates.
  • the amount of the CF in each brain region was calculated from a calibration curve of CF, taking into consideration the weight of the tissue and the dilution done during the homogenization. Each bar represent an average value obtained from 5 mice +/- SEM.
  • FIG. 31 depicts the results of this experiment.
  • the highest amount of CF was found in the striatum of animals that were injected with DAT ligand- coated vesicles.
  • the largest difference in CF concentrations between uncoated vesicles and coated vesicles was observed in the striatum, then in the cortex and lastly in the cerebellum, where there was almost no difference between the amounts of the CF that were measured in animals that received uncoated vesicles versus those that received DAT ligand-coated vesicles.
  • Free CF did not penetrate into the brain in significant amounts.
  • mice Prior to delivering GDNF to the brain, pilot experiment, in vivo, studies were carried out with the model protein - tiypsinogen, which was labeled for this purpose with AlexaFluor®-488. The experiment was intended to determine whether the labeled protein can be seen in brain sections directly by histofluorescence. Mice were pretreated with pyridostigmine 15 min prior to vesicle injection (to inhibit peripheral ChE) and 30 min after the injection of the vesicles, the mice were sacrificed, perfused with 10 ml PBS and tissues were dissected out, frozen in isopentane that was cooled by liquid nitrogen, sectioned by cryomicrotime and fluorescence was observed by confocal microscopy. The fluorescence that was seen in three different tissues: a) brain; b) liver; c) kidney; is shown in FIG 32.
  • FIG. 32 provides representative histofluorescence slides showing AlxaFlour-488- labeld trypsinogen in brain (A-C); liver (D-F) and kidney (G-I) of mice that were injected with the labeled protein encapsulated in CS-coated vesicles or with the free protein.
  • Panels A, D and G are micrographs taken from control untreated mice.
  • Panels B,E and H are micrographs taken from mice injected with 200 ⁇ g of free tiypsinogen labeled with AlexaFluor®-488 and C,F and I are micrographs taken from mice that were injected with 200 ⁇ g of encapsulated trypsinogen labeled with AlexaFluor®-488.
  • the labeled trypsinogen is found in the brain only when it was injected encapsulated in the bolavesicles. Also, in the liver, the injection of encapsulated trypsinogen resulted in higher fluorescence than was obtained after injection of the free labeled protein. In the kidney, high fluorescence was observed also after injection of the free labeled protein. Quantification of these results was done by imaging software and is shown in FIG. 33.
  • FIG. 33 depicts that distribution of trypsinogen labeled with
  • This figure presents quantification of the data obtained in the experiment described in FIG. 32.
  • Each bar represent an average value of 5 mice +/- SEM.
  • GDNF-biotin Alomone Lab Inc., Jerusalem, IL
  • GDNF-biotin a derivative protein that maintains all the properties of GDNF, including full GDNF activity.
  • the GDNF-biotin was introduced inot vesicles that were made from a formulation that contained all the components described above, including GLH-55a and GLH-57, bolas that contain CS and DAT ligand head groups, and the GDNF-biotin-loaded vesicles were injected (i.v.) into mice.
  • mice were sacrificed, perfused with 10 ml PBS, to remove the GDNF-biotin from blood vessels, and brains were removed and frozen in isopentane immersed in liquid nitrogen. The frozen brains were ciyosectioned and the sections were stained with DAPI (to visualize the nuclei of the cells for orientation purposes).
  • avidine-AlexaFluor®-488 was added to the slides, which were then washed and observed using a confocal microscope.
  • the avidine binds specifically to the GDNF-biotin and only the sites in the brain that contained the delivered GDNF-biotin showed fluoresce.
  • the avidine-AlexaFluor ⁇ 3 ⁇ 4-488 was added also to brain sections taken from mice that were injected with PBS.
  • mice were pretreated with 0.5 mg/kg (i.m.) pyridostigmine, then injected i.v. with vesicles coated with CS groups and DAT ligand with encapsulated GDNF-biotin. After 30 min, animals were sacrificed, perfused with 10 ml PBS, brains removed and striata, cortex and cerebella were dissected out, frozen and cryosectioned. Brain sections from these mice were stained with DAPI (blue) and avidine-AlexaFluor®-488 (green) and observed using confocal microscopy at a magnification of 10X.
  • DAPI blue
  • avidine-AlexaFluor®-488 green
  • A Stiatum from a mouse treated with PBS
  • B striatum from a mouse injected with GDNF-biotin encapsulated in vesicles
  • C cortex from a mouse injected with PBS
  • D cortex from a mouse injected with GDNF-biotin encapsulated in vesicles
  • E cerebellum from a mouse injected with PBS
  • F cerebellum from a mouse injected with GDNF-biotin encapsulated in vesicles.
  • FIG. 34 sections of the striatum from animals that were injected with vesicles with encapsulated GDNF-biotin, show a sharp focused fluorescence arranged in a circular shape around and within the striatum, whereas less fluorescence was seen in the cortex and even less fluorescence was seen in the cerebellum.
  • the small amount of the fluorescence seen in the cortex was diffused and as focused as in the striatum. No fluorescence was seen in the control mice, indicating that the fluorescence which is seen in the brain section is specific for GDNF-biotin. Localization of the fluorescence in the brain section were also examined under higher magnification, and these results are shown in FIG. 35.
  • FIG. 35 It is clear from FIG. 35 that the GDNF-biotin is concentrated around many cells in the striatum and is found to a lesser extent in the cortex and the cerebellum.
  • the micrographs of high magnification, (60X) of FIG. 35 were taken from brain sections obtained from the mice used in the experiment described in FIG. 34. The nuclei of the cells appear in blue, due to DAPI staining, and the GDNF-biotin appears in green, due to the binding of the avidine-AlexaFluor®-488. (A) ).
  • Stiatum from a mouse treated with PBS (B) striatum from a mouse injected with GDNF-biotin encapsulated in vesicles; (C) cortex from a mouse injected with PBS; (D) cortex from a mouse injected with GDNF-biotin encapsulated in vesicles; (E) cerebellum from a mouse injected with PBS; (F) cerebellum from a mouse injected with GDNF-biotin encapsulated in vesicles. Whether all the cells which stained for GDNF-biotin are dopaminegic neurons will be answered from co- localization studies that performed using antibodies against tyrosine hydroxylase (TH) to stain specifically the TH expressing cells.
  • TH tyrosine hydroxylase
  • vesicles have been prepared from bolas and coated with CS groups and DAT ligands. It is also apparent that these vesicles are capable of delivering GDNF to brain regions affected in Parkinson's disease.
  • building blocks bolas
  • vesicles that were made from these building blocks were characterized.
  • GDNF encapsulation in these vesicles was optimized, and it has been demonstrated that they have a controlled release mechanism enabling the vesicles to release their content via the hydrolysis of the ACh head groups by brain ChE.
  • the vesicles are targeted to dopamine transporter expressing cells, but not to cells that do not express the dopamine transporter.
  • these experiments had demonstrated that the vesicles described herein are capable of transporting GDNF to the brain following systemic administration, and targeting the neurotrophin to brain regions that are affected in PD.
  • the present disclosure further provides a method for controlling the rate of drug release from bolaamphiphilic vesicles with acetylcholine head groups by changing the length of an alkyl chain adjacent to the head group.
  • Bolaamphiphilic compounds with acetyl choline (ACh) head groups with two different alkyl chains adjacent to the head groups were investigated for their ability to form vesicles that release the encapsulated material upon the introduction of a triggering event.
  • bolaamphiphiles which was synthesized from vernolic acid, has an alkyl chain with 5 methylene groups adjacent to the ACh head group and the other, which was synthesized from oleic acid, has an alkyl chain with 8 methylene groups adjacent to the ACh head group.
  • Both bolaamphiphiles formed stable vesicles with a diameter of about 100 nm.
  • the ACh head groups of both bolaamphiphiles were hydro lyzed by acetylcholine esterase (AChE), however, the hydrolysis rate was significantly faster for the bolaamphiphile with the shorter aliphatic chain pendant.
  • the starting materials for bolaamphiphile synthesis are functional vegetable oils and their corresponding fatty acids.
  • Vernolic acid a naturally epoxidized fatty acid (cis-12, 13 epoxy, cis-9 octadecenoic acid) that constitutes the main constituent of vernonia oil was used for the synthesis of the bolaamphile GLH-20, noted above, which has a head group hydro lyzed by AChE.
  • a second bolaamphiphile was prepared from oleic acid.
  • the FT-IR spectrum of the diester 3 showed the disappearance of the absorption band at 1700 cm-1, which is related to the carboxylic acid group, and the appearance of the absorption band at 1727 cm-1, characteristic of the new ester group.
  • the new alkoxy methylene group CH2-0-C(0)- appeared at 4.04 ppm in the 1H-NMR spectrum and at 64 ppm in the 13C-NMR.
  • the epoxy group remained unchanged
  • the head groups were attached in a two-stage reaction (Scheme 7b), involving (1) opening the epoxy ring with chloroacetic acid to give the dichloroacetate, derivative 4, and (2) quaternization stage of ⁇ , ⁇ -dimethylaminoethyl acetate with 4 to give the bolaamphiphile 5 with two acetylcholine head groups bound to the hydrophobic chain through the nitrogen of the choline moiety.
  • the diepoxy distearate 3 was reacted with an excess of chloroacetic acid in dry toluene at 85°C for 48 h.
  • the last stage of the synthesis is the quaternization reaction of N,N-dimethylamino ethyl acetate with the dicholoro acetate 4 (scheme 7B) that yields the final bolaamphiphile 5 with two acetyl choline head groups.
  • the reaction was carried out with a large excess of the amine at 45°C for 6 h followed up by repeated washings with ether to remove the excess of the tertiary amine and the desired bolaamphiphile was obtained as a yellow viscous product.
  • the X H-NMR of the bolaamphiphilic compound (Fig 4) can distinguish the new peaks of the ACh head group.
  • the methyl (24) of the acetate CH3-C(0)-0 appear as a singlet at 2.12 ppm.
  • the methylene group (22) N + -CH2-CH2-0- near the quaternary nitrogen appeared at 4.25 ppm and the methylene group (23) N + -CH 2 -CH2-0-C(0)- near the oxygen appeared at 4.60 ppm.
  • the two methyl groups (21) of the quaternary nitrogen appeared as two singlets at 3.61 and 3.62 ppm, while the two different protons of the methylene group (20) between the quaternary nitrogen and the carbonyl -0-C(0)-C3 ⁇ 4-N + - appeared each one as a multiplet at 4.79 and 5.46 ppm.
  • Amphiphiles in general, and specifically bolaamphiphiles can form micelles, multilayered sheets, vesicles, rings, or a variety of microstructures with cylindrical geometry, such as rods, tubules, ribbons, and helices.
  • the morphology of the self-aggregate structure is a function of the molecular parameters of the specific bolaamphiphile.
  • bolaamphiphiles was studied by transmission electron microscopy (TEM), and showed spherical aggregate nanostructures for both bolaamphiphilic compounds (FIG. 38).
  • the vesicles were fairly heterogeneous in size with diameters ranging between 50-300 nm.
  • the size distribution was determined by dynamic light scattering (DLS) and the data are shown in Table 1 1.
  • the average diameter of the vesicles that were made from GLH-20 was 368 nm, whereas the average diameter of vesicles made from GLH-32 was 345.
  • vesicles made from GLH-32 were more heterogeneous in size as only 68% of the main peak was within the range of the average diameter.
  • Table 11 DLS measurements of vesicles from GLH-20 and GLH-32
  • GLH-20 and GLH-32 are symmetrical bolaamphiphiles forming monolayer membranes. Due to differences in the void spaces between the bolaamphiphiles at the inner versus the outer surfaces of the vesicle's membrane, the aggregation of the bolaamphiphiles into a stable vesicle structure requires relatively large diameter vesicles to reduce the relative large differences in surface areas the inner and the outer surfaces.
  • One way of stabilizing smaller vesicles made of symmetrical bolaamphiphiles is by incorporating additives that act as membrane stabilizers that will be situated among the outer parts of the bolaamphiphiles and thus, will be used as spacers that stabilize a higher curvature between the bolaamphiphiles.
  • Membrane stabilizers such as cholesterol (CHOL) and cholesteryl hemysuccinate (CHEMS), may be used for this purpose.
  • cholesterol cholesterol
  • CHEMS cholesteryl hemysuccinate
  • such compounds raise the order-disorder transition temperature and make the membrane more stable at higher temperatures.
  • Table 3 DLS measurement of vesicles made from GLH-20 and GLH32 formulated with CHOL and CHEMS at a ratio of 2: 1 : 1.
  • Vesicle stability was evaluated by measuring both changes in the concentration of encapsulated carboxyfluorescein (CF) and vesicles size as a function of time when incubated in PBS at room temperature.
  • CF carboxyfluorescein
  • both vesicles started to release their encapsulated material immediately after the addition of AChE to the vesicle suspension.
  • the release rate was biphasic with a more rapid release rate seen immediately after the addition of the enzyme and then, after about 20-50 seconds, the release stabilized at a slower but constant rate.
  • the rate of release from GLH-20 vesicles was more rapid than the release rate from GLH-32 vesicles.
  • the percent release from each vesicle preparation was calculated at 4 times after the addition of AChE, which were taken during the second phase.
  • the results of this analysis show that the slope of the curve that represents the release as a function of time is significantly greater for GLH-20 vesicles, compared to GLH-32 vesicles.
  • the greater slope represents a faster release rate; indeed, at 400 seconds after the exposure of the vesicles to the enzyme, GLH-20 vesicles released about 44% of their content, whereas GLH-32 vesicles released only about 20% of their content (FIG. 44).
  • the present disclosure is directed to compounds, compositions, and method of the treatement of neurological diseases including, for illustrative purposes amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • the present disclosure is directed to testing demonstrate in a mouse model of ALS beneficial effects of systemically administered GDNF, encapsulated in novel nano-sized vesicles provided herein.
  • the present disclosure provides vesicles that will be designed to target sites in the
  • Targeting of the vesicles to sites in the CNS where motor neurons degenerate will be achieved by coating the vesicles with manose pendants that will direct the vesicles to activated microglia, which over express manose receptors.
  • Selective release of the encapsulated GDNF is achieved by enzymatic hydrolysis of the head groups at the sites where the vesicles accumulate; in the case of the proposed vesicles - in regions of the CNS where activated microglia accumulate due to motor neurons degeneration in the ALS mouse.
  • Specific elements of this approach include: 1) synthesis of bolaamphiphiles (bola) - the vesicle's building blocks; 2) formation of vesicles coated with manose pendants and encapsulation of GDNF in these vesicles; 3) testing the nano-sized vesicles for brain delivery and for targeting to activated microglia pharmacokinetic (PK) studies); and 4) demonstrating the beneficial effects of the delivered GDNF in an ALS mouse model.
  • bola bolaamphiphiles
  • the GDNF-loaded vesicle system disclosed herein may be a breakthrough in the treatment of ALS for which there is no effective treatment. Moreover, developing the presently- disclosed nanovesicle platform for GDNF has wider implications for additional neurotrophic factors with the potential for ALS therapy as well as for other neurodegenerative diseases that may benefit from neurotrophic factors.
  • the present disclosure provides nano-sized vesicles made from bolaamphiphiles (bolas) that were designed drug delivery and were synthesized as described herein along with vesicles made of monolayer membrane that provides stability (due to high energy barrier for lipid exchange); high encapsulation capacity (due to their thin membrane that makes vesicles with a large inner volume), good brain penetrability (due to surface pendants that induce transcytosis via the brain microvessels endothelial cells) and an efficient controlled release mechanism (due to specific hydrolysis of the head groups at the target site).
  • bolaamphiphiles bolaamphiphiles
  • vesicles made of monolayer membrane that provides stability (due to high energy barrier for lipid exchange); high encapsulation capacity (due to their thin membrane that makes vesicles with a large inner volume), good brain penetrability (due to surface pendants that induce transcytosis via the brain microvessels endothelial cells
  • ALS For the treatment of ALS, we propose to coat similar vesicles with manose pendants that will direct the vesicles to sites in the brain where motor neurons degenerate.
  • the targeting concept is based on the notion and findings that in brain regions in which motor neurons degenerate, there is an accumulation of activated microglia [Xiao et al, 2007; Corcia et al, 2012; Liao et al, 2012; Hovden et al, 2013] that overexpress manose receptors [Galea et al, 2005].
  • GDNF will be encapsulated in the vesicles described herein.
  • vesicles will be prepared that are coated with mannose pendants will include encapsulated GDNF. That is, another bola that will be needed for the in vivo studies, i.e., a bola with the manose head groups that will be added to the vesicle formulation to coat the vesicles with manose pendants for targeting to activated microglia.
  • a bola with the manose head groups that will be added to the vesicle formulation to coat the vesicles with manose pendants for targeting to activated microglia.
  • the synthesis of this bola will be based on the methods used with many other bolas described herein. In various approaches, at lest two different vegetable oils can be used as the starting material (see below), including castor oil and vernonia oil.
  • the manose head groups are expected not to affect vesicle properties except for targeting them to cells that express manose receptors.
  • vesicles made from GLH-19 are quite stable in whole serum (GLH-
  • the ratio between GLH-19 and GLH-20 will be gradually adjusted to provide the most stable vesicles that still release their content upon exposure to choline esterase and this basic formulation will be used for all future studies.
  • the bola GLH-55B an asymmetric bola with a CS head group on one side and acetylcholine head group on the other side
  • the last stage of optimizing the vesicle formulation will be an introduction of the bola with manose head groups into the vesicle formulation and test the proportion of this bola that does not affect vesicle properties.
  • endocytosis of vesicles with and without manose pendant into macrophage cell line that express manose receptors will be tested.
  • vesicles loaded with a fluorescent marker will be used as a model system for initial experiments and the initial P studies will be carried out with control mice.
  • Mice will be injected with vesicles loaded with a fluorescent marker (vesicles with and without manose pendants) and the amount of the fluorescent dye in the brain will be measured.
  • the proportion of the manose-bola trying will be varied (along with other parameters) optimize targeting efficiency without losing penetration into the brain in normal mice. In normal mice biodistribution of the encapsulated fluorescent dye in various tissues will also be tested.
  • PK studies will be carried with ALS mice, using vesicles loaded with GDNF. Mice will be injected with GDNF-loaded vesicles with and without manose pendant and the distribution and the quantities of the GDNF in the brain will be determined using ELISA and immunohistochemical techniques (for details see method section).
  • mice will be injected with the optimal vesicle formulation and efficacy parameters will be assessed.
  • the following experimental groups will be used (10 animals per group): 1) Mice injected with empty vesicles as control; 2) Mice injected with optimal vesicles containing encapsulated GDNF; 3) Mice injected with free GDNF as a negative control.
  • the mice will receive multiple injections of the test material during 45 days and the intervals between injections will be determined in the PK studies (see above), whereas the criteria for the intervals will be the time period that takes for the clearance of the GDNF from the brain.
  • the treatment period changes in body weight, test motor behavior, and performance of
  • the second stage is the esterification of the secondary hydroxyl groups of the ricinoleic moiety of the diricinoleate 3 with a dicarboxylic acid in the presence of an acidic catalyst under azeotropic conditions.
  • the attachment of the mannose head group will be performed again by a chemoenzymatic esterification, in order to obtain selective binding to the primary hydroxylic position.
  • This is a consecutive nucleophilic substitution reaction, which will likely yield a mixture of monoester 4 and diesters 5, allowing the formation of a symmetric and asymmetric bolacompounds that will be further separated by flash chromatography and their effect on vesicle formation, vesicle stability and targeting will be investigated.
  • GDNF encapsulation determination of GDNF activity, determination of vesicle stability, and determination of GDNF relaease in vitro will be carried out as described hereinabove.
  • Macrophages that express manose receptors on their surface will be grown in 24- well plates the medium will be replaced with culture medium without serum and samples of carboxyfluorescein-loaded vesicles with and without manose pendants or free (non-encapsulated) carboxyfluorescein (equivalent to the encapsulated carboxyfluorescein) will be added to the cells and incubated for 1-5 h at 4 °C or at 37 °C. At the end of the incubation, cells will be washed and either detached from the plates using cell detachment medium and analyzed by FACS

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Abstract

L'invention porte sur des composés bolaamphiphiles selon la formule (I) dans laquelle HG1, HG2 et L1sont tels que définis dans la description. Les composés bolaamphiphiles divulgués et les compositions pharmaceutiques les comprenant sont utiles pour délivrer GDNF ou NGF dans un cerveau d'animal ou d'humain.
PCT/US2013/057956 2012-09-04 2013-09-04 Composés bolaamphiphiles, compositions et leurs utilisations WO2014039501A1 (fr)

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IL237541A IL237541B (en) 2012-09-04 2015-03-03 Amphiphilic compounds, their preparations and uses
US15/099,956 US20160367678A1 (en) 2012-09-04 2016-04-15 Bolaamphiphilic compounds, compositions and uses thereof
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IL280316A IL280316B (en) 2012-09-04 2021-01-20 Amphiphilic compounds, preparations and their use
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WO2016168580A1 (fr) * 2015-04-16 2016-10-20 Lauren Sciences Llc Compositions, composés bolaamphiphiles, et leurs utilisations
WO2021026647A1 (fr) * 2019-08-12 2021-02-18 Integrated Nanotherapeutics Inc. Lipides pour l'administration d'un matériau chargé, leurs formulations et leur procédé de fabrication
CN114945555A (zh) * 2019-08-12 2022-08-26 集成纳米治疗股份有限公司 用于荷电材料递送的脂质、其制剂及其制备方法

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