US20200009067A1

US 20200009067A1
IN
(19) United States
(12)HOFFMAN
Patent Application Publication ( 10) Pub. No.: US 2020/0009067 A1
et al. (43 ) Pub . Date : Jan. 9 , 2020
(54 ) FORMULATION AND METHOD FOR 61/615,457, filed on Mar. 26 , 2012, provisional ap
INCREASING ORAL BIOAVAILABILITY OF plication No. 61 /588,341, filed on Jan. 19, 2012.
DRUGS
(71) Applicant: Yissum Research Development Publication Classification
Company of the Hebrew University of (51) Int. CI.
Jerusalem Ltd., Jerusalem (IL ) A61K 9/51 (2006.01 )
(72) Inventors : Amnon HOFFMAN , Jerusalem (IL ); A61K 9/10 (2006.01)
A61K 31/05 (2006.01)
Abraham J. DOMB, Efrat ( IL ); Anna A61K 31/17 (2006.01)
ELGART, Mevaseret Zion (IL ); Irina A61K 31/343 (2006.01 )
CHERNIAKOV, Gan Yavne (IL ) A61K 31/436 (2006.01)
(73 ) Assignee : Yissum Research Development A61K 47/14 ( 2006.01 )
Company of the Hebrew University of A61K 47/22 ( 2006.01 )
A61K 47/24 (2006.01)
Jerusalem Ltd., Jerusalem (IL ) (52) U.S. CI.
( 21 ) Appl. No .: 16 /577,609 CPC A61K 9/5123 (2013.01); A61K 9/10
(2013.01); A61K 31/05 (2013.01); A61K 31/17
(22) Filed : Sep. 20 , 2019 (2013.01); A61K 47/24 (2013.01 ); A61K
31/436 ( 2013.01 ); A61K 47/14 ( 2013.01) ;
Related U.S. Application Data A61K 47/22 (2013.01 ); A61K 31/343
(62 ) Division of application No. 14 /371,819 , filed on Jul. ( 2013.01 )
11, 2014 , now abandoned , filed as application No.
PCT /IL2013 /050047 on Jan. 17 , 2013 . (57 ) ABSTRACT
(60 ) Provisional application No. 61/ 704,893 , filed on Sep.
24 , 2012, provisional application No. 61/696,540 , Provided is a formulation and method for increasing bio
filed on Sep. 4 , 2012 , provisional application No. availability of an orally administered drug .
Patent Application Publication Jan. 9 , 2020 Sheet 1 of 16 US 2020/0009067 A1
100
80
60 pH 6.8
40 + pH 1.2
20
0
0 20 40 60 80 100 120
Figure 1
50
40
AM-naolipshers AM
2Figure
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Time
(
h)
20
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0.72 0.6 0.5 0.4 0.34 0.1- 0.1 0

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)ml/ug( .Conc
300
250 CBD Nano- lipospheres
200 CBD
150
100
50
0
T
HH Y
0 50 100 150 200 250
Figure 3
16
14
12 Talinolol Nano
10
lipospheres
Conc
.
ng
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ml
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Figure 4
4
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heart liver
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0.8 AM nano
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0.6 AM
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Figure 6A
0.8
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Figure 6B
0.87 + AM nano - lipospheres

0.6 AM + Blank nano-lipospheres
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Figure 6C
1.2 + AM nano - lipospheres

1
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Figure 6D
Blank nano-lipospheres + AM after 2h
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0.1
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Figure 7
juared uonezyddy iqnd ?uoge Jan. 9 , 2020 Sheet 8 of 16 US 2020/0009067 A1
250
240
230
220
210
200
AMN-lipaosnheor s
190
180 Figure
8
170
SHAM 160
150
140
130
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AMBn-lipaosnpheorks 70
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baseline the from change %
juared uonezyddy iqnd ?uoge Jan. 9 , 2020 Sheet 9 of 16 US 2020/0009067 A1
250
240
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200
AMN-lipaospnheor s -
190
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170
SHAM 160
150
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AMBn-lipaospnheorks 70
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AM
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0 10 20 30 40 50 60
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Figure 10
2.00E + 05
1.80E + 05 nanolipospheres + talinolol
Pc(cm/sec)eormfeaibcleinty 1.60E + 05
1.40E + 05 talinolol
1.20E + 05
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8.00E + 06
6.00E + 06
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Figure 11
110
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60 testosterone
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0 20 40 60 80 100 120
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Figure 12
500 TEER
450
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Figure 13
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Figure 14
160+ CBD - Piperin - PNL

140 CBD - SNEDDS
120
100 CBD
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40
20
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Figure 15
PPNLiperine --CBD-c2%-PNLurcumin samymcCBD--PNL1%urcumin CBD-RPNLesveratrol

CBD
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US 2020/0009067 A1 Jan. 9. 2020
FORMULATION AND METHOD FOR tools in pharmacokinetics, as it must be considered when

INCREASING ORAL BIOAVAILABILITY OF calculating dosages for none intravenous routes of admin
DRUGS istration
[0008 ] Despite the great advancements in the area of
FIELD OF THE INVENTION various drug delivery systems such as nano-lipospheres ,
[0001 ] The present invention pertains to a formulation and
many drugs are prone to poor oral bioavailability due to
biological barriers at the enterocyte level, termed “ intestinal
method for increasing the oral bioavailability of drugs by first pass metabolism ” . These biological processes include
utilizing advanced pro - nano lipospheres (PNL ) and PNL Phase I metabolism , namely oxidative enzymes , and Phase
incorporating piperine. II metabolism including conjugation , sulphation and glu
curonidation by intestinal enzymes . In addition , the poor
BACKGROUND OF THE INVENTION oral bioavailability is attributed to efflux transporters e.g.
permeability- glycoprotein (P - gp ) at the enterocyte luminal
[ 0002 ] Many dispersion systems are currently in use as, or membrane [ 3 ].
being explored for use as carriers of substances, particularly [0009 ] P -gp can efflux out a variety of drugs from cells
biologically active compounds. These systems are designed which ultimately lead to unsuccessful drug therapy . It also
to protect the substance from the environment during deliv affects various pharmacokinetic parameters of drugs, such as
ery and to provide a controlled release of the substance to a P -gp substrates like absorption , distribution ,metabolism and
targeted area . In some cases , the goal is to target specific excretion from the body which leads to modified bioavail
sites in the body using the dispersion . In other cases, the goal ability and possible adverse drug reactions. Thus, it is
is to prepare a drug carrier system that acts as a reservoir at believed that the P - glycoprotein efflux pump prevents cer
the site of injection . Dispersion systems used for pharma tain pharmaceutical compounds from transversing the
ceutical and cosmetic formulations can be categorized as mucosal cells of the small intestine and , therefore, from
either suspensions or particles ranging in size from a few being absorbed into the systemic circulation .
nanometers up to hundreds of microns , dispersed in an [0010 ] Knowledge of the approaches to circumvent P - gp
aqueous or non -aqueous medium using suspending agents . efflux pump is critical for targeted drug therapy and formu
Solid particles include microspheres, microcapsules, nano lation design and development of dosage forms. It is estab
particles and nanospheres.
lished that drugs and excipients interact with P - gp in a
[0003 ] One of the in situ methods of preparation of complicated procedure and information of its mechanism of
lipospheres with a particle size below 100 nanometers was interaction are still unclear.
developed by using a dispersible pre -concentrate system [ 1 ] . [0011 ] While there are several methods to inhibit meta
This delivery system , termed pro -nanoliposphere (LIPO bolic and efflux processes by pharmacological agents, there
SPHERES), is based on a solution containing a drug, a are limited pharmaceutical solutions to overcome this prob
triglyceride , a phospholipid and other additives, in a mixture lem .Much more difficult is the case of Phase II metabolism ,
of common surfactants , and an organic solvent that is where there are currently no effective technologies that
miscible with all components . This solution spontaneously control this biological process in the clinical setting .
forms nanoparticles when in an aqueous media, and even in [0012 ] Piperine is an alkaloid responsible for the pun
vivo , e.g., the upper GI lumen content. gency of black pepper (Piper nigrum ), and long pepper
[0004 ] U.S. Pat. No. 7,919,113 [ 2 ] discloses dispersible (Piper longum ), along with chavicine (an isomer of piper
concentrate preparations (nano -lipospheres) for the solubi ine ). The active compound in both Piper longum and Piper
lization of lipophilic drugs to enhance the bioavailability of nigrum is piperine (1 -piperoyl piperidine), which has been
water insoluble drugs. shown to possess bioavailability enhancing activity with
[0005 ] When given orally, a drug is absorbed into the various structurally and therapeutically diverse drugs. It has
enterocyte monolayer in the basolateral side of the intestine , been found that piperine bioavailability-enhancing property
where it can undergo metabolism and/or efflux back into the may be attributed to increased absorption ,which may be due
lumen by trans-membranal transporters. From the apical to alteration in membrane lipid dynamics and change in the
side ofthe entrocytes the drug is delivered via the portal vein conformation of enzymes in the intestine [4 ].
to the liver and thereafter into the systemic blood circulation . [0013 ] Kulkarni S K , et al., [5 ] examined the antidepres
Bioavailability is defined as the fraction of an administered sant effect of curcumin with piperine and concluded that the
dose of unchanged drug that reaches the systemic circula combination of piperine (2.5 mg/kg , i.p., 21 days ), with
tion . By definition, when a medication is administered
intravenously , its bioavailability is 100 % curcumin ( 20 and 40 mg/kg, i.p.) showed significant poten
tiation of its anti - immobility , neurotransmitter enhancing
[0006 ] Research in the field of drug absorption has and monoamine oxidase inhibitory (MAO - A ) effects as
focused on ways to increase drug efficacy by increasing drug compared to the effect curcumin alone.
absorption . To this end , methods have been used to increase [0014 ] Pattanaik S, et al., [6 ] examined the effect of
drug absorption using liposomes as carriers and by design simultaneous administration of piperine (20 mg, p.o.) on
ing more lipophilic drugs. However, these methods have not plasma concentration of carbamazepine (300 mg or 500
been successful in circumventing liver biotransformation mg), twice daily, in epileptic patients and found that piperine
and biliary secretion of drugs. significantly increased the mean plasma concentrations of
[0007 ] Thus, when a medication is orally administered , its carbamazepine in both dose groups.
bioavailability generally decreases due to incomplete [0015 ] U.S. Pat.No. 5,439,891 [7 ] discloses that the active
absorption and first-pass metabolism and also may vary constituentof Piper longum and Piper nigrum , piperine, was
from patient to patient.Bioavailability is one of the essential shown to increase bio -availability of certain anti - tubercular
US 2020/0009067 A1 Jan. 9. 2020
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and anti- leprosy drugs like rifampicin , isoniazid , pyrazina spontaneously forms drug encapsulated nano -particles with
mide , ethambutol and dapsone . a particle diameter of 500 nm or less.
[ 0016 ] U.S. Pat. No. 5,616,593 [8 ] discloses use of pip [0032 ] The piperine - PNL utilizes this concept and in a
erine to improve bioavailability of substances used to treat synergistic manner with piperine , a natural food derived
disease of cardiovascular system , central nervous system , component and adds to the absorption enhancement prop
gastrointestinal treatment or hemopoetic system . erties of the PNL disclosed in U.S. Pat. No. 7,919,113 [2 ],
[0017] U.S. Pat. No. 5,536,506 [ 9 ] and U.S. Pat. No. herein incorporated by reference .
5,972,382 [ 10 ] disclose compositions and methods for the [0033 ] Furthermore, due to piperine's poor aqueous solu
improvement of gastrointestinal absorption and systemic bility and fairly good lipid solubility , it can be readily
utilization of nutrients and nutritional supplements , wherein incorporated into the lipid PNL core . Incorporating piperine
the compositions comprise a minimum of 98 percent ofpure into PNL presents an opportunity to manipulate the critical
alkaloid piperine . systems accounted for the marked first pass effect of various
drugs ( e.g. cannabidiol and tetrahydrocannabinol). The
REFERENCES incorporation of piperine increases the piperine's concen
[ 0018 ] [ 1 ] Bekerman , T., J. Golenser, and A. Domb. J tration that reaches the intestine and the liver, and potentially
Pharm Sci., 93 (5 ): 1264-70 , 2004. competitively inhibits the drugs' metabolism in those meta
[0019 ] [2 ] U.S. Pat. No. 7,919,113 . bolic sites. As a result the oral bioavailability of the various
[0020] [3 ] Lipinski, C. A., et al., Adv Drug Deliv Rev., drugs could be further enhanced as compared to the admin
46 ( 1-3 ): p . 3-26 , 2001. istration of the drugs in PNL not containing piperine.
[0021 ] [4 ] PATIL UK , International Journal of Recent [0034 ] As known to a person of skill in the art, the increase
Advances in Pharmaceutical Research , 4 : 16-23 , October in drug bioavailability is defined as an increase in the Area
2011. Under the Curve (AUC ); being the integrated measure of
[0022] [5 ] Kulkarni S. K., Bhutani M. K., Bishnoi M systemic drug concentrations over time, in units of mass
Psychopharmacology (Berl), 201 :435-442 , 2008 . time/ volume. The AUC from time zero (the time of dosing )
[ 0023 ] [6 ] Pattanaik S , et al., Phytother Res. 23 (9 ): 1281-6 , to time infinity (when no drug remains in the body ) follow
2009. doi: 10.1002/ptr.2676 . ing the administration of a drug dose is a measure of the
[ 0024 ] [ 7 ] U.S. Pat. No. 5,439,891 .
exposure of the patient to the drug .
[ 0025] [ 8 ] U.S. Pat. No. 5,616,593 . [0035 ] The inventors of the present invention have sur
[0026 ] [ 9 ] U.S. Pat. No. 5,536,506 .
prisingly found that administration of cannabidiol (CBD ) in
piperine- PNLs resulted in significantly higher oral relative
[ 0027 ] [ 10 ] U.S. Pat. No. 5,972,382 . bioavailability in comparison to administration of canna
[0028 ] [11 ] Koul S , et al. Bioorg Med Chem ., 8 ( 1 ):251-68, bidiol in PNLs without piperine. Oral administration of
2000 . CBD -piperine -PNL resulted in significantly higher AUC and
SUMMARY OF THE INVENTION
Cmax values by 5 fold and 15 fold , respectively, as compared
to control, (as further described hereinbelow ) attesting to the
[0029 ] The present invention is concerned with optimiza increase in the oral bioavailability of drugs that is attributed
tion of drug bioavailability. It is the purpose of the present to the piperine-PNLs system of the present invention.
application to provide a superior nano -particulate formula [0036 ] Thus, in one aspect of the present invention , there
tion , e.g., delivery system , for enhancing the oral bioavail is provided a composition (formulation or a drug delivery
ability of a broad range of drugs , having a low oral bio system ) for oral administration of at least one drug, said
availability, by combining its administration with pro -nano composition comprising :
lipospheres (PNLs) or with PNL loaded piperine (herein [0037 ] a ) a dispersible concentrate, characterized by
referred to in short as piperine -PNL ). The PNL or piperine being capable of forming , upon contact with an aque
PNL technology enhances the oral bioavailability of drugs, ous solution , particles of a size of less than about 500
in particular lipophilic drugs , which bioavailability is lim nm , said dispersible concentrate comprising :
ited not only due to Phase I metabolism and /or P - gp efflux [0038 ] (i) at least one surfactant;
but also due to direct Phase II metabolism in the intestine . [0039 ] ( ii) at least one solid component at room
[ 0030 ] The inventors of the present invention have devel temperature ; and
oped a PNL and piperine -PNL systems that has multiple [0040 ] ( iii) an amphiphilic solvent;
concerted productive activities that altogether synergisti [ 0041 ] b ) an amount of at least one piperine, piperine
cally enhances bioavailability of poor bioavailable drugs and analog or isomer thereof, e.g., in an amount sufficient
results in more stable , and less variable, absorption of the to increase the bioavailability of said composition .
drugs from the gastrointestinal tract and thereby also to a [0042 ] The " dispersible concentrate ” is a composition
reduction of the required dose of the drug . which spontaneously forms a nano -particulate dispersion in
[0031 ] The development of piperine -PNL is based on a an aqueous medium , for example in water upon dilution, or
PNL pro -nano -dispersion system which employs an orally in the gastric juices after oral administration . The " dispers
administered mixture of lipids, surfactants and co -solvent, ible concentrate ” includes those compositions that form
herein referred to as a " dispersible concentrate ” . This con solid particles having a mean diameter of less than about 500
centrate may be administered orally in combination with an nm upon contact with an aqueous medium .
active agent which is administered either within the pro [0043 ] The “ aqueous medium ,” refers to a water based
nanodispersion formulation or provided shortly before or medium , i.e., a liquid medium in which water is the major
after the administration of the PNL dosage unit ( e.g. soft component. In accordance with the present invention , the
gelatin capsule ). Thus,when reaching the aqueous phase of aqueous medium may be the digestive fluid formed in the
the gastro intestinal (GI) tract , the PNL is released and stomach (e.g., gastric fluid formed by cells lining the stom
US 2020/0009067 A1 Jan. 9. 2020
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ach ), GI tract fluids or any liquid medium , in vivo or ex vivo [0057 ] In some embodiments, the amphiphilic solvent is
in which the herein defined dispersible concentrate is dis selected from methyl lactate , ethyl lactate, propyl lactate ,
solved . spironolactone and N -methylpyrrolidone.
[0044 ] The “ surfactant” is any amphiphilic compound [0058 ] In some embodiments, the amphiphilic solvent is a
generally recognized in the art as having surface active combination of a lower alkyl ester of lactic acid with
qualities . Surfactants generally include anionic , cationic , N -methylpyrrolidone .
nonionic, and zwitterionic compounds, as further described [0059 ] In some embodiments, the amphiphilic solvent
herein . In some embodiments , the surfactant is a combina comprises a combination of a solvents selected from the
tion of at least one high HLB (hydrophilic /lipophilic bal family of lower alkyl esters of lactic acid together with a
ance ) surfactant having an HLB of at least about 8, with at solvent selected from the family of alkyl lactone esters or
least one surfactant being a low HLB surfactant having an N -methylpyrrolidone .
HLB of less than about 5 . [0060 ] In other embodiments, the amphiphilic solvent is
[ 0045 ] In some embodiments, the surfactant is selected combined with a hydrophilic organic solvent such as ethyl
from polyoxyethylene, sorbitanmonolaurate, sorbitanmo ene glycol, glycofurol or PEG 400 .
nooleate and mixtures thereof. [0061] As known , “ Piperine” is (E ,E ) 1- [5-( 1,3 -benzodi
[0046 ] In other embodiments, the composition further oxyl-5 -yl)-1 -oxo -2,4 -pentadenyl] piperidine being the main
comprises an ethoxylated fat and fatty compounds. Some constituent of many Piper species. In accordance with the
non -limiting examples being selected from polyethylenegly present invention , piperine may be obtained commercially
col-hydrogenated castor oil ( e.g. cremophor and cremophor or from a number of species of peppers from the Piperaceae
RH ). family ( e.g., Piper nigrum , P. longum , P. tuberculatum , P.
[ 0047 ] In still other embodiments, the composition further hancei, P. hispidum , P. retrofactum , P. attenuatum , P.
comprises a phospholipid selected from an egg phospho genueense , P. chaba, P. aurantiacum , P. auritum , P. cubeba ,
lipid , a soy phospholipid and lecithin of various grades and P. peltatum , P. umbellatum and P. mystheticum , P. nigrum ).
purities. [0062 ] In some embodiments , piperine utilized in accor
[0048 ] In still yet other embodiments , the composition dance with the invention is obtained from Piper longum or
further comprises a fatty acid ester, e.g., a fatty acid ester Piper nigrum .
that is a solid fat at room temperature such as tricaprin . [0063] In other embodiments, piperine utilized in accor
[0049 ] In some embodiments, the composition of the dance with the invention is in pure form (e.g. piperine at a
purity of > 95 % ) .
present invention is of a particle size that is less than about [0064 ] In other embodiments, piperine is a piperine analog
200 nm .
[ 0050 ] In other embodiments , the composition is of a (e.g., derivatives in which the piperidine ring is substituted ,
e.g., by an amino group , or an ester group ( e.g., C1-6 alkyl
particle size that is less than about 100 nm . esters ) ofmetabolites containing an OH group as described ,
[0051 ] In still other embodiments, the composition is of a for example in Koul S , et al . [11 ]. The piperine analogues
particle size that is less than about 50 nm . may be synthetically obtained or produced from a natural
[ 0052] As used herein , the term “ solid component” refers source .
to solid materials that are solid at room temperature (defined [0065 ] In other embodiments , piperine is a piperine isomer
herein as 25 ° Celcius) and that dissolve in the dispersible ( e.g. chavicine)
concentrate and which upon dispersion in aqueous medium [0066 ] In some embodiments, the amount of piperine in
becomes part of the formed solid nanoparticles . the composition of the invention is in the range of about 0.1
[0053] Some non- limiting examples of solid components, to about 10 mg/kg body weight.
in accordance with the present invention include fatty acids, [0067 ] In accordance with the present invention , the pip
fatty amines and fatty alcohols or their respective esters or erine , piperine analog or isomer thereofmay or may not be
amides that melt at a temperature above 25 ° C .; polymers incorporated into the pro - nano lipospheres formed upon
that are solids at 25 ° C., and paraffins and waxes that are contact of the dispersible concentrate with an aqueous
solid at 25 ° C. solution and may also be partially incorporated into said
[0054 ] In some embodiments, the fatty acid esters are lipospheres.
selected from mono-, di-, and triglycerides and fatty acid [0068 ] The composition comprising piperine ( and /or a
esters with long and short chain alcohols that are solids at piperine analog or isomer thereof) and referred to in short as
25 ° C. In other embodiments, the solid components are piperine -PNL is also related to hereinafter as an empty
selected from tricaprin , trilaurin , trimyristine , tripalmitin , composition , as compared to a composition comprising a
tristearin and mixtures thereof that are solids at 25 ° C. drug (described hereinbelow ).
[0055 ] In still other embodiments , the solid components [0069] The increase in drug bioavailability attributable to
are compounds that solidify in situ upon dispersion in administration of the piperine , as described herein , can be
aqueous medium , e.g., partially or fully hydrogenated veg determined by measuring total systemic drug concentrations
etable oil that are solid at 25 ° C. over time after administration of a composition comprising
[0056 ] The " amphiphilic solvent” utilized in a composi piperine and the drug as compared to after administration of
tion of the invention is selected from lower alkyl (having the drug alone , defined as an increase in the Area Under the
between 1 and 8 carbon atoms) esters of lactic acid , lower Curve (AUC ) as described hereinabove.
alkyl (having between 1 and 8 carbon atoms) lactone esters [0070 ] Thus, when the piperine, piperine analog or isomer
and N -methylpyrrolidone . Some non - limiting examples of thereof are used in sufficient amounts in the composition of
lower alkyl esters include methyl, ethyl, propyl, isopropyl, the present invention , the activity of P -gp and Cytochrome
butyl, hexyl, pentyl and octyl esters . P450 3 A4 (abbreviated CYP3A4) is reduced.
US 2020/0009067 A1 Jan. 9. 2020
4
[0071] The sufficientamounts ofpiperine , piperine analog [0081 ] Some non -limiting examples for compounds sub
or isomer thereof are the amounts necessary to elevate the jected to P - gp efflux and which are not subjected to CYP3A4
systemic concentrations of the drug over time and can be metabolism substrates are talinolol and fexofenadine.
determined by measuring total systemic drug concentrations [0082 ] In some embodiments, the lipophilic compound is
as readily recognized by the skilled artesian . a BCS Class IV compound (e.g. hydrochlorothiazide ,
[0072 ] The increase in drug bioavailability achieved by amphothericin B ).
the use of the composition of the invention and by practicing [0083] Examples for additionalpoorly water soluble com
the methods described herein also results in reduction in pounds with oral bioavailability affected by intestinal and
inter -individual and intra -individual variation in oral bio hepatic first pass metabolism and subjected to P -gp efflux
availability of the drug , enabling the practitioner to better are amlodipine, diltiazem , felodipine, nicardipine, nife
standardize drug administration regimens using the piper dipine, nimodipine, nisoldipine , verapamil , sirolimus,
ine-PNL system of the present invention . tacrolimus, atorvastatin , lovastatin , simvastatin , fexofena
[0073 ] In some embodiments, the empty composition as dine, buspirone , carbamazepine , pimozide, midazolam , tri
defined herein comprises at least one drug. azolam , albendazole, itraconazole, cisapride, colchicine ,
[0074 ] The " drug " is generally any chemical capable of sildenafil, amphotericine B , steroids, polyphenols and can
administration to a mammal , which modifies or alters the binoids .
mammal’s physiology. Thus, in accordance with the present [0084 ] In some embodiments, the lipophilic compound is
invention the “ drug ” is any substance intended for use in the a nutraceutical, a food product or a homeopathic agent ( e.g.,
treatment or prevention of disease or a disorder. Drugs polyphenols , carotenoids, cyclopsporine ).
include synthetic and naturally occurring substances as well [0085 ] In other embodiments, the drug is a lipophilic
as recognized pharmaceuticals, such as those listed in the compound or a mixture of at least two lipophilic compounds.
United States Pharmacopeia , (USP36 NF31, 2013: U.S. [0086 ] In some embodiments, the lipophilic compound is
Pharmacopoeia National Formulary United States Pharma Amiodarone .
copeial Convention 36th Edition 2012; ISBN 978 [0087 ] In other embodiments, the lipophilic compound is
3769258844 ). Tacrolimus .
[0075 ] In some embodiments, the drug is a lipophilic [0088 ] In still other embodiments , the lipophilic com
compound or a mixture of two or more different lipophilic pound is Talinolol.
compounds. As used herein , the “ lipophilic compound " is a [0089] In still yet other embodiments, the lipophilic com
compound that in its non -ionized form is more soluble in pound is cyclosporine A.
lipid or fat than in water. [0090] In additional embodiments , the lipophilic com
[0076 ] In some embodiments , the lipophilic compound is pound is a cannabinoid , a derivative or a synthetic analog
a compound having a LOG P22 (Log P being an estimate of thereof, or a mixture of cannabinoids. Some non -limiting
a compound's overall lipophilicity as recognized by the examples of cannabinoids are tetrahydrocannabinol, canna
skilled artesian ). bidiol (CBD ), cannabinol, cannabigerol, tetrahydrocannabi
[0077 ] The lipophilic compounds, as described herein , are varin , cannabidivarin and cannabichromene .
generally compounds which have limited oral bioavailabil [0091 ] In accordance with the present invention , when the
ity due to first pass metabolism , by (1) Phase I metabolic composition comprises a drug , as defined herein , the drug
enzymes e.g. CYP3A4 (2 ) intra -enterocytes phase II meta may or may not be incorporated into the pro -nano lipo
bolic enzymes (e.g. UDP- glucuronosyltransferases , sul spheres formed upon contact of the dispersible concentrate
fotransferases , N - acetyltransferases, glutathione S -trans with an aqueous solution and may also be partially incor
ferases and methyltransferases; (3 ) efflux from the porated into said lipospheres.
enterocytes back to the gastrointestinal lumen by efflux [0092 ] In some embodiments, the composition of the
pumps ( responsible for the export of many lipophilic and invention further comprises a pharmaceutically acceptable
ampiphilic drugs that impedes their intracellular absorption ) carrier and /or excipient, which physiologically tolerable and
such as P -gp (that is extensively distributed and expressed in do not typically produce an allergic or similar untoward
the intestinal epithelium ), and thus are associated with poor reaction, such as gastric upset , dizziness and the like , when
and variable oral bioavailability administered to a mammal such as a human .
[0078 ] In some embodiments, the lipophilic compounds [0093] The composition of the present invention ( either
are compounds categorized as Class II of the Biopharma empty composition or composition comprising a drug ) is a
ceutical Classification System (BCS) proposed by Amidon , composition suitable for administration by the oral route
G. L., et al. Pharm Res., 12( 3 ):413-20 , 1995. BCS is an (e.g. buccal, sublabial , sublingual) using a swallowable
experimentalmodel that measures permeability and solubil capsule or any other medicament formed in a relatively
ity under prescribed conditions. BCS Class II compounds stable shell ( e.g. soft gel capsule, pill, tablet, granule ).
are considered to have low solubility and high permeability [0094 ] In some embodiments , the composition and drug
and Class IV compounds characterized as having low solu are administered in the same pharmaceutical formulation ,
bility and low permeability. i.e., in a single unit dosage form .
[0079 ] Some non -limiting examples of lipophilic com [0095 ] In other embodiments, the composition and drug
pounds that are subjected to both CYP3A4 and P -gp efflux are administered in a different pharmaceutical formulation ,
include amiodarone , tacrolimus, cannabidiol, cyclosporine , i.e., in a separate unit dosage form .
indinavir , nicardipine , quinidine and verapamil . [0096 ] In accordance with the present invention , the
[0080 ] Some non - limiting examples of compounds that herein defined composition may also be diluted or combined
are subjected to intestinal CYP3A4 metabolism and which in a food or beverage prior to feeding to a subject.
are not subjected P -gp efflux substrates include felodipine, [0097 ] In another aspect of the present invention there is
midazolam , nifedipine and propafenone . provided a method for increasing the bioavailability of a
US 2020/0009067 A1 Jan. 9. 2020
5
drug , the method comprising orally co -administering to a [0111 ] (b ) adding at least one surfactant to the solution
mammal (human or non -human ) in need of treatmentby said obtained in step (a ) (and optionally gently heating and
pharmaceutical drug: stirring to obtain a homogenous solution.
[0098 ] ( 1 ) said drug, and [0112 ] In some embodiments, said process further com
[ 0099 ] (2 ) an empty composition , according to the present prises a step (c ) of adding the drug (e.g. as a powder) to the
invention , wherein said at least one piperine , piperine analog pre - concentrate formed in step (b ) and optionally gently
or isomer thereof being present in said composition in a heating (e.g. at between about 30 ° C. and between about 50 °
sufficient amount to provide bioavailability of said drug C.) and stirring to obtain a homogenous solution which upon
greater than bioavailability of said drug in the absence of gentle agitation in aqueous phase, spontaneously forms drug
said at least one piperine , piperine analog or isomer thereof. encapsulated nano - dispersion .
[0100 ] The herein described method can be used to modify [0113 ] When a piperine comprising composition is
the pharmacokinetics of drugs by multiple mechanisms, desired , piperine (e.g. as a powder ) may be added with the
including inhibition of phase I and phase II metabolism , in drug .
the intestine and in the liver and intestinal inhibition of P - gp [0114 ] In some embodiments, the ratio of amphiphilic
efflux pumps. solvent and phospholipid in step (a ) of said process is
[0101] In some embodiments, the empty composition is between 8 and 1, respectively .
co -administered substantially simultaneously with the drug [0115 ] In some embodiments, the ratio of amphiphilic
(either less than 20 min . before , less than 20 min after or solvent and phospholipid I step (a ) of said process is about
together with the drug ). 8 : 1, respectively.
[ 0102 ] In other embodiments, the empty composition and [0116 ] The present invention also contemplates the use of
drug are sequentially administered . the composition of the invention for the preparation of a
[0103] The term “ sequentially ” or “ sequentially adminis medicament for increasing the bioavailability of a drug .
tered ” as used herein is intended to mean that an empty [0117] The medicament may include pharmaceutically
composition is administered before one or more subse acceptable carriers, for example, vehicles, adjuvants , excipi
quently administered drugs or that the drug is administered ents, or diluents, which are well -known to those who are
before the empty composition . skilled in the art and are readily available to the public. It is
[0104 ] In some embodiments , the empty composition is preferred that the pharmaceutically acceptable carrier be one
administered 0 minutes, 20 minutes , before the subsequently which is chemically inert to the piperine -PNL and active
administered drug. drug and one which has no detrimental side effects or
[0105 ] In other embodiments, the drug is administered 0 toxicity under the conditions of use.
minutes, 20 minutes, before the subsequently administered [0118 ] Medicament formulations suitable for oral admin
empty composition . istration may comprise (a ) liquid solutions, such as an
[0106 ] In some embodiments , the drug is administered in effective amount of the composition dissolved in a non
at least one dose within 24 hrs after administration of the aqueous diluent; (b ) capsules, sachets , tablets , lozenges, and
dose of the empty composition ; i.e., the empty composition troches, each containing a predetermined amount of the
is not administered again before or with every administra composition , as solids or granules ; (c) powders; (d ) suspen
tion of the drug , but may be administered intermittently sions in an appropriate non -aqueous liquid ; and (e ) suitable
during the course of treatment. non -aqueous emulsions. Liquid formulations may include
diluents , such as alcohols, for example , ethanol, benzyl
[0107 ] In some embodiments , when the drug is adminis alcohol, and the polyethylene alcohols , either with or with
tered before the empty composition , clinical evaluation of out the addition of a pharmaceutically acceptable surfactant,
the drug's effect (e.g. on a disease state ) and of the drug's suspending agent, or emulsifying agent. Capsule forms may
plasma concentration are measured ( e.g. using methods be of the ordinary hard- or soft-shelled gelatin type contain
well-established as common practice in the art) and based on ing, for example, surfactants, lubricants, and inert fillers ,
said measured parameters , a decision of the amount of
empty composition to be administered thereafter or before such as lactose , sucrose , calcium phosphate, and corn starch .
Tablet formsmay include one or more of lactose, sucrose ,
the administration of the drug, is made . According to such mannitol, corn starch , potato starch , alginic acid , microc
embodiments, depending on the type of disease to be treated , rystalline cellulose , acacia , gelatin , guar gum , colloidal
the condition of the patient or any other parameters that are silicon dioxide , talc, magnesium stearate, calcium stearate ,
readily apparent to the skilled artesian , the amount of empty zinc stearate , stearic acid , and other excipients, colorants,
composition to be administered and/or amount of piperine in diluents, buffering agents , disintegrating agents , moistening
said empty composition are determined . agents, preservatives, flavoring agents , and pharmacologi
[0108 ] Thus, in accordance with the present invention , the cally compatible carriers. Lozenge formsmay comprise the
empty composition and drug may be administered together, composition in a flavor, usually sucrose and acacia or
alternately or intermittently in all of the various aspects of tragacanth , as well as pastilles comprising the composition
the invention , depending on the type of drug, the type of in an inert base, such as gelatin and glycerin , or sucrose and
disease to be treated with the drug and /or any other consid acacia , emulsions, gels, and the like containing .
erations that are readily apparent to skilled artesian . [0119 ] In another one of its aspects the present invention
[0109 ] In still another aspect of the present invention , provides the use of the composition of the invention for
there is provided a process for preparing the composition of increasing the bioavailability of a drug.
the invention, said process comprising : [0120] In still another one of its aspects the present
[0110 ] (a) dissolving the amphiphilic solvent and option invention provides the use of the composition of the inven
ally phospholipid (and optionally gently heating to achieve tion for the preparation of a medicament for the treatment of
complete dissolution of the phospholipid ), at least one disease and/ or disorder .
US 2020/0009067 A1 Jan. 9 , 2020
6
[0121] In an additional aspect, the present invention pro said other therapeutic modality results in more efficacious
vides the composition of the invention for use in the treat treatment (e.g. shortening of the time period to achieve
ment of at least one disease and/ or disorder. remission , treatment and /or amelioration ) of the disease or
[0122 ] The present invention also contemplate the use of disorder treated with said other therapeutic modality than
the composition of the invention for treating at least one would have been obtained without the administration of the
disease and/or disorder composition of the invention .
[0123 ] In some embodiments , the disease is a disease [0134 ] Generally , when used in an add -on therapy, the
treatable with at least one cannabinoid and/ or a derivative composition of the invention is administered less than 24
thereof. hours after the initiation of treatment with said other thera
[0124 ] The “ disease treatable with at least one cannabi peutic modality .
noid or a derivative thereof" generally refers to a disease or [0135 ] By yet another aspect, the present invention pro
disorder selected from intractable cancer pain , neuropathic vides a kit for oral administration of the composition of the
and chronic pain , postoperative pain , rheumatoid arthritis, invention , said kit comprising:
multiple sclerosis and spasticity , fibromialgia , inflammation , [ 0136 ] a ) an empty composition as defined herein ;
gastrointestinal disorders (e.g. nausea and vomiting, motility [0137 ] b ) instructions of use .
disorders ), acute schizophrenia , cancer, tics and behavioral [0138] In some embodiments, the herein defined kit fur
problems experienced by patients with tourette's syndrome, ther comprises at least one drug, as defined herein .
parkinson's disease, huntington's disease, diabetes and dia [0139 ] The components composed in any of the kits of the
betic complications, cerebrovascular disorders and glau invention , may be contained in a single vessel or holding
coma .
unit or in separate vessels and contain a label attached to or
[0125 ] The term “ treatment” or any lingual variation packaged with the container that describes the contents of
thereof is used herein to denote treating the disease , disorder the vessels and provides indications and/or instructions
or condition , or ameliorating, alleviating, reducing, or sup regarding administration of contents of the vessels to a
pressing symptoms of the disease, or slowing or stopping the mammalian subject in need of treatment with said kit(s ). The
progress of the disease . kit form is particularly advantageous when the components
[0126 ] In still another one of its aspects , the present are contained in different vessels for administration in
invention provides the use of the composition of the inven different dosage amounts or when titration of the individual
tion in combination with other therapeutic modalities. components of the kit (e.g., dispersible concentrate , piperine
[0127 ] As used herein , the terms “ therapeutic modalities” , and drug ) is desired by the prescribing physician .
generally refers to any therapeutic agent, treatment or pro [0140 ] The present invention also provides a composition
tocol/method that can be used in the treatment or alleviation for oral administration of at least one drug, said composition
of a disease or disorder or at least one symptom thereof. comprising a dispersible concentrate , characterized by being
[0128 ] In some embodiments , the therapeutic modality is capable of forming, upon contact with an aqueous solution ,
selected from chemotherapy, hormonal therapy, radiation particles of a size of less than about 500 nm , said dispersible
therapy, surgery, biological therapy and immunotherapy. concentrate comprising:
[0129 ] In some embodiments, the therapeutic modality is [ 0141 ] (i) at least one surfactant;
a non - drug therapy. [0142 ] ( ii ) at least one solid component at room tem
[0130 ] Thus, according to this aspect of the invention, a perature ; and
subject may be administered with a therapeutic agent (per [0143] ( iii) an amphiphilic solvent.
taining to the other therapeutic modality ) in accordance with [0144 ] It should benoted that where various embodiments
the therapeutic regimen that is dictated by said other thera are described by using a given range, the range is given as
peutic modality while the composition of the invention may such merely for convenience and brevity and should not be
be sequentially or substantially simultaneously administered construed as an inflexible limitation on the scope of the
in single or in multiple unit dosage forms. invention . Accordingly , the description of a range should be
[0131 ] In accordance with the present invention , the considered to have specifically disclosed all the possible
administration of the therapeutic agent pertaining to said sub-ranges as well as individual numerical values within that
other therapeutic modality can be carried out by any appro range .
priate route including , but not limited to , oral routes, intra [0145 ] It is appreciated that certain features of the inven
venous routes, intramuscular routes or any other adminis tion , which are , for clarity, described in the context of
tration route that is suitable to achieve the desirable effect of
the other therapeutic modality . separate embodiments,may also be provided in combination
[0132 ] In some embodiments, the composition of the in a single embodiment. Conversely , various features of the
invention is used to treat at least one symptom of a disease invention , which are , for brevity , described in the context of
or disorder treated by said other therapeutic modality. For a single embodiment,may also be provided separately or in
example , when the disease is cancer and the therapeutic any suitable sub -combination or as suitable in any other
modality is indicated to eradicate the cancer, the herein described embodiment of the invention . Certain features
defined composition may be used to treat the pain symptoms described in the context of various embodiments are not to
associated with the cancer. be considered essential features of those embodiments,
[0133] In other embodiments , the composition of the unless the embodiment is inoperative without those ele
ments.
invention is used as an add -on (adjuvant) therapy to said
other therapeutic modality to augment the treatment of the BRIEF DESCRIPTION OF THE DRAWINGS
disease or disorder to be treated by said other therapeutic
modality . In accordance with such embodiments the admin [014 ] In order to better understand the subjectmatter that
istration of the composition of the invention together with is disclosed herein and to exemplify how it may be carried
US 2020/0009067 A1 Jan. 9. 2020
7
out in practice , embodiments will now be described , by way [0159 ] FIG . 13 presents TEER values (outer line chart)
of non -limiting example only , with reference to the accom ( USD ) of Caco - 2 cells monolayers in the presence of blank
panying drawings, in which : nano -lipospheres vs. buffer, which was used as a control
[0147] FIG . 1 presents comparative dissolution profile of (n = 3 ). The embedded column chart represents Papp values
AM (Amiodarone ) nano lipospheres containing soft gelatin ( USD ) of mannitol across the Caco -2 monolayers in the
capsules at pH 6.8 and 1.2 (n = 3 for each pH ). presence of blank nano - lipospheres vs. buffer.
[0148 ] FIG . 2 presents plasma AM concentration - time plot [0160 ] FIG . 14 presents the percentof LDH release (rela
(mean + S.E.M .) following PO administration of AM and tively to 100 % release control) upon incubation of Caco -2
AM -nano - lipospheres, 12.5 mg/kg (n = 6 for each group ) . cellwith medium containing various concentrationsofblank
[0149] FIG . 3 present plasma CBD concentration - time nano - lipospheres . Nano - lipospheres increased LDH release
plot (mean : S.E.M .) following PO administration of CBD in concentration dependant manner with highest toxicity
and CBD nano lipospheres, 12.5 mg/kg (n = 6 for each obtained at 10 % nano - lipospheres concentration and negli
group ). gible toxicity produce by nano - lipospheres concentration of
[0150 ] FIG . 4 presents plasma talinolol concentration 1 % and less .
time plot (mean + S.E.M .) following per os (PO ) administra [0161 ] FIG . 15 presents plasma CBD concentration - time
tion 4 mg/kg of talinolol vs. AM -nano - lipospheres (n = 5 for plot (mean S.E.M.) following PO administration of CBD ,
each group ). CBD -PNL and CBD - Piperine- PNL to rats , CBD - 15 mg/kg ;
[0151] FIG . 5 presents plasma (m /mL ) and tissue (m /g ) Piperine 10 mg/kg (n = 6 for each group ). (* ) Significant
concentrations of AM following PO administration of 12.5 difference (p < 0.05 ) from CBD corresponding value is found .
mg/kg of AM and AM nano - lipospheres for 5 days. Signifi [0162 ] FIG . 16 presents plasma concentration vs. time plot
cant differencewas found both in plasma and in correspond for CBD and Cannabidiol (CBD )-PNL with and without
ing heart and liver concentrations. (n = 3 for each group ). various absorption enhancers : Resveratrol (CBD -Resvera
[0152] FIGS. 6A -D present plasma AM concentration trol-PNL ), Curcumin 1 % & 2 % (CBD -curcumin 1 % -PNL
time plot (mean : S.E.M .) following PO administration 12.5 and CBD -curcumin 2 % -PNL , respectively ) and CBD -Pip
mg/kg of AM vs. AM -nano - lipospheres ( A ), AM vs. erine-PNL to rats , CBD - 15 mg/kg; (n = 6 for each group).
AM + blank nano - lipospheres (B ), AM -nano -lipospheres vs. * The concentration of piperine and resveratrol in the for
AM + blank nano - lipospheres (C ), and AM -nano -lipospheres mulation was 2 % ; ** PNL - Pro -Nano-Lipospheres .
vs. AM -nano - lipospheres + blank nano - lipospheres in the ***CBD Cannabidiol. (* ) Significant difference (p < 0.05)
dose of 12.5 mg/kg (D ). (n = 6 for each group). from CBD corresponding value is found . (†) Significant
[0153 ] FIG . 7 presents plasma AM concentration -time plot difference (p < 0.05 ) from CBD -PNL corresponding value is
(mean : S.E.M .) following PO administration of blank nano found .
lipospheres following AM (12.5 mg/kg ) administration after
2 h or water following AM (12.5 mg/kg) administration after DETAILED DESCRIPTION OF EMBODIMENTS
2 h ( n = 3 for each group ). [0163] The present invention is thus based on the realiza
[0154 ] FIG . 8 presents mean IBI changes ( as percentage tion that it is possible to increase the oral bioavailability of
from baseline ) following 12.5 mg/kg AM administration in a drug, such as a drug containing a lipophilic compound , by
3 different formulations: AM , AM nano - lipospheres, administering the drug in a composition comprising pro
AM + Blank nano lipospheres and administration of blank nano lipospheres incorporating piperine .
nano -lipospheres. The drug was administered at t = 60 min [0164 ] The following specific examples illustrate various
(n = 6 for each group ) aspects of the present invention , and are not intended to be
[0155 ] FIG . 9 presents mean HR changes ( as percentage limiting in any way . For all examples, all the ingredients
from baseline ) following 12.5 mg/kg AM administration in were dissolved in the solvent using magnetic stirrer, and
3 different formulations : AM , AM nano-lipospheres , heated gently (~ 40-45 ° C.) for 15-45 minutes until the
AM + Blank nano lipospheres and administration of blank ingredients were completely dissolved . To obtain particles ,
nano -lipospheres. The drug was administered at t = 60 min the concentrate formulation was diluted in at least 10
(n = 6 for each group ). volumes of water in aqueous solution .
[0156 ] FIG . 10 presents plasma concentration - time profile [0165 ] For all experiments described below , the particle
( USE ) obtained following IV administration of 12.5 ug/kg size of the diluted formulation was measured with VLA
Amiodacore® and AM nano - lipospheres. particle size analyzer (Coulter N4 MD Submicron Particle
[0157 ] FIG . 11 presents papp ( SEM ) values of talinolol Size Analyzer), suitable for submicron particle size deter
vs. talinolol + blank nano -lipospheres in Caco - 2 model in A mination . About 75 ul of the concentrate formulation were
to B and B to A directions (n = 3 for each group). Signifi added to five milliliters of water. The particle size of the
cantly higher (p < 0.001) Papp values of talinolol + blank diluted formulation did not change when it dispersed in five
nano -lipospheres vs. talinolol were obtained in A to B milliliters of 0.1N HCl solution . For all experiments
direction . Significant (p < 0.001) reduction in Papp values described below , unless otherwise stated , the active phar
obtained for talinolol+ blank nano - lipospheres vs. talinolol in maceutical ingredient (API) was in the base form (and not in
B to A direction . the salt form ).
[0158 ] FIG . 12 presents the difference (p < 0.01) found
between intact AM concentrations remaining following 120 Preparation of a Composition According to the Invention
min . incubation of AM nano - lipospheres vs. AM in isolated
rat CYP3A4 microsomes . Significant difference (p < 0.05 ) [0166 ] The composition containing amiodarone HCl or
was found between testosterone with blank nano - lipo tacrolimus, solvent, TRC (tricaprin ) or alternatively TRL
spheres vs. testosterone alone incubated under the same ( trilaurin ), egg phospholipid ( Avanti, USA ), TweenTM 20 ,
conditions (n = 3 for each study ). SPANTM 80 and CremophorTM was prepared as given in
US 2020/0009067 A1 Jan. 9. 2020
8
Table 1 (all amounts of ingredients , in Table 1 as well as in TABLE 2B - continued

the following tables, are given in milligrams). All compo
sitions had a particle size of less than 100 nm . The particle Formulations of various hydrophobic API and Vitamins
in different amounts
size decreased as the amount of either ethyl lactate or Poly
Ethylene Glycol (PEG ) 400 was increased . Ethyl lactate was Ingredient 1 2 3 4 5 6 7 8 10
generally more effective than Poly Ethylene Glycol (PEG )
400 for providing smaller size particles . SPAN TM 80
Tween TM 20
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
Cremophor TM 40 40 40 40 40 40 40 40 40 40
TABLE 1 Ethyl lactate 140 140 140 140 140 140 140 140 140 140
Sildenafil 20 40
Amiodarone Pre -concentrate Basic Formulations Tadalafil 30 60
Vardenafil 20 40
Ingredient 1 2 ??? 4 5 6 Maxiletine 20 40
Vitamin D2 40
Amiodarone HCL 10 10 10 10 20 20 Vitamin D3 40
Tricaprin 40 40 52
Trilaurin 40 40 52
Egg phospholipids 40 40 40 40 20 20
Tween TM 20 60 60 60 60 52 52
SPAN TM 80 40 40 40 40 52 52 Amiodarone HCL Pre -Concentrate Formation
Cremophor TM 52 20 20 20 52 52
[0169 ] The final Amiodarone HCL composition was based
Ethyl lactate 160 160 190 180
Poly Ethylene Glycol 160 160 on preliminary formulation optimization studies and
(PEG ) 400 selected according to optimal solubilization capacity of the
active ingredient and smallest particle size obtained upon
dilution in aqueous phase, as described above . Initially
Effect of Various Hydrophobic Pharmaceutical Active amphiphilic co -solvent (ethyl lactate) and phospholipid
Ingredients (API) and Vitamins on Particle Size (lecithin ) at the ratio of 8 :1 respectively were placed in a
[0167] Different compositions containing various hydro clean scintillation tube and heated to 40 ° C. until the lecithin
phobic Pharmaceutical Active Ingredients (API) and Vita was completely dissolved . Then triglyceride ( trilaurin ),
mins were prepared as described in Tables 2A and 2B . The polyoxyl 40 -hydroxy castor oil, Tween 20 , and Span 80 at
effect of the API's on the particle size when dispersed in the ratio of 1: 1:1 :1 were added ; the mixture was gently
water was examined Briefly , the droplet size of compositions stirred and heated to 45 ° C. until a homogenous solution was
which included hydrophobic API had small particle sizes formed . Further, Amiodarone HCL powder was added ,
(less than 100 nm ) in cases of low API concentration but the forming the AM pre -concentrate containing 3 % AM . This
particle size was increased in cases that the API concentra pre -concentrate was gently stirred and heated to 45° C. till
tion was higher (and was above 100 nm in some cases ). homogenous solution was formed . Upon gentle agitation in
Similar phenomenon was received with the vitamins. aqueous phase , this pre-concentrate spontaneously forms
[0168 ] Other hydrophobic API's and Vitamins were also drug encapsulated O /W nano -emulsion . Amiodacre?
tested and similar results were obtained ( not shown in the ampoules content (50 mg/mlAmiodarone HC1, 20.2 mg/mL
next table). In all cases, the pharmaceutical active ingredient benzyl alcohol and 100 mg/mL Tween 80 in water) was used
(API) was in the base form (and not in the salt form ). as control AM throughout all in - vivo and in - vitro studies .
[0170 ] Talinolol pre -concentrate was prepared by the
TABLE 2A same method as amiodarone HCL pre - concentrate, except
for the different triglyceride that was used . For the prepa
Formulations of various hydrophobic API in different amounts ration of the pre -concentrate a triglyceride; lecithin was
Ingredient 1 2 3 4 5 6 7 8 9 10 used . The concentration of the compound i.e. talinolol in the
pre -concentrate was 1 % .
Trilaurin
Egg phospholipids
40
20
40
20
40
20
40
20
40
20
40
20
40
20
40
20
40
20
40
20
[0171 ] Blank pre -concentrate was prepared by the same
SPAN TM 80 40 40 40 40 40 40 40 40 40 40 method as miodarone HCL preconcentrate , except no active
Tween TM 20 40 40 40 40 40 40 40 40 40 40 ingredient was added .
Cremophor TM
Ethyl lactate
40 40 40 40 40 40 40 40 40 40 [0172 ] Tacrolimus nano lipospheres were prepared by the
Soltanol
140
20
140
40
140 140 140 140 140 140 140 140
same method to produce preconcentrate containing 5 %
Flecaininde 20 40 tacrolimus . Prograf® capsules were used as control tacroli
Propafenone 20 40 mus throughout in -vivo studies .
Ezetimibe 20 40
Doxorudicin 20 40
Cannabidiol (CBD ) Nano Lipospheres
[0173 ] CBD nano lipospheres formulation was prepared
TABLE 2B
by pre -concentrate preparation method . The final nano lipo
spheres composition was based on preliminary formulation
Formulations of various hydrophobic API and Vitamins optimization studies and selected according to optimal solu
in different amounts bilization capacity of the active ingredient and smallest
Ingredient 1 2 3 4 5 6 7 8 9
particle size obtained upon dilution in aqueous phase . Prior
10
to adding active ingredient, two kinds of mixtures were
Trilaurin 40 40 40 40 40 40 40 40 40 40 prepared . The first is a mixture of amphiphilic co -solvent
Egg phospholipids 20 20 20 20 20 20 20 20 20 20 ( ethyl lactate ) and phospholipid (lecithin ) at the ratio of 8 : 1,
respectively . The second is a mixture of a triglyceride
US 2020/0009067 A1 Jan. 9. 2020
9
( tricaprin ), polyoxyl 40-hydroxy castor oil, Tween 20, and dissolution paddle rotating at 50 rpm . The emulsification
Span 80 at the ratio of 1: 1: 1 :1 . Both mixtures were gently time was visually assessed as previously reported .
stirred and heated to 40 ° C. till homogenous solutions were
formed . Both mixtures were added at the ratio of 1 : 1 w / w to Emulsification Time and Drug Release Results
a clean scintillation tube containing CBD powder, forming [0177 ] In both tested pH environments, pro nano - lipo
the CBD nano lipospheres preconcentrate containing 3 % spheres presented similar dissolution profile, where the drug
CBD . This pre-concentrate was gently stirred and heated to was entirely released within about 10 minutes ( FIG . 1 ). The
40° C. till homogenous solution was formed . Upon gentle emulsification time obtained for pro nano -lipospheres was
agitation in aqueous phase, this preconcentrate spontane ~ 10 sec .
ously forms drug encapsulated 0 / W nanoemulsion . CBD
solution in Propylen Glycol/EthOH /Water 30/30/40 respec In - Vivo PK Studies
tively was used as control throughout all in - vivo studies .
[0178] Animals:
Characterization of the Formulations [0179 ] Male Wistar rats weighing 300-350 g were used for
[ 0174 ] The mean particles diameter, zeta potential and the in vivo PK and ex vivo permeability studies. The project
polydispersity index of the formed formulations by 1:10 adhered to the principles of Laboratory Animal Care . All
dilution of the pre -concentrate in water are listed in Table 3 . animals were deprived of food but not water 12 h prior to the
Amiodarone HCL , talinolol, tacrolimus, CBD and blank experiments. All surgical and experimental procedureswere
formulations were administered to animals by 1:10 dilution reviewed and approved by the Animal Experimentation
of the pre -concentrate in water. Thus, the characterizations Ethics Committee of the Hebrew University Hadassah
were AM assessed following this dilution . Medical School Jerusalem . An indwelling cannula was
placed in the right jugular vein of each animal for systemic
TABLE 3 blood sampling, by a method described by Hoffman , A. and
G. Levy., J. Pharmacol Exp Ther., 249( 1 ):117-22 , 1989.
The mean particles diameter, zeta potential and
polydispersity index of the formed formulations Amiodarone (AM ) Bioavailability Studies
by 1:10 dilution of the pre - concentrate in water
[0180 ) AM nano - lipospheres were freshly prepared 30
Size
Zeta
potential min before each experiment, by vortex -mixing 1 mL of the
Formulation (d · nm ) (mv) Pdi pre - concentrate in 9 mL water pre -heated to 37 C0 for 30
Amiodarone
sec. The obtained AM concentration was 3 mg/mL . AM
HCL
10 35.2 0.48 nano - lipospheres (12.5 mg/kg ) were administered to the
CBD 26 0.07 animals by oral gavage (n = 6 ). The control group received
Talinolol 45 5.9 0.45 12.5 mg/kg AM solution prepared from Amiodacore®
Blank 52 13.2 0.38 ampoules content ( AM 50 mg/mL ) dissolved in water to
obtain 5 mg/mL concentration ( n = 6 ) . Systemic blood
samples ( 0.35 mL ) were taken at 5 min pre -dose , 0.5 , 1 2 ,
Drug Release Studies 4 , 8 , 12 , 24 , 36 and 48 h post -dose .
[0181] For tacrolimus bioavailability studies , tacrolimus
[0175 ] Soft gelatin capsules containing AM nano - li nanolipospheres were freshly prepared 30 min before each
posheres (0.5 g ) were assessed for in - vitro release using experiment,by vortex -mixing 1 mlof the pre -concentrate in
standard USP 24 method , apparatus II (paddles ) equipment 9 ml water pre -heated to 37 C0 for 30 sec . The obtained
(Caleva ST7, G.B. Caleva Ltd., Dorset, UK ). A stirring concentration was 0.5 mg/ml. tacrolimus nano - lipospheres
speed of 75 rpm was used at 370 C in 500 ml of release ( 1 mg/kg) were administered to the animals by oral gavage
medium ( either pH 6.8 or pH 1.2 ). At predetermined time (n = 6 ). The control group received 1 mg/kg tacrolimus
points samples of the release medium ( 0.5 mL ) were with suspension prepared from Prograf® capsules content sus
drawn and analyzed by HPLC (analytical methods section ) pended in water to obtain 0.5 mg/ml concentration (n = 6 ).
for AM content. Gastric buffer pH 1.2 was prepared as Systemic blood samples (0.3 ml) were taken at 5 min
following: 250 ml of NaCL 0.2M were mixed with 425 ml predose, 0.25 , 0.5 , 0.75 , 1, 1.5 , 2 , 4 , 8 , and 12 h post -dose .
of HCL 0.2M then distilled water was added to make a total [0182 ] For talinolol relative bioavailability studies: nano
volume of 1 L. Duodenal buffer pH 6.8 was prepared as lipospheres were freshly prepared 30 min before each
following : 112 ml of NaOH 0.1 M were mixed with 250 ml experiment, by vortex -mixing of the pre -concentrate in
of potassium dehydrogenphosphate then distilled water was water (1 :9 ) pre -heated to 37 C0 for 30 sec . The obtained
added to make a total volume of 1 L. talinolol concentration was 1 mg/mL . Talinolol nano - lipo
spheres (4 mg/kg) were administered to the animals by oral
Emulsification Time Study gavage (n = 6 ). The control group received 4 mg/kg talinolol
dissolved in PEG400 :water : ethanol 25:60:15 to obtain 1
[0176 ] To assess the self-emulsification properties, the mg/mL concentration (n = 6 ).
emulsification time of the pro nanolipospheres was evalu [0183 ] For CBD relative oral bioavailability studies, nano
ated according to United States Pharmacopeia (USP ) XXIII , lipospheres was freshly prepared 30 min before each experi
dissolution apparatus II (Caleva ST7 , G.B. Caleva Ltd., ment, by vortex -mixing of the pre -concentrate in water ( 1:9 )
Dorset, UK ). Briefly , 0.5 mL of the pro nano -lipospheres pre -heated to 37 C0 for 30 sec . The obtained CBD concen
was added drop wise to 500 mL of distilled water at 37 ° C. tration was 3 mg/mL. CBD nano lipospheres ( 15 mg/kg) was
Gentle agitation was provided by a standard stainless steel administered to the animals by oral gavage (n = 6 ). The
US 2020/0009067 A1 Jan. 9. 2020
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control group received ( 15 mg/kg ) CBD solution in propyl TABLE 5 - continued

ene glycol/EthOH /water 30/30/40 respectively (1 mg/mL )
( n = 6 ). Coefficient of variation ( % CV ) in various time points during
[0184] Systemic blood samples (0.35 mL) were taken at 5 two hours following oral administration of tacrolimus and tacrolimus
min pre -dose, 20 min, 40 min , 1 h 1.3 h , 1.6 h , 2 h , 3 h and nano - lipospheres 1 mg/kg (n 5 for each group ).
4 h post-dose .
AM PK Results tacrolimus-nano
[0185 ] The plasma concentration time profiles for AM and tacrolimus lipospheres time (h )
AM - nano -lipospheres following oral administration of dose
equivalent to 12.5 mg/kg AM are shown in FIG . 2. The 83.5 22.0 (* ) 1
corresponding AUC and Cmax parameters obtained in these 70.5 30.7 (* ) 1.5
in vivo experiments are listed in Table 4. The bioavailability 103.5 45.4 (* ) 2
of AM nano -lipospheres was significantly greater in com
parison to AM alone . Similar results were obtained for the (* ) Significantly lower % CV than the corresponding values at the same time point in the
Cmax values. group that received tacrolimus alone.
[0186 ] Another finding is that the erratic absorption that
characterizes Amiodarone becomes more regular when the [0190 ] These findings demonstrate that Amiodarone and
drug is administered in the nano- formulation . The reduced tacrolimus bioavailability increases not only when it is
fluctuations in Cmax are of high importance since Amio encapsulated in nano-lipospheres , but even the simultaneous
darone is characterized by narrow therapeutic window and presence of empty ( i.e. drug-less) nano - lipospheres in the
life- threatening side effects. Reduction of such fluctuations intestine. Moreover , encapsulation into nanolipospheres
may improve the safety of Amiodarone therapy . In addition , reduces the high variability in plasma concentrations , typical
the overall dose reduction which may be achieved by higher for Amiodarone, tacrolimus and other BCS class II com
bioavailability might result in reduction of side effects of this pounds .
drug.
Tacrolimus PK Results CBD PK Results
[ 0187 ] The tacrolimus concentration vs. time plot follow [0191] The plasma concentration time profiles for CBD
ing oral administration of the drug alone or compounded in and CBD nano lipospheres following oral administration of
nano - lipospheres (1 mg/kg) is presented in FIG . 3, and the 15 mg/kg CBD to rats are shown in FIG . 3. The correspond
derived PK parameters are presented in Table 6 . ing AUC and Cmax parameters obtained in these in vivo
[0188 ] The bioavailability of tacrolimus nano - lipospheres experiments are listed in Table 6 .
was significantly greater in comparison to tacrolimus alone .
Similar results were obtained for the Cmax values. [0192 ] The bioavailability of CBD nano lipospheres was
[0189 ] Table 5 clearly demonstrates the reduced variabil significantly greater in comparison to CBD alone . Similar
ity in plasmas tacrolimus concentrations obtained following results were obtained for the Cmax values .
tacrolimus-nano - lipospheres administration . Table 5 repre
sents the coefficient of variation ( % CV ) in various time TABLE 6
points during two hours following administration . PK parameters derived from PO administration of CBD and
CBD nano lipospheres 15 mg/kg (n 6 for each group ).
TABLE 4
PK parameters derived from PO administration of tacrolimus AUC (min *ng/mL) Cmax (ng /mL)
and tacrolimus - SNEDDS 1 mg/kg (n = 5 for each group ). 15533 + 11679 105 + 103 CBD
46356 = 14567 (* ) 337 + 100 (* ) CBD nano - lipospheres
Cmax (ng/mL) AUC ( hr */ ng/ mL)
14.7 + 4/11 40.8 + 10.6 tacrolimus
(* ) Significant difference (p < 0.05 ) from CBD corresponding value is found
23.8 - 2.66 (* ) 69.4 + 8.96 (* ) Tacrolimus- nano
lipospheres [0193 ] Thus , incorporation of CBD into nano lipospheres
(* )Significant difference (p < 0.05) from tacrolimus corresponding value is found . is a promising strategy to overcomemajor obstacles in the
way of development oral treatments with CBD such as first
pass hepatic metabolism , instability in the acidic gastric pH
TABLE 5 and/or low water solubility , leading to incomplete absorp
tion
Coefficient of variation (% CV ) in various time points during
two hours following oral administration of tacrolimus and tacrolimus
nano -lipospheres 1 mg/kg (n 5 for each group ). Talinolol PK Results
tacrolimus- nano
tacrolimus lipospheres time ( h ) [0194 ] Significantly higher plasma talinolol concentra
95.1 31.5 (* ) 0.25 tions were obtained following talinolol nano lipospheres
90.8 35.8 (* ) 0.5 administration in comparison to free talinolol (FIG . 4 ).
73.2 9.1 (* ) 0.75 Corresponding increase in AUC and Cmax values are shown
in Table 7 .
US 2020/0009067 A1 Jan. 9. 2020
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TABLE 7 the same route (n = 6 ). The blank nanolipospheres volume

was equal to AM nano-lipospheres volume administered in
Summary of PK parameters derived from PO administration the bioavailability studies and the AM dose was 12.5 mg/kg
of talinolol vs. talinolol nano - liposheres at the (approximately 150 ul of 25 mg/mL AM solution ).
dose of 4 mg/kg ( n 5 for each group ).
[0199 ] For assessment of blank nano - lipospheres co -ad
Talinolol
Talinolol nano
lipospheres
ministration effect on AM nano - lipospheres bioavailability ,
AM nano -lipospheres were prepared as described above and
11.9 = 2.71 (* ) 35.98 + 8.09 AUC ( Hr * ng/ mL) administered by oral gavage. The administered dose was
5.55 + 1.65
1.15 + 0.19
15.28 £ 4.7 Cmax (ng /mL) 12.5 mg/kg (n = 6 ). Blank nano - lipospheres were freshly
1.31 + 0.23 T1/2 ( hr ) prepared and 1 mL was administered by the same route .
Systemic blood samples (0.35 mL ) were taken at 5 min
[0195 ] Based on this finding we conclude that nano pre -dose , 0.5 , 1 2, 4 , 8 , 12 , 24 , 36 and 48 h post-dose. To
lipospheres possess the ability to inhibit intestinal P -gp prevent dehydration equal volumes of physiological solution
efflux and thus to increase the oral bioavailability of intra were administered to the rats following each withdrawal of
enterocyte P - gp efflux substrates upon oral administration . blood sample. Plasmawas separated by centrifugation (4000
g , 7 min , 4 ° C.) and stored at -20 ° C. pending analysis
AM Tissue Distribution Studies (FIGS. 6A - D ).
[0196 ] The rats were randomly assigned into two groups Blank Nano Lipospheres Co-Administration Results
(n = 3 in each group ) and received AM or AM nano -lipo
spheres (12.5 mg/kg) for 5 days, once daily by oral gavage . [0200 ] The bioavailability of AM nano - liposheres,
Two hours after the last dose the rats were euthanized by AM + blank nano - liposh?res, and AM nano - liposh?res + blank
CO2 and liver and heart were removed and weighted . Plasma nano- liposheres was significantly greater in comparison to
samples were collected as well. AM alone. Similar results were obtained for the Cmax
[0197] The tissue distribution results (FIG . 5 ) corroborate values. Moreover, the AUC value following AM nano
the increased plasma AUC obtained following single AM liposh?res + blank nano -liposh?res administration was sig
nano - lipospheres administration . Following chronic oral nificantly higher than other delivery systems. There was no
administration of AM nano-lipospheres and AM , we significant difference found between the obtained AUC and
observed increased tissue (heart and liver ) and correspond Cmax values following AM nano - liposh?res and AM + blank
ing plasma concentrations of AMin the AM nanolipospheres nano -liposh?res administration . These results demonstrate
group. Interestingly , the extent of increase was similar in that co -administration of blank nano-liposh?res with the
both tissues and plasma (about 1.5 folds). Since AM has a drug has the same effect on its bioavailability as the incor
high volume of distribution and is extensively bound to poration into the nano -liposh?res.
TABLE 8
Summary of PK parameters derived from PO administration of AM , AM nano
liposh?res, AM + blank nano-liposh?res and AM- nano -liposheres +
blank nano -liposh?res in the dose of 12.5 mg/kg (n 5 for each group ).
AM nano
liposheres + AM + blank
AM nano blank nano nano
AM lipospheres lipospheres lipospheres
AUC 4.54 + 2.6 9.52 = 0.47 (* ) 13.0 + 4.78 (**) 10.65 4.38 (* )
(hr * ug /ml)
Cmax 0.36 + 0.07 0.66 + 0.08 1.32 0.54 (**) 0.73 + 0.16 (* )
(ug/mL )
T1/2 (hr) 11.3 + 4.26 18.0 + 5.41 16.6 + 6.36 12.3 + 2.21
V /F (mL /kg) 34320 + 5447 33774 + 8638 24421 + 8099 22451 + 4950
CL / F 2412 + 1428 1315 + 66.3 1061 + 396 1309 + 454
(mL/hr/kg)
F (% ) 23.66 49.60 (* ) 67.96 (* ) 55.50 (* )
(* ) Significant difference (p < 0.05) from amiodarone corresponding value is found .
(* )Significant difference (p < 0.05 ) from (amiodarone + blank nano - liposh?res ) and (nano - liposheres + Blank
nano- liposh?res) corresponding value is found
tissues, the correlation between single and multiple dosing [0201 ] Absolute bioavailability ( F ) values were calculated
results emphasizes that the enhanced bioavailability of AM using the following equation
nanolipospheres is a constant and profound effect.
Blank Nano Lipospheres Co-Administration Studies AUC v * Dosepo
[0198 ] For the assessment of blank nano - lipospheres co AUCpo * Doseiv
administration effect on AM bioavailability , blank nano
lipospheres not containing AM were freshly prepared as
described above and administered by oral gavage. AM and utilizing the AM AUCi, values from our preliminary IV
(Amiodacore® ) was administered immediately after that by studies .
US 2020/0009067 A1 Jan. 9. 2020
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[0202 ] In addition , the absolute CL values were back contrast, a lower AM dose (25 mg/kg ) triggered a brief
calculated by multiplying the CL /F obtained for each group tachycardia , indicating that only higher AM dose was able to
by its corresponding F value calculated as described above . counteract the baroreflex allowing the reflex tachycardia to
No significant difference was found between the CL values occur.
of the different study groups and the CLiv value from our [ 0208 ] The deferential effect of AM on heart rate was used
preliminary study, implying that nano -liposh?res adminis as a pharmacodynamic marker for assessment of AM phar
tration did not affect AM clearance rate and extent. macological effect following oral administration .
Investigation ofDuration of Nano - Lipospheres Effect on GI Transmitter Implantation
Absorption and Enterocyte Recovery [0209 ] Male Wistar rats (Harlan , Israel) weighing 300-350
[0203 ] Freshly prepared as described above blank nano g were kept under a 12 h light/dark cycle with free access to
lipospheres were administered by oral gavage . The blank water and food ( standard rat chow ) Animals were anesthe
nano - lipospheres volume was equal to AM nano - lipospheres tized for the period ofsurgery by intraperitoneal injection of
volume administered in the bioavailability studies . After 2 1 mL /kg of ketamine -xylazine solution ( 90 / 10 % , respec
hours 12.5 mg/kg AM solution prepared from Amiodacore® tively ) .
ampoules content as described above was administered [0210 ] An ECG transmitter (TA11CA -F40 , Data Sciences
(n = 3 ) . The control group received 12.5 mg/kg AM solution International Inc., St. Paul, Minn .) was implanted subcuta
prepared from Amiodacore® ampoules at the same time neously over the scapula with the leads in a Lead 2 con
point (n = 3 ). In the investigation of duration of nano -lipo figuration by tunneling subcutaneously from the dorsal
spheres effect on GI absorption of AM , the last sampling incision using a trocar. The negative lead was placed in the
point was 12 hours , assuming the effect will not last beyond area of the right shoulder and the positive lead was placed
this stage. immediately to the left of the xyphoid space and caudal to
the rib cage .
Duration of Nano -Liposh?res Effect on GI Absorption and [0211 ] After the surgery the animals were left to recover
Enterocyte Recovery Results for 5 days. During the first 72 hours of the recovery period
[0204 ] The plasma concentration time profiles for AM the animals were treated with Bitryl (5 mg/kg) and Tramadol
(5 mg/kg ).
following oral administration at a dose of 12.5 mg/kg 2
hours after administration of blank nano - lipospheres or Data Recording and Analysis
water are shown in FIG . 7. There was no significant differ
ence in the obtained AM plasma concentrations between the [0212 ] A night before the recording the animals were
groups . These results suggest that the effect of nano- lipo relocated in their cages to the recording room in order to
spheres on AM bioavailability is reversible and lasts no adjust to the new environment. The experiment started after
more than 2 hours . the animals were at least 8 hours with food deprivation .
[0213] The signal from the transmitters was received by
AM Pharmacodynamic (PD ) Study two RLA 2000 receivers and transmitted via a Data
Exchange Matrix to a Dataquest PCI card. The data were
[0205 ] Most of the investigations described so far have recorded continuously during the whole experiment at 1000
evaluated the pharmacokinetics of the drug when incorpo Hz rate with no filter cutoff and full scale at 10 mV.
rated in lipospheres and very few investigations have dem [0214 ] The collected data were interpreted by Dataquest
onstrated pharmacodynamic efficacy. Although pharmacoki A.R.T program . The IBI (R -R duration , sec ) and HR (bpm )
netic studies are sufficient to establish proof of concept for were calculated in average values of each 10 min of the
lipospheres, the results of the pharmacokinetic study should recording
preferably be corroborated by pharmacodynamic studies . [0215 ] At the beginning the baseline were recorded at least
This is particularly important for drugs which do not show for 120 min .After establishing stable recording the rats were
pharmacokinetic - pharmacodynamic correlation . Such treated by oral gavage. During the treatment the data was
aspects should be carefully considered while planning inves collected and afterwards extracted in further analysis .
tigations on the lipospheres .
[0206 ] Bradicardia and hypotension have been reported as Dosing Protocol
the most common haemodynamic effects caused by acute
AM administration . Several studies assessed the effect of [0216 ] Two groups of three rats received the treatment in
AM on heart rate in freely moving rats using radiotelemetry a cross over manner, meaning each rat received each treat
method following IV and PO AM administration . Radiote ment once . The treatments were as follows:
lemetry is the “ state of the art” for monitoring physiological [0217 ] AM : 12.5 mg/kg AM solution prepared from
functions in awake and freely moving animals , while mini Amiodacore® ampoules content (AM 50 mg/mL) dissolved
mizing stress artifacts and effects of anesthesia . in water to obtain 2.5 mg/mL concentration
[0207 ] Da Silva et al. ( Am J Physiol Regul Integr Comp [0218 ] AM Nano -Lipospheres :
Physiol., 283 ( 2):R543-8m 2002) reported a significant [0219 ] AM containing nano - lipospheres were freshly pre
increase in R - R interval 5 min post AM IV administration pared 30 min before each experiment, by vortex -mixing of
(50 mg/kg ). Such bradicardia, observed in several studies the pro nano -dispersion containing AM in water (1: 9) pre
following single high IV AM dose can be attributed to heated to 37 ° C. for 30 sec . The obtained AM concentration
several mechanisms; for instance, Na+ and Ca2 + channel was 3 mg/mL . The AM nano - lipospheres ( 12.5 mg/kg ) were
blocking actions depressing the automaticity of sinus node, administered to the animals by oral gavage .
a non -competitive beta -adrenoreceptor blockade, a reserpine [0220 ] After each recordingphase the rats were left for the
like sympatholytic action and direct K * channel blockade. In washout period of 7 days before the next recording phase .
US 2020/0009067 A1 Jan. 9. 2020
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[0221] AM + Blank Lipospheres : adequate R wave amplitudes and QRS complexes for deter
[ 0222 ] Blank nanolipospheres not containing AM was minations of heart rate (HR ) and intra -beat R -R interval
freshly prepared as described above and was administered (IBI) variability .
by oral gavage. AM ( Amiodacore® ) was administered [0230 ] In order to inspect separately the effect of each
immediately after that by the same route . The blank nano delivery method , we first calculated the mean baseline IBI
lipospheres volume was equal to AM nano - lipospheres duration throughout 60 min prior the administration . The
volume administered in to the AM group and the dose was drug was administered at t= 60 min Post administration the
12.5 mg/kg identical. data was calculated throughout 180 min . The net change was
[0223 ] Blank Nano -Lipospheres with AM Nano - Lipo calculated as the percentage of the change for each drug
spheres: administration mode relative to the baseline recording . The
changes from base - line in two parameters : IBI and HR were
[ 0224 ] AM -nano-lipospheres were prepared as described analyzed in two points: 90 min . post administration (mid
above and was administered by oral gavage . The adminis phase ) and 180 min post administration ( end phase ) ( Tables
tered dose was 12.5 mg/kg . Blank nano - lipospheres were 9A - B and 10A - B , respectively ).
freshly prepared and 1 mL was administered by the same [0231] Sham operated group that received only the blank
route . nano - lipospheres without any active compound , didn't
[0225 ] Sham Group exhibit any significant change in the IBI duration and HR .
[0226 ] received equivalent volume of nano -liposphere [0232 ] Following AM (12.5 mg/kg ) administration , the
mean IBI values decreased from 0.175 + 0.006 sec to
without any active ingredient. 0.143 + 0.0074 sec and the heart change increased 344 : 13.58
[0227 ] After each recording phase the rats were left for the BPM to 452.916 + 20.01 as expected .
washout period of 7 days before the next recording phase . [0233 ] When the drug was incorporated into the nano
lipospheres (AM nano - lipospheres ) the mean IBI increased
Pharmocodynamic Results from 0.179 + 0.0026 sec to 0.197 + 0.009 sec and the heart rate
decreased from 390.9 + 9.4 BPM to 365.334 + 7.089 BPM .
[0228] One week following transmitter implantation all Thus, at same when the drug at the same dosage was
animals did not exhibit significant changes of body weight incorporated into the nano -lipospheres, a reverse effect was
as compared to the pre-surgery status . A normal behavioral shown . This effect, bradycardia , of the drug is characteristic
pattern was observed 48-72 hours after the surgery, includ to the high dosage of AM meaning that the drug incorpo
ing circadian rhythm , grooming, exploration of the cage and ration to the nano -lipospheres increases the bioavailability
reactivity to handling . of the drug and the drug concentrations at the active site
[0229 ] Initially , baseline recording were performed in (cardiac tissue). Similar bradycardic effect was created when
order to obtain the signal pattern and to assure that the ECG AM was co -administered with blank nano - lipospheres
leads were stable at their insertion points . ECG signals were (AM + Blank nano -lipospheres): the mean IBI increased from
not filtered or smoothed neither during the recording session 0.175 + 0.002 sec to 0.184 + 0.003 sec and the HR decreased
nor during analysis . All of the animals showed fine signal from 377.54 + 4.82 BPM to 342.522-13.94 BPM (FIGS. 8
strength and ECG wave pattern . The recording yielded and 9 ).
TABLE 9
Mean HR ( A ) and IBI (B ) changes (as percentage from baseline ) following 12.5 mg/kg
AM following administration in 3 different formulations: AM , AM nanolipospheres
( SNEDDS), AM + Blank nano lipospheres and administration of blank nanolipospheres,
as measured at the mid phase (90 min following oral administration )
A.
HEART RATE (BPM )
CHANGE % MID PHASE BASELINE
131.28 452.916 + 20.01 344 + 13.58 AM

93.46 365.334 + 7.089 390.9 + 9.4 AM SNEDDS
90.73 342.522 13.93 377.54 + 4.82 AM + BLANK SNEDDS
105.72 354.605 18.43 335.42 + 12.89 SHAM
HEART RATE (BPM )

CHANGE % END PHASE BASELINE
128.43 443.071 + 11.06 344 + 13.58 AM

95.37 372.839 + 11.54 390.9 + 9.4 AM SNEDDS
92.61 349.626 + 8.00 377.54 + 4.82 AM + BLANK SNEDDS
106.09 355.870 + 11.63 335.42 + 12.9 SHAM
B.
IBI (sec )
88.86 0.1435 + 0.008 0.175 + 0.006 AM
110.01 0.197 + 0.009 0.179 + 0.0026 AM SNEDDS
US 2020/0009067 A1 Jan. 9 , 2020
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TABLE 9 - continued
Mean HR ( A ) and IBI (B ) changes (as percentage from baseline ) following 12.5 mg/kg
AM following administration in 3 different formulations : AM , AM nanolipospheres
(SNEDDS), AM + Blank nano lipospheres and administration of blank nanolipospheres ,
as measured at the mid phase (90 min following oral administration )
104.04 0.182 + 0.003 0.175 + 0.002 AM + BLANK SNEDDS
98.57 0.173 + 0.01 0.175 + 0.001 SHAM
IBI (sec)
89.79 0.143 = 0.007 0.175 + 0.006 AM
108.76 0.195 + 0.008 0.179 + 0.0026 AM SNEDDS
105.39 0.184 + 0.003 0.175 + 0.002 AM + BLANK SNEDDS
98.39 0.172 + 0.01 0.175 + 0.001 SHAM
TABLE 10
Mean HR (A ) IBI (B ) and changes (as percentage from baseline)
following 12.5 mg/kg AM following administration in 3 different
formulations: AM , AM nanolipospheres, AM + Blank nano lipospheres
and administration of blank nanolipospheres, as measured at the
end phase ( 180 min following oral administration ).
A.
HEART RATE (BPM )
131.28 ( * ) 452.916 + 20.01 344 + 13.58 AM

93.46 (* ) 365.334 + 7.089 390.9 + 9.4 AM SNEDDS
90.73 ( * ) 342.522 + 13.94 377.54 + 4.82 AM + BLANK SNEDDS
105.72 354.605 + 18.43 335.42 + 12.89 SHAM
HEART RATE
128.43 ( * 443.07 + 11.06 344 + 13.58 AM
95.37 ( * ) 372.839 = 11.54 390.9 + 9.4 AM SNEDDS
9.61 (* ) 349.626 + 8.00 377.54 + 4.82 AM + BLANK SNEDDS
106.09 355.870 + 11.63 335.42 + 12.89 SHAM
B.
IBI (sec)
88.86 (* ) 0.1435 + 0.008 0.175 + 0.006 AM

110.01 (* ) 0.197 + 0.009 0.179 + 0.0026 AM SNEDDS
104.04 (* ) 0.182 + 0.003 0.175 + 0.002 AM + BLANK SNEDDS
96.57 0.173 + 0.01 0.175 + 0.001 SHAM
IBI (sec)
89.79 (* 0.143 + 0.007 0.175 + 0.006 AM
108.76 (* ) 0.195 + 0.008 0.179 + 0.0026 AM SNEDDS
105.39 (* ) 0.184 + 0.003 0.175 + 0.002 AM + BLANK SNEDDS
98.39 0.172 + 0.01 0.175 + 0.001 SHAM
[0234 ] These findings demonstrate that Amiodarone bio AM + blank nano -lipospheres, which is significantly different
availability increases not only when it is encapsulated in from the PD effect obtained upon administration of free AM .
nano - lipospheres , but even the simultaneous presence of
empty ( i.e. drug -less ) nano -lipospheres in the intestine. Parenteral Administration of the Nano -Lipospheres
Moreover, encapsulation into nano -lipospheres reduces the
high variability in plasma concentrations, typical for amio [0235 ] As for IV administration , the nano -liposphere for
darone and other BCS class 2 compounds . Our PD studies mulation alters the biodistribution parameters of a given
results corroborate the PK findings and demonstrate similar molecule , as well as its disposition . FIG . 10 demonstrates
PD effect upon administration ofAM nano - lipospheres and the plasma concentration - time profile obtained following IV
US 2020/0009067 A1 Jan. 9. 2020
15
administration of 12.5 ug/kg Amiodacore® and AM nano marker for passive paracellular permeability , was used for
lipospheres. It can be seen that the administration of nano further evaluation of proper carrying out of each study.
lipospheres resulted in higher plasma concentrations of AM [0239] In -vitro permeability studies of talinolol through
at the central compartment during the first disposition phase . Caco - 2 monolayers resulted in significantly higher (p < 0 .
The AUCs were 22.1 and 17.1 ug /mL after AM nano 001) permeability coefficient values of talinolol + blank nano
lipospheres and Amiodacore® administration respectively . lipospheres vs. talinolol (1.73x10-6 and 7.66x10–7 respec
[0236 ] It can be seen that the nano - lipospheres also alter tively ) in A to B direction . As expected for P -gp substrate ,
the PK parameters of the administered drug . Significant both talinolol and talinolol+ blank nano - lipospheres Papp
reduction in volume of distribution and in clearance of values obtained in the B to A direction are higher than the
amiodarone when administered as nano -lipospheres are corresponding Papp values obtained in the A to B direction .
observed ( Table 11). The reduction in Vss is a result of better B to A permeability resulted in statistically significant (p < 0 .
solubility of the drug in plasma while encapsulated in 001 ) reduction in Papp values obtained for talinolol +blank
nanolipospheres . Thus, more drug is retained in the central nano - lipospheres vs. talinolol (7.36x10-6 and 1.77x10-5
compartment. As drug encapsulated in the formulation respectively ), as expected for P - gp substrate when P - gp
reaches P - gp containing barriers ( such as the blood -brain activity is inhibited (FIG . 11 ).
barrier ), it might possess improved penetration qualities due [0240 ] It should be emphasized that talinolol was not
to the P - gp inhibition ability of the formulation . As a result, incorporated into nano -lipospheres, but administered simul
penetration of parenteral drugs to P - gp expressing tissues taneously . These results suggest that nano -lipospheres pos
will be improved . sess the ability to inhibit P - gp efflux and thus to increase the
oral bioavailability of compounds subjected to extensive
TABLE 11 intra -enterocyte efflux . Furthermore, nano -lipospheres can
inhibit P -gp not only by incorporation of the compound into
PK parameters obtained following IV administration of 12.5 ug/kg
Amiodacore ® and AM nano -lipospheres .
this delivery system but also when nano - lipospheres e.g.
blank nanolipospheres are administered simultaneously
Cl (mL /hr (kg ) Vss (ml/kg ) AUC (hr* ug/mL ) HL (h ) with the active compound . As expected for a P - gp substrate ,
both values obtained in the basal-to -apical direction are
549 8002 22.9 17.1 nano
lipospheres higher than the Papp obtained in the apical-to -basal direc
818 18138 15.3 22.1 Amiodacore tion . Interestingly , basal-to -apical permeability resulted in
significant reduction in Papp values obtained for talinolol
nano lipospheres vs. talinolol. This pattern is typical for P -gp
substrates in the presence of a P -gp inhibitor; while apical
In - Vitro Mechanistic Investigation to -basal efflux is increased due to P -gp inhibition , the
opposite direction transport is decreased as a result of
In Vitro Permeability Studies Using Caco- 2 Cells: reduced P - gp contribution to drug transport from the entero
cyte to the lumen .
[0237 ] Caco - 2 cell line is a broadly used in vitro model to [0241 ] To comprehend the effects of nano - lipospheres on
study various aspects of intestinal permeability of drug intra -enterocyte metabolic activity , we tested its effect on
molecules, which was established by Fogh in 1974. The CYP3A4 mediated AM and testosterone (control CYP 3A4
Caco - 2 cell line is derived from a human colon carcinoma. substrate ) metabolism in isolated rat CYP 3A4 microsomes
These cells spontaneously differentiate to enterocytes under (FIG . 12 ). Significant difference (p < 0.01 ) was found
conventional cell culture conditions. Caco - 2 cells exhibit between intact AM concentrations remaining following 120
morphological as well as biochemical similarities to intes min . incubation of AM -nano -lipospheres vs. AM (102.4 + 5.
tinal (absorptive ) enterocytes. The cells form tight intercel 61 % and 68.57-1.17 % respectively ). In order to distinguish
lular junctions and microvilli, express several transporters between mechanistic protection of nano -lipospheres from
(e. g. sugar carrier, bile acid carrier, large neutral amino acid enzymatic degradation and the effect of nano -lipospheres
carrier, and P - gp ) and metabolic enzymes ( e g aminopepti ingredients on enzymatic activity, blank nano -lipospheres
dases, esterases, sulfatases, and cytochrome P450 enzymes ). were added to testosterone and incubated in microsomes .
The unusually high degree of differentiation , morphological Significant difference (p < 0.05 ) was found between testos
and functional structure resembling enterocytes has resulted terone with nano - lipospheres vs. testosterone alone incu
in Caco -2 becoming one of the most popular cell culture bated at the same conditions (91.26_5.31 % and 75.39+ 8 .
models to study intestinal epithelial integrity and drug 82 % respectively ).
transport. [0242 ] These results imply that increased bioavailability
[0238 ] For determination of nano -lipospheres ability to achieved by nanolipospheres partially results from reduced
inhibit intestinal P -gp efflux pumps, Caco - 2 transport study first pass intra -enterocyte metabolism . Moreover , similarly
was performed using talinolol as a model molecule. As to our in vivo results , the incorporation of the drug into
nano - lipospheres was not necessary to gain the effect of
previously stated , talinolol is a known P - gp substrate and is increased bioavailability or reduced first pass metabolism ,
not metabolized by the intra-enterocyte CYP enzymes. In and co -administration of the drug with the blank nano
this case, the donor compartment of the transwells contained lipospheres resulted in comparable effects .
either 10 ug/mL talinolol , or 10 ug /mL talinolol with blank
nanolipospheres (amount equivalent to 100 ug/mL AM -nano Safety and Toxicity Studies
lipospheres ). At fixed time points (0 , 30 , 60, 90 , 120 , and In - Vitro Paracellular Transport Studies:
150 min ), 150 uL samples were withdrawn from the receiver
side, and similar volumes of blank buffer were added to [0243] Caco - 2 cells permeability studies were performed
maintain constant volume. C14 -Mannitol, a commonly used with paracellular transport marker C14 -Mannitol (2 uCi/
US 2020/0009067 A1 Jan. 9. 2020
16
mL) . Samples (200 uL ) were withdrawn from the basolateral concerns a composition being the nano - lipo formulation as
side at the same fixed time points described above, and described above , but devoid of a drug ( empty composition )
similar volumes of blank buffer were added following each for oral administration for increasing the absorption of a
withdrawal. drug with low bioavailability due to first pass intestinal
metabolism or substrate of intestinal efflux transporters ( e.g.
Transepithelial Electrical Resistance ( TEER ) Studies: P -gp ) administered simultaneously , or in a close time win
[0244 ] Caco -2 cells were grown on 12 -transwell plate and dow to the nano lipo formulation .
cultured for 21 days The apical bufferwas replaced with 0.6 [0249 ] Based on these finding it is concluded that the
mL pre -warmed (37° C.) apical buffer containing blank nano - formulation serve as a platform for administration of
nano -lipospheres (amount equivalent to 100 ug/mL AM drugs which are P - gp and CYP3A4 substrates that are
nano - lipospheres ). In order to evaluate the effect of the characterized by low oral bioavailability and erratic absorp
nano - lipospheres on the paracellular transport across the tion . Examples of P -gp and CYP3A4 substrates that are
cells (i.e. the extent to which the tight junctions are opened ), suitable candidates for administration in nanolipospheres are
listed in Table 12 .
TEER values were measured at the above time points and
compared to the TEER values measured in control cells TABLE 12
containing only buffer.
[ 0245 ] In FIG . 13, the outer line represents the TEER Examples of P - gp and CYP3A4 substrates at the
values of Caco -2 monolayers in the presence of blank gut-wall and the liver, which are suitable candidates
for administration in nanolipospheres
nano-lipospheres or control. There was no difference in the
TEER values measured throughout the experiment both in Amlodipine Calcium channel blockers
the presence and absence of blank nano -lipospheres (p = 0 . Diltiazem
Felodipine
19 ), indicating that the integrity of the monolayer was Nicardipine
remained . The embedded chart represents the Papp values of Nifedipine
mannitol (a paracellular transport marker ) following incu Nimodipine
bation with blank nano - lipospheres or control in Caco - 2 Nisoldipine
model. There was no significant difference in the perme Verapamil
ability ofmannitol in the presence of blank nano - lipospheres Cyclosporine when administered with Immunosuppressants
" empty ” lipo -nanosphere formulation
compared to buffer (p = 0.51). These observations indicate Sirolimus
that nano - lipospheres neither affect paracellular permeabil Tacrolimus
ity, nor disrupt the enterocyte monolayer integrity . Atorvastatin
Lovastatin
Statins
Simvastatin
Cellular Toxicity Studies: Fexofenadine Antihustamins
Buspirone CNS drugs
[0246 ] Lactate dehydrogenase (LDH ) is a cytoplasmic Carbamazepine
enzyme which is released into the culture supernatant when Pimozide
the plasma membrane is damaged . Nano - lipospheres toxic Midazolam
Triazolam
ity was assessed over a range of concentrations (0.1 , 1 , 5 , Albendazole Anti - fungal/antibiotics
and 10 % in medium ) in Coco - 2 cells grown as described Itraconazole
above using LDH cytotoxicity detection kit, after the incu Erythromycin
Amiodarone Miscellaneous
bation of the cell culture with blank nano -lipospheres con Cisapride
tainingmedium for 2 h . Percent of cell damage is calculated Colchicine
so that low control was the spontaneous LDH release Sildenafil
measured in untreated cells medium and high control was
100 % LDH release measured in cells incubated with 1 %
Triton X - 100 containing medium . [0250 ] Moreover, nano lipospheres of the present inven
[0247 ] As shown in FIG . 14 , nano -lipospheres increased tion affect intestinal CYP3A4 enzyme in a reversible and
LDH release in concentration dependant manner with high non selective manner, such that it is possible that the nano
est toxicity obtained at 10 % nano -lipospheres concentration lipospheres of the present invention inhibit other metabolic
(7.27 % ) and negligible toxicity (< 1 % ) produce by nano enzymes in the intestine e.g. CYP2C19 and CYP2C9.
lipospheres conc entration of 1 % and less compared to 100 % Cannabidiol and Cannabidiol-Piperine Nano -Liposphere
toxicity standard . These results suggest that no significant Formation (CBD - PNL and CBDpiperine-PNL ,
damage is caused to the apical cell membrane of the entero Respectively )
cyte by nano - lipospheres administration .Moreover, an over
estimation of cytotoxicity might be obtained in Caco - 2 [0251] CBD nano lipospheres formulation was prepared
model since it is lacking the repair and recovery mechanisms by pre - concentrate preparation method . The final nano
present in the intact tissue. lipospheres composition was based on preliminary formu
[ 0248 ] Thus , it is shown that nano - lipospheres improve lation optimization studies and selected according to optimal
oral bioavailability of Class II compounds by multi -pro solubilization capacity of the active ingredient. Initially
cesses mechanism : increased GI milieu solubilization , amphiphilic co -solvent (ethyl lactate) and phospholipid
reduced intra-enterocyte metabolism and reduced P - gp ( lecithin ) at the ratio of 8 :1 , respectively were placed in a
efflux activity. In addition to increased bioavailability , nano clean scintillation tube and heated to 40 ° C. until the lecithin
lipospheres reduces the high variability typical for Class II was completely dissolved . Then triglyceride ( tricaprin ),
compounds. Nano - lipospheres do not cause tissue or cell polyoxyl 40 -hydroxy castor oil, Tween 20 , and Span 80 at
membrane damage. Therefore , the present invention further the ratio of 1: 1 : 1 :1 were added ; the mixture was gently
US 2020/0009067 A1 Jan. 9 , 2020
17
stirred and heated to 40° C. till homogenous solution was PNL previously developed by our group have been success
formed . Further, CBD powder was added , forming the CBD fully utilized to enhance the oral absorption of drugs sub
preconcentrate containing 3 % CBD . This pre -concentrate jected to intraenterocyte Phase I “ first-pass” metabolism and
was gently stirred and heated to 40 ° C. till homogenous P -gp efflux . The innovation of our current approach is
solution was formed . Upon gentle agitation in aqueous adding piperine into PNL .
phase, this pre -concentrate spontaneously forms drug encap [0258 ] The utilization of piperine in addition to PNL
sulated nano -dispersion . For the preparation of CBD -Piper synergistically enhances bioavailability and may increase
ine -PNL piperine powder was added with CBD powder to the oral absorption to greater extent than the utilization of
form the CBD -Piperine -PNL . CBD solution in Propylen PNL alone . The result is a platform that can be employed for:
Glycol/EthOH /Water 30/30/40 respectively was used as [0259 ] 1. Drugs which are subjected to Phase I intra
control throughout all in -vivo studies . enterocyte metabolism and/or P - gp efflux .
[0260 ] 2. Various drugs which are also directly eliminated
In - Vivo PK Studies by phase II metabolism e.g. glucuronidation process.
[0252 ] Animals : Male Wistar rats weighing 300-350 g [0261 ] Thus, the present invention applies the Piperine
were used for the in vivo studies. The project adhered to the PNL concept to compounds suffering from significant first
principles of Laboratory Animal Care (NIH publication no . pass Phase 1, phase II metabolism and /or P - gp efflux at the
85-23 , revised 1985 ). All animals were deprived of food but intraenterocyte level . Suitable candidates for administration
not water 12 h prior to the experiments. All surgical and in Piperine - PNL are listed in Table 14 .
experimental procedures were reviewed and approved by the TABLE 14
Animal Experimentation Ethics Committee of the Hebrew
University Hadassah Medical School Jerusalem . An Examples of P - gp , CYP3A4 and UDP - glucuronosyltransferase (UGT)
indwelling cannula was placed in the right jugular vein of enzymes substrates at the gut-wall and the liver, which are
each animal for systemic blood sampling . suitable candidates for administration in Piperine -PNL .
[ 0253 ] CBD Relative Oral Bioavailability Studies , Amlodipine Calcium channel blockers
[0254 ] nano lipospheres was freshly prepared 30 min Diltiazem
Felodipine
before each experiment, by vortex -mixing of the pre -con Nicardipine
centrate ( CBD -PNL and CBD - Piperine -PNL ) in water ( 1:9 ) Nifedipine
pre-heated to 37 CO for 30 sec . The obtained CBD and Nimodipine
piperine concentrations were 3 mg/mL and 2 mg/ml, respec Nisoldipine
Verapamil
tively. CBD - Piperine-PNL (15 mg/kg of CBD and 10 mg/kg Phenytoin Antiepileptics
ofpiperine) was administered to the animals by oral gavage Cyclosporine Immunosuppressants
(n = 6 ). The control groups received (15 mg/kg) CBD solu Sirolimus
tion in propylene glycol/EthOH /water 30/30/40 respectively Tacrolimus
(1 mg/mL ) (n = 6 ) or CBD - PNL (( 15 mg/kg of CBD ). Atorvastatin
Lovastatin
Statins
[0255 ] Systemic blood samples (0.35 mL ) were taken at 5 Simvastatin
min pre -dose , 20 min , 40 min , 1 h 1.3 h , 1.6 h , 2 h , 3 h and Fexofenadine Antihustamins
4 h post - dose . Buspirone CNS drugs
Results :
[0256 ] 1.1 The results indicate that in rats the Piperine Results of CBD -Pro -Nano Lipospheres Incorporating
PNL elevated the bioavailability of CBD by 6 fold ( Table 13 , Curcumin , Resveratrol and Piperine as Absorption
FIG . 15 ). The contribution of the Piperine componentwas 2 Enhancers
fold increased bioavailability due to its phase- II metabolism [0262 ] The results in FIG . 16 and Table 15 show that
inhibition . administration of CBD -piperine-PNL resulted in signifi
TABLE 13
cantly higher oral relative bioavailability in comparison to
all the tested formulations, when administered to rats . Oral
PK parameters (mean - S.E.M.) derived from PO administration of CBD -piperine -PNL resulted in signifi
administration of CBD , CBD -PNL and CBD - Piperine -PNL cantly higher AUC and Cmax values by 5 fold and 15 fold ,
15 mg/kg; Piperine 10 mg/kg (n 6 for each group ). respectively , as compared to control.
A?C ( h * ng/ mL) Cmax (ng/mL) TABLE 15
90 + 21 39 + 8 CBD
300 + 95 (* ) 137 = 43 (* ) CBD - PNL PK parameters (mean = S.E.M.) derived from PO administration of CBD
570 = 23 (*** ) 170 + 13 (* ) CBD - Piperine -PNL and CBD -Piperine 2 % -PNL to rats ; (n 6 for each group ).
=
(* ) Significant difference (p < 0.05 ) from CBD corresponding value is found Cmax (ng /mL) AUC ( h * ng/ mL)
) Significant difference (p < 0.05) from (CBD ) and ( CBD - PNL ) corresponding value is CBD 39 + 8 90 + 21
found .
CCBD -Piperine 170 + 13 (* ) 570 + 23(**)
[0257] Currently , there are limited pharmaceutical solu 2 % -PNL
tions in the clinical setting to overcome the absorption (* ) Significant difference (p < 0.05) from CBD corresponding value is found .
barriers accounted for poor oral drug availability. As men (*) Significant difference (p < 0.05 ) from (CBD -PNL ) corresponding value is found .
tioned hereinabove , “ First pass metabolism ” (both by Phase
I and Phase II enzymes at the enterocyte level) and efflux [0263] It is noted that curcumin could not be effectively
transporters ; P -gp hamper the oral delivery of many drugs. incorporated into PNL system . The incorporation of cur
US 2020/0009067 A1 Jan. 9. 2020
18
cumin resulted in its precipitation following the introduction [0268 ] Incorporation of curcumin into all five formula
of the pre -concentrate (containing piperine) into the water tions formed a pre -concentrate in which curcumin was fully
phase in -vitro . The same effect most probably occurs in dissolved . However , upon dilution with water phase, cur
vivo . Moreover, as the absorption of CBD following CBD cumin at the concentration of 1 % and 2 % precipitated
curcumin 1 % or 2 % -PNL is lower as compared to CBD immediately from pre -concentrates 2 , 3 , 4 and 5. The same
PNL it is reasonable to assume that the incorporation of effect most probably occur in -vivo . Only pre -concentrate 1
curcumin damages the proper formation of the CBD nano following dilution with water phase formed a nanodisper
lipospheres resulting in poorer oral bioavailability of CBD . sion system which was stable , i.e. no precipitation of cur
[0264 ] Though the incorporation of both resveratrol and cumin at the concentration of 1 % and 2 % was observed , for
piperine results in homogeneous and visually clear pre 10 min at 37 ° C., giving sufficient time window to perform
concentrate the oral bioavailability ofCBD is enhanced only pre -clinical studies. Thus CBD 3 % was added to these
when the delivery system contains piperine . formulations to form two pre -concentrates ; CBD -curcumin
[ 0265 ] Curcumin , resveratrol and piperine were reported , 1 % -PNL and CBD - curcumin 2 % -PNL . Following introduc
in the literature, to successfully enhance the bioavailability tion of these two pre-concentrates to water phase nano
of several lipophilic compounds. The proposed mechanisms dispersions were formed . No precipitation both of the active
behind this phenomenon are: CYP P450 inhybition , p -gly ingredient (CBD ) and of the absorption enhancer (curcumin )
coprotein (p -gp ) efflux reduction and inhibition of phase 2 was observed for 10 min at 37° C. following dilution . Thus
metabolism . Due to its poor aqueous solubility, curcumin CBD - curcumin 1 % and CBD - curcumin 2 % -PNL were
can be incorporated into the lipid Self-nanoemulsifying drug tested in pre -clinical trials . The oral administration of CBD
delivery systems (SNEDDS) core. This incorporation curcumin 1 % and 2 % -PNL resulted in significantly lower
increases curcumin concentration that reaches the intestine AUC and Cmax values as compared to the oral administra
and the liver, and competitively inhibit CBD metabolism in tion of CBD -PNL to rats. Moreover, the oral administration
those metabolic sites . of CBD -curcumin 1 % and 2 % -PNL didn't enhance the oral
[0266 ] Thus, incorporation of piperine into SNEDDS bioavailability of CBD as compared to the administration of
increases the plasma concentrations of THC and CBD . CBD alone. Thus , it is reasonable to assume that the
incorporation of curcumin damages the proper formation of
A Selection of an Absorption Enhancer for the Development the CBD nano -lipospheres upon introduction to water phase ,
of Various Pro -Nano -Lipospheres Containing an Absorption i.e. the fluids of the gastrointestinal tract, resulting in poorer
Enhancer and Several Different Lipophilic Drugs oral bioavailability of CBD .
[0267 ] The development of each of the formulations, in TABLE 17
terms of a selection of the different components, was con
ducted by the following method . A combination of certain Resveratrol and piperine -ProNanoLiposphere development
components was used as a reference starting point (as per Pre Pre
Table 16 ). The choice of these certain triglyceride , organic % (w /w ) concentrate 1 concentrate 2
amphiphilic co -solventand the combination of surfactants in resveratrol ?
the detailed portions in Table 16 was based on the inventors ' piperine
2
2
previous experience with various lipophilic drugs. Thus, an tween 20 12.5
absorption enhancer was added to this specified prototype span 80 12.5
formulation to form a pre -concentrate . In case this prototype lecitin 9
formulation was failed to efficiently dissolve the absorption HC0 40
ethyl lactate
12.5
36
enhancer or in case a precipitation of an absorption enhancer tricaprine 12.5
was observed following the introduction of the pre- concen
trate to a water phase , a replacement of triglyceride or/and
organic co -solvent was performed . [0269] The incorporation of both resveratrol and piperine
formed homogeneous and visually clear pre -concentrate . No
TABLE 16 precipitation of both resveratrol and piperine were observed
upon dilution of these pre -concentrates with water. Thus
Curcumin - ProNanoLipospheres development CBD 3 % was added to these formulations to form two
Pre Pre Pre Pre Pre pre -concentrates ; CBD -resveratrol- PNL and CBD -piperine
% concen concen concen concen concen PNL . Following introduction of these two pre -concentrates
(w /w ) trate 1 trate 2 trate 3 trate 4 trate 5 to water phase a stable and visually clear nano-dispersions
curcumin 2
were formed . No precipitation both of the active ingredient
tween 20 12.5 ? ( CBD ) and of the absorption enhancers (resveratrol and
span 80 12.5 piperine ) was observed for up to two month following
lecithin
HC0 40
9
12.5
dilution. Thus , these two formulations were tested in pre
ethyl
clinical trials .
37 36 V
lactate [0270 ] All the selected and tested materials curcumin ,
N -methyl ? resveratrol and piperine were reported to enhance the oral
pyrolidone
trilaurin 12.5
bioavailability of various lipophilic compounds in -vivo , thus
tripalmitin 12.5 are referred to as “ absorption enhancers” . Moreover, both
trimyristin 12.5 y CBD -resveratrol-PNL and CBD -piperine-PNL showed
tricaprine 12.5 y ? favorable stability characteristics as compared to CBD
curcumin 1 % and 2 % -PNL . However, results of pre -clinical
studies indicated that only CBD -piperine-PNL oral admin
US 2020/0009067 A1 Jan. 9. 2020
19
istration resulted in significant enhance in the bioavailability large mean droplet size. No precipitation of the drug was
of CBD as compared to the administration of CBD -PNL and observed up to 2 h post dissolution .
the administration of CBD alone .
[ 0271] Testing the Compatibility of Various BCS Class II TABLE 19
and Class IV Drugs with the Pre -Concentrate:
[0272 ] In order to determine the compatibility of the Particle size and polydispersity index (PdI) of the different
developed PNL delivery system with various lipophilic PNLs following 1:10 dilution with water. Particle size
and potential were determined using Zetasizer Nano
drugs, we have tested the ability of the piperine -PNL to ZS ZEN 3600 (Malvern Instruments Ltd , Malvern , UK ) .
dissolve various BCS class II and IV drugs. Incompatibility
was determined if the pre -concentrate were unable to fully Size nm
dissolve the tested drug or if the tested drug -piperine-PNL formulation (diameter) PdI
were not able to self-emulsify upon gentle agitation in CBD -piperine -PNL 30 0.23
aqueous environment or resulted in unstable dispersion Cyclosporine-piperine - PNL 26 0.2
system .
TABLE 18
Testing the compatibility of various BCS class II and class IV drugs with the
pre - concentrate .
% (w /w ) tacrolimus amiodarone Amphotericin B simvastatin cyclosporine Tetrahydrocan -nabinol (THC )

piperine 2 ? ? ? V
tween 20 13
span 80 13 ?
lecitin 9 ?
HC0 40 13 ?
ethyl lactate 37 ? ?
trilaurin 13 ? ?
tricaprine 13 ?
[0273 ] Amphotericin B was found to be incompatible with TABLE 19 -continued

the pre -concentrate . The rest of the drugs tested were found Particle size and polydispersity index (PdI) of the different
to be compatible with the pre -concentrate and upon dilution PNLs following 1:10 dilution with water. Particle size
with water formed clear and transparent nano -dispersion and potential were determined using Zetasizer Nano
systems with desirable range ( less than 200 nm ) of the size ZS ZEN 3600 (Malvern Instruments Ltd , Malvern , UK ) .
of the particles formed . CBD //CBD - THC //tacrolimus and
cyclosporine piperine-PNL formed nano - dispersion systems formulation
Size nm
( diameter) PdI
of optimal particle size < 50 nm . The exception was pre
concentrate incorporating Amiodarone which when dis Simvastatin-piperine -PNL 76 0.53
persed formed particles of 276 nm ( Table 18 ). Tacrolimus-piperine -PNL 38 0.34
[0274 ] A Detailed Description of the Compatibility Char THC - CBD -piperine-PNL 40 0.25
acteristics of the Tested Compounds with the Piperine-PNL Amiodarone -piperine -PNL 276 0.41
CBD - resveratrol- PNL 65 0.5
[0275 ] Amphotericin - Piperine-PNL :
[ 0276 ] The drug did not dissolve in the pre -concentrate . *** Polysorbate 20 ( Tween ® 20 ), Sorbitan monooleate 80 (Span ® 80 ), Polyoxyethylene
hydrogenated castor oil 40 (HCO -40 ).
[ 0277 ] Cyclosporine -Piperine-PNL :
[0278 ] the drug was fully dissolved in the pre - concentrate . 1.-31. ( canceled )
Upon dissolution of the pre - concentrate with water, a clear 32. A method for increasing the bioavailability of at least
and transparent dispersion was formed . one orally administered drug in a subject in need of said
[0279 ] THC -CBD -Piperine -PNL : drug, the method comprising orally administering to said
[ 0280 ] the drug was fully dissolved in the pre -concentrate . subject:
Upon dissolution of the pre - concentrate with water, a clear ( 1 ) said drug , and
and transparent dispersion was formed . (2 ) a composition , comprising :
[0281] Tacrolimus - Piperine -PNL : a dispersible concentrate comprising :
[0282 ] the drug was fully dissolved in the pre -concentrate . (i) at least one surfactant;
Upon dissolution of the pre -concentrate with water , a clear ( ii ) at least one solid component at room temperature ,
and transparent dispersion was formed. the at least one solid component having a melting
[0283] Simvastatin -Piperine - PNL : temperature above 25 ° C. and is selected from the
[ 0284 ] the drug was fully dissolved in the pre -concentrate. group consisting of fatty acids, fatty acid esters , fatty
Upon dissolution of the pre -concentrate with water, clear amines, fatty amides and fatty alcohols; and
opal white dispersion was formed . No precipitation of the (iii ) an amphiphilic solvent,
drug was observed up to 2 h post dissolution . wherein upon contact of said concentrate with the
[0285 ] Amiodarone -Piperine -PNL : gastrointestinal (GI) tract fluids, the concentrate
[0286 ] the drug was fully dissolved in the pre -concentrate . transforms into a dispersion of nanoparticles having
Upon dissolution of the pre-concentrate with water , milky a size of less than about 500 nm , the nanoparticles
white dispersion was formed , implying an insufficiently comprising the at least one surfactant, the at least one
US 2020/0009067 A1 Jan. 9 , 2020
20
solid component at room temperature, and the 50. The method according to claim 32, wherein said drug
amphiphilic solvent; such that the bioavailability of is a cannabinoid being selected from the group consisting of
said at least one orally administered drug is tetrahydrocannabinol, cannabidiol ( CBD ), cannabinol, can
increased , as determined by measuring the total nabigerol, tetrahydrocannabivarin , cannabidivarin and can
systemic drug concentrations over time after admin nabichromene .
istration of said composition as compared to after 51. The method according to claim 32, wherein the
administration of the drug alone , surfactant is a combination of at least one high HLB
and wherein the composition does not comprise cur (hydrophilic/ lipophilic balance ) surfactant, having an HLB
cumin . of at least about 8, with at least one surfactant being a low
33. The method according to claim 32 , wherein said HLB surfactant having an HLB of less than about 5 .
composition being administered substantially simultane 52. The method according to claim 32 , wherein said
composition further comprises a phospholipid .
ously with the drug .
34. The method according to claim 32 , wherein said 53. The method according to claim 32, wherein the
composition and drug are sequentially administered . amphiphilic solvent is selected from the group consisting of
35. The method according to claim 32 , wherein the drug lower alkyl esters of lactic acid , lower alkyl lactone esters
is administered in at least one dose within 24 hrs after and N -methylpyrrolidone .
administration of the composition . 54. The method according to claim 32 , wherein the
36.- 38 . (canceled ) composition and at least one drug are formulated for admin
39. The method according to claim 32 , for treatment of at istration in the same pharmaceutical formulation .
least one disease or disorder. 55. The method according to claim 32 , wherein the
40.-48 . (canceled ) composition and said at least one additional drug are for
49. The method according to claim 32 , wherein said at mulated for administration in different pharmaceutical for
least one drug is a cannabinoid , a derivative or a synthetic mulations.
analog thereof, or a mixture of cannabinoids .

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0.72 0.6 0.5 0.4 0.34 0.1- 0.1 0

0 50 100 150 200 250

0.87 + AM nano - lipospheres

1.2 + AM nano - lipospheres

Blank nano-lipospheres + AM after 2h

130 125 120 115 110 105

) line base the from change %

160+ CBD - Piperin - PNL

PPNLiperine --CBD-c2%-PNLurcumin samymcCBD--PNL1%urcumin CBD-RPNLesveratrol

160 140 120 100 80 60 40 20

FORMULATION AND METHOD FOR tools in pharmacokinetics, as it must be considered when

Table 1 (all amounts of ingredients , in Table 1 as well as in TABLE 2B - continued

control group received ( 15 mg/kg ) CBD solution in propyl TABLE 5 - continued

TABLE 7 the same route (n = 6 ). The blank nanolipospheres volume

131.28 452.916 + 20.01 344 + 13.58 AM

HEART RATE (BPM )

128.43 443.071 + 11.06 344 + 13.58 AM

131.28 ( * ) 452.916 + 20.01 344 + 13.58 AM

88.86 (* ) 0.1435 + 0.008 0.175 + 0.006 AM

% (w /w ) tacrolimus amiodarone Amphotericin B simvastatin cyclosporine Tetrahydrocan -nabinol (THC )

[0273 ] Amphotericin B was found to be incompatible with TABLE 19 -continued

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