U.S. patent application number 09/795979 was filed with the patent office on 2002-12-19 for novel vitamin b12 - biodegradable micro particulate conjugate carrier systems for peroral delivery of drugs, therapeutic peptides/proteins and vaccines.
Invention is credited to Chalasani, Kishore Babu, Diwan, Vamanrao, Jain, Sanjay Kumar, Raghavan, Kondapuram Vijaya, Rao, Kollipara Koteshawar, Russell-Jones, Gregory John.
Application Number | 20020192235 09/795979 |
Document ID | / |
Family ID | 26324106 |
Filed Date | 2002-12-19 |
United States Patent
Application |
20020192235 |
Kind Code |
A1 |
Chalasani, Kishore Babu ; et
al. |
December 19, 2002 |
NOVEL VITAMIN B12 - BIODEGRADABLE MICRO PARTICULATE CONJUGATE
CARRIER SYSTEMS FOR PERORAL DELIVERY OF DRUGS, THERAPEUTIC
PEPTIDES/PROTEINS AND VACCINES
Abstract
The invention relates to a novel complex for oral delivery of
drugs, therapeutic protein/peptides and vaccines which are loaded
in a Vitamin B.sub.2 (VB.sub.12) coupled particulate carrier system
with spacers in between, the carrier system with spacers having a
formula VB.sub.12-R'/R"-N wherein, R' or R" is spacer and/or agents
for derivatization of VB.sub.12 to provide either NH.sub.2 or COOH
or SH groups, and N is the micro or nano particle carriers for the
delivery of injectable drugs, therapeutic protein/peptides and
vaccines.
Inventors: |
Chalasani, Kishore Babu;
(Hyderabad, IN) ; Diwan, Vamanrao; (Hyderabad,
IN) ; Raghavan, Kondapuram Vijaya; (Hyderabad,
IN) ; Russell-Jones, Gregory John; (Roseville,
AU) ; Jain, Sanjay Kumar; (Sagar, IN) ; Rao,
Kollipara Koteshawar; (Hyderabad, IN) |
Correspondence
Address: |
Bernhard D. Saxe
FOLEY & LARDNER
Washington Harbour
3000 K Street, N.W., Suite 500
Washington
DC
20007-5109
US
|
Family ID: |
26324106 |
Appl. No.: |
09/795979 |
Filed: |
March 1, 2001 |
Current U.S.
Class: |
424/227.1 ;
424/261.1; 424/85.5; 514/1.2; 514/10.3; 514/14.1; 514/2.8; 514/4.3;
514/5.9; 514/53; 514/56; 514/7.7 |
Current CPC
Class: |
A61K 9/1635 20130101;
A61K 9/5026 20130101; A61K 9/5153 20130101; A61P 3/10 20180101;
A61K 9/5031 20130101; A61K 9/5036 20130101; A61K 9/167 20130101;
A61K 9/1641 20130101; Y02A 50/472 20180101; A61K 9/1652 20130101;
Y02A 50/484 20180101; A61K 47/54 20170801; Y10S 977/906
20130101 |
Class at
Publication: |
424/227.1 ;
424/85.5; 514/3; 514/7; 514/53; 514/56; 424/261.1 |
International
Class: |
A61K 039/106; A61K
039/29; A61K 038/28; A61K 038/21; A61K 038/17; A61K 031/714; A61K
031/727; A01N 043/04 |
Claims
1. A novel complex for oral delivery of drugs, therapeutic
protein/peptides and vaccines which are loaded in a Vitamin
B.sub.12 (VB.sub.12) coupled particulate carrier system with
spacers in between, the carrier system with spacers having a
formula VB.sub.12-R'/R"--N wherein, R'or R" is spacer and/or agents
for derivatization of VB.sub.12 to provide either NH.sub.2 or COOH
or SH groups, and N is the micro or nano particle carriers for the
delivery of injectable drugs, therapeutic protein/peptides and
vaccines.
2. A complex as claimed in claim 1 wherein, the drugs to be
delivered are the traditional drugs administered exclusively by
parenteral routes, therapeutic peptides/proteins, other
biopharmaceuticals such as heparin and vaccines for
immunization.
3. A complex as claimed in claim 1 wherein, the drugs to be
delivered are the traditional drugs administered exclusively by
parenteral routes selected from gentamycin and Amikacin,
therapeutic peptides/proteins selected from insulin, EPO, G-CSF,
GM-CSF, Factor VIII, LHRH analogues and Interferons, other
biopharrnaceuticals such as heparin and vaccines selected from
Hepatitis `B` surface antigen, typhoid and cholera vaccine for
immunization
4. A complex as claimed in claim 1 wherein, the carrier system is a
pharmaceutically acceptable carrier comprising VB.sub.12 or its
analogs or their derivatives to be coupled to biodegradable
polymeric particulate carriers, which are, loaded with
drugs/vaccines given parentally.
5. A complex as claimed in claim 1 wherein, the carrier system is
transcytosed after receptor mediated endocytosis at or around ileum
by the natural uptake VB.sub.12.
6. A complex as claimed in claim 1 wherein, VB.sub.12 is the
targeting ligand for the intestine to systemic and/or lymphatic
destiny.
7. A complex as claimed in claim 1 wherein, VB.sub.12 is native
cynocobalamine (VB.sub.12) or various analogs of VB.sub.12 viz.,
aquocobalimin, adenosylcobalamin, methylcobalamin, hydroxycobalamin
and/or their derivatives or alkylcobalamines in which alkyl chain
is linked to cobalt of VB.sub.12 or cynocobolamin with chloro,
sulphate, nitro, thio or their analide, ethylamide, propionamide,
monocarboxylic and dicarboxylic acid derivatives of VB.sub.12 and
its analogues or monocarboxylic, dicarboxylic and tricarboxylic
acid derivatives and prominamide derivatives of `e` isomer of
monocarboxy VB.sub.12 and analogues of VB.sub.12 in which cobalt is
replaced by other metals (zinc or nickel etc) and various spacers
attached to these derivatives or any derivatives which retains IF
affinity after coupling to biodegradable particulate carriers.
8. A complex as claimed in claim 7 wherein, the derivatives of
VB.sub.12 also include derivatization at primary 5'-hydroxyl and
2'-hydroxyl groups of ribose moiety of VB.sub.12 and various
spacers attached at this site useful for coupling with particulate
carriers.
9. A complex as claimed in claim 1 wherein, R'-R"-the spacers
and/or agents for derivatization of VB12 to provide either NH.sub.2
or COOH or SH groups and combination thereof are selected from
diacids or alkyl diacids (COOH--COOH, (COO(CH.sub.2)nCOO)) alkyl
diamines (NH.sub.2(CH.sub.2)n-NH.sub.2) or alkyl diamides (NHCO
(CH)n CONH.sub.2) or hydrazides (NH.sub.2NH.sub.2) or alkyl
dihydrazides (NH.sub.2NHCO(CH.sub.2)n CONHNH.sub.2 or substitution
SH group containing agents (N-succinymidyl3-(2-pyridyldithio)
propionate or its long chain alkyl derivatives or acid anhydrides
[(CH)n CO CO O)] or acid halide spacers (R(CH.sub.2)n COCl) or
anhydroxy activated ester functional groups (NHS) which are used
for the peptide bonds or surfactant derivatives or polymeric
spacers, and in all cases `n` is an integer from 1,2,3. .
.infinite.
10. A complex as claimed in claim 1 wherein, the microspheres or
nanoparticles are made up of biodegradable and pharmaceutically
acceptable polymers.
11. A complex as claimed in claim 1 wherein, the drug loaded
polymeric particulates degrade in-vivo to release and deliver a
protein pharmaceutical/vaccine for its bioactive response.
12. A complex as claimed in claim 1 wherein, the particulate
carriers are biodegradable, hydrophobic or hydrophilic polymeric
microspheres/nanoparticles containing surface COOH, anhydride,
NH.sub.2, SH groups and combination thereof.
13. A complex as claimed in claim 1 wherein, the particulate
carriers systems are of both monolithic (matrix type, cross-linked)
and/or reservoir type (microcapsule and nanocapsule or multi
particulate type (particles within the particles) or particles
formed by co-addition the ligand VB.sub.12 to the polymer i.e.,
conjugate polymer particulates.
14. A complex as claimed in claim 1 wherein, the particulate
carriers include polysaccharide polymers viz., starch and their
derivatives, pectin, Amylose, guar gum and their derivatives,
dextran of different molecular weights and their derivatives,
chitosan and their derivatives, chondriotan sulphate and their
derivatives and finally other natural and semi synthetic
derivatives of polysaccharides and such polymers thereof.
15. A complex as claimed in claim 14 wherein, the cross linking
agents for polysaccharides include epichlorohydrin, POCl.sub.3,
borax (guar gum), aldehydes (e.g., glutaraldehyde) and other cross
linking agents thereof which cross links polysaccharide polymers to
give particulate carriers.
16. A complex as claimed in claim 12 wherein, less hydrophilic to
hydrophobic particulate carriers include biodegradable polymers of
poly(methylmethacrylate), poly(hydroxybutyrate),
polylactide(coglycolic acid), poly(anhydride) microspheres of
(fumaric acid: sebacic acid and poly fumaric acid) and poly
(lactide co-glycolide), fatty acylated particulates (hydrophilic
core surrounded by fatty acyl layer), LDL carriers,
multiparticulate systems (polymeric particles encapsulated in
polymers of different hydrophilicity or hydrophobicity) and finally
different compositions of these polymers.
17. A complex as claimed in claim 1 wherein, the particulate
carriers also include natural protein polymers such as albumin,
gelatin, semi synthetic or peptide based synthetic polymers and
derivatives thereof.
18. A complex as claimed in claim 15 wherein, the cross-linking
agents such as terephthloyl chloride, glutaraldehyde and such cross
linkers which cross-links protein polymers to give particulate
carriers.
19. A complex as claimed in claim 1 wherein, the particulates,
which are formed by polymerization of monomers such as
glutaraldehyde to poly (glutaraldehyde), and such particles formed
by polymerization of monomers and their coupling to VB.sub.12.
20. A complex as claimed in claim 1 wherein, the size of the system
for the systemic delivery of drugs, proteins and vaccines are
ranging from few nanometers to 10 .mu.m or more.
21. A complex as claimed in claim 1 wherein, these particulate
systems which are modified or activated on the surface to suit
VB.sub.12 linkage and/or complexation.
22. A complex as claimed in claim 21 wherein, the particulate
system is surface modified with VB.sub.12, and includes coupling of
VB.sub.12 to biodegradable particulate carriers.
23. A complex as claimed in claim 21, wherein, coupling of
VB.sub.12 derivative and particulates include both biodegradable
and non biodegradable bonds with and without spacers in
between.
24. A complex as claimed in claim 1 wherein, coupling between
VB.sub.12 derivative and particulate carriers or surface
modified/activated particulates is by amide bond with carbodiinides
such as 1-ethyl 3-3dimethyl amino propyl carbodiimide (EDAC) or
1,1' carbonyl diimidazole (CDI) or N, N' diisopropyl carbodiimide
(DIPC) and other peptide coupling agents or N-hydroxy succinimide
activated coupling (NHS) or periodate coupling or glutaraldehyde
activated coupling or CNBR mediated coupling or acid halide induced
amide coupling or by the use of hydrophilic spacer ethylene glycol
bis (succinimidyl succinate) (EGS) or hydrophobic spacers
disuccinimidyl suberate (DSS) or via a thiol cleavable spacer with
N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and any such
conjugates which retains IF affinity for VB.sub.12.
25. A complex as claimed in claim 1 wherein, the coupling involves
physical adsorptive type or complexation.
26. A complex as claimed in claim 1 wherein, the drugs, therapeutic
peptides/proteins and vaccines are entrapped within the highly
dense VB.sub.12 coupled biodegradable particulate carriers.
27. A complex as claimed in claim 25 wherein, the loading step of
the bioactive agent in question is in the preformed particulate
carrier conjugate or within the carrier during the preparation of
the particulate spheres or during the coupling between the
particles and VB.sub.12 derivative.
28. A complex as claimed in claim 26 wherein, the bioactives to be
delivered are 1) physically entrapped with in the particles
before/after/or during the conjugation or 2) adsorbed or 3)
covalently coupled or 4) by ionic interaction or 5) complexation
and any other methods there off to load VB.sub.12 particulate
systems.
29. A complex as claimed in claim 1 wherein, the use of
surfactants, aggregation minimizers, protease inhibitors and
permeation enhancers as adjuvants in the formulations.
30. A complex as claimed in claim 1 wherein, the delivery systems
are co-administered with exogenous intrinsic factor (IF) of
VB.sub.12, especially for IF deficient species.
31. A complex as claimed in claim 1 wherein, the complex is for
oral delivery of injectable drugs therapeutic protein/peptides and
vaccines.
32. A complex as claimed in claim 1 wherein, the said delivery
systems are formulated into dosage forms suitable for oral
delivery.
33. A complex as claimed in claim 32 wherein, the oral dosage forms
include solutions, suspensions, gels, pastes, elixirs, viscous
colloidal dispersions, tablets, capsules and/or oral control
release types and finally any such delivery forms for oral route.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a complex for oral delivery of
drugs, therapeutic protein/peptides, vaccines, which are loaded in
Vitamin B.sub.12 coupled particulate carriers system with spacers
in between. Typically, VB.sub.12 acts as ligand for endocytosis
through intestine and the particulate systems will act as a cargo
protecting the intestinal labile drugs to deliver them to systemic
destination and/or to the specific site required.
BACKGROUND ART
[0002] Although many peptide/proteins, pharmaceuticals and vaccines
are currently administered by injection, this method of delivery
has a number of disadvantages which has led the scientific
community to strive to develop an alternative oral delivery system
for these bioactives. The inherent limitations to parenteral
delivery include (1) patient compliance (there is a need for
repeated injections due to the short half-life of these molecules)
(2) discomfort from this form of delivery caused by the need for
repeated prolonged dosage regimen (3) highly variable
bioavailability both within and between subjects for molecules such
as subcutaneous insulin and (4) the non-physiological delivery
pattern particularly of subcutaneous injections. Further, small
increases in actual drug delivered due to changes in dosage and/or
mode of delivery may cause down-regulation of the desired response
to most of these bioactives. In contrast most often a pulsatile or
flat delivery profile is required which mimics the normal
physiological rhythm. Apart from the problem described, parenteral
vaccines are of limited efficacy due to the need for repeated
vaccination and due to the fact that they elicit only humoral
immunity.
[0003] To address the above problems, various non-invasive delivery
systems have been attempted. The major delivery barriers common to
these routes are (1) poor intrinsic permeability due to large size
and hydrophilicity of the bioactive (2) enzymatic degradation in
the hostile environment of GIT by lumenal proteases and cellular
peptidases, unlike some of the traditional drugs. Although these
odds are formidable for the per oral route, it enjoys advantages in
terms of convenience and patient compliance as well as safety, less
stringent quality control, cost of therapy and for vaccines, the
potential for unlimited frequency of boosting. Furthermore, oral
vaccines offer the potential to protect against not only enteric
pathogens (by producing localized sIgA), but also a wide range of
pathogens infecting other mucosa (respiratory and genital) by
producing a common disseminated mucosal immune response.
Furthermore oral vaccines may prove particularly useful in the
elderly because mucosal immunity, unlike systemic immunity does not
seem to be an age associated dysfunction. This mode of immunization
may also be beneficial in the very young, because mucosal immunity
develops earlier in ontogeny than systemic immunity. Over the past
few decades significant efforts have been made to develop oral
protein/vaccine systems using permeation enhancers (ZOT, which has
proven to be toxic on long term use), enzyme inhibitors,
surfactants, emulsions, colon targeted systems, bioadhesive systems
and particulate systems. Although the challenges to develop oral
systems are numerous, the potential therapeutic need remains high,
particularly with increased identification of novel peptides and
increased production of existing protein drugs from the
biotechnology arena.
[0004] Peptide/protein drugs and vaccines are mostly given by
parenteral routes. The problems for the oral delivery are:
[0005] 1) Degradation of the above bioactives in the harsh
environment of intestine and by gut proteases.
[0006] 2) Their large size and hydrophilicity causes permeability
problem across the intestine.
[0007] 3) The fragility of these bioactives precludes to be
formulated as oral dosage forms.
[0008] 4) And finally their short in-vivo half lives.
[0009] Over the past 3-4 decades efforts have been made to deliver
them by oral route using various formulation approaches such as
emulsions, microspheres, nanoparticles, vasicular carriers such
liposomes, using permeation enhancers and protease inhibitors and
by protein-carrier conjugates.
[0010] Recently it has been shown that the problem of poor
intrinsic permeability can be possibly overcome by delivery via a
specific carrier mechanism that transports pharmaceuticals from the
intestine into the circulation. In this regard, Russell-Jones and
co workers [1994] have found the possibility of coupling these
bioactives with Vitamin B.sub.12 (VB.sub.12) in a manner that does
not interfere with intrinsic factor (IF) mediated uptake of the
VB.sub.2 carrier to the systemic circulation. Some interesting
results have been achieved with oral VB.sub.12 conjugates of LHRH
(Russell-Jones 1998), interferon (Habberfield and Jensen-Pippo PPO,
1996) G-CSF and erythropoietin (Habberfield, 1996). However, such
successful oral delivery of conjugates cannot be obtained with many
other bioactives because of the limited uptake of VB.sub.12 (1
nmol/dose), the loss of bioactivity due to covalent linkage, loss
of IF affinity of VB.sub.12 (steric factor) and finally the
liability of such conjugates to GIT degradation. To address the
above problems we have made endeavors to make an oral delivery
system (VB.sub.12-sphere conjugate) that could be targeted to the
systemic circulation through VB.sub.12-IF-IFR ligand-mediated
endocytosis via ileocytes of the intestine following oral
administration. In vitro transport of VB.sub.12 coated polystyrene
nanoparticles (non-biodegradable, hydrophobic) has been
demonstrated across Caco-2 cell cultures (Russell Jones, 1997,
1999). However, the level of transport cannot be extended to other
polymeric particulates in general. This can be explained by the
differing physicochemical characteristics of polymers such as
hydrophobicity, as hydrophobic materials may be more readily taken
up by cell surface lipid bilayers. Also one of the prerequisites
for drug delivery is that the polymer used to make the
nanoparticles should readily or slowly be degraded in the systemic
environment or by enzymes which release the pharmaceutical molecule
and thereby initiate its bioactive response. Hence, the use of
nonbiodegradable polymers for transport studies is not necessarily
indicative, of uptake by other biodegradable polymers in general.
Further, developing a system of--loading of hydrophilic
peptide/protein or vaccine bioactives into hydrophobic polymeric
carriers is definitely not obvious.
[0011] Among all these approaches, there was some enhancement of
delivery, but major break through of achieving therapeutically
relevant dose delivery is not achieved. In the recent past, a
delivery system was invented by one of the co-inventors using
VB.sub.12 as carrier molecule which is coupled with protein drug to
be delivered i.e. VB.sub.12-protein conjugates. VB.sub.12 binds
with intrinsic factor of the intestine
(VB.sub.12+IF.fwdarw.VB.sub.12 IF) in the duodenum and the whole
VB.sub.12 IF complex binds to intrinsic factor receptor (IFR) at
the ileum of the small intestine. From there, it is endocytosed and
subsequently transcytosed by transcobolamin (TcII) to reach
systemic circulation.
[0012] Therefore, if protein is coupled with VB.sub.12, it will be
co-transported along with VB.sub.12 molecule to reach systemic
circulation.
[0013] Two conditions are to be satisfied for this approach
i.e.
[0014] Case 1: VB.sub.12 molecule must retain its IF affinity after
being coupled to protein to be delivered.
[0015] Case 2: Also protein molecule must retain its bioactivity
after coupling to VB.sub.12.
[0016] For case 1: Native VB.sub.12 (cynocobolamin) cannot be
directly coupled to protein, i.e. VB.sub.12 losses its IF affinity
by such coupling.
[0017] Therefore, in the above technology VB.sub.12 is hydrolyzed,
where it forms 3 isomers (a, b and e isomers). Among the 3 isomers,
the `e` isomer retains considerable IF affinity after coupling to
other molecules.
[0018] Case 2: For the ease of coupling to provide suitable groups
and to retain full bioactivity of protein drug, various derivatives
of e-VB.sub.12 using various spacers. These derivatives are coupled
to protein drugs to be delivered. Such spacers also increases IF
affinity of e isomer. About this technology, one of the inventors
of the present application namely Dr. Gregory Russell Jones written
a chapter in a book in 1995 (peptide based drug design Chapter 8).
In this chapter, the author elaborated this technology and
summarized the results of VB-LHRH analogs and VB.sub.12 vasopressin
and also mentioned the possibility of conjugating other bioactives
such as EPO and G-CSF. Later in 1995, two research papers appeared
in bioconjugate chemistry (1) VB.sub.12-LHRH analogs (Volume 6, No.
1, 1995, 34-42), (2) VB.sub.12-EPO and VB.sub.12-G CSF (Volume 6,
No.4, 1995, 461-465). Later in 1996, one of the authors patented
VB.sub.12-EPO and VB.sub.12-G CSF conjugates (U.S. Pat. No. 548068,
August 1998. In this patent he claimed any derivative of VB.sub.12
which retains IF affinity after being coupled protein drug to be
delivered. Later, in November 1996 there is another patent (U.S.
Pat. No. 5,574,018) on the similar system (Habber Field, who is
associated D. Russell on some research paper works earlier) i.e.
VB.sub.12-EPO, VB.sub.12-G SCF and VB.sub.12-Interferon. But the
derivative used is different i.e. at hydroxy site of ribose of
VB.sub.12. This derivatization at this site and derivatives coupled
to protein drug also retain IF affinity of VB.sub.12. But in the
earlier patent any derivative of VB.sub.12, which retains IF
affinity after being coupled to protein drugs is claimed. Protein
drugs used in the other patent are also same with exception of
interferon.
[0019] Two U.S. Pat. No. 5,589,463 and 5,807,832 wherein, the one
of the coinventor of the present application is also main inventor
describe VB.sub.12 -bioactive chemical (covalent) conjugate. The
bioactive may be protein, vaccine or antigen (may be of
polysaccharide nature) or other biopharnaceutical (heparin) or
traditional drug (neomycin). The above chemical complexes they
claim to formulate in known dosage forms suitable for oral delivery
gels, dispersions, emulsions, capsules, tablets etc. Technology of
both these patents is somewhat similar to other U.S. Pat. Nos.,
5,548,064 and 5,574,018, which are, referred above.
[0020] In spite of all these, the conjugates of VB.sub.12 have the
following drawbacks.
[0021] 1) Protein is not protected from degradation by proteases in
gut.
[0022] 2) All proteins cannot be coupled, as some protein
bioactives may loose active molecular confirmation due to its
coupling to bulky VB.sub.12 (steric factors).
[0023] 3) Uptake of VB.sub.12 transport system (1 nano gram g/dose)
is not sufficient for proteins which have very short half lives
i.e. therapeutically relevant dose cannot be achieved.
[0024] The present invention is entirely different in that
bioactive (protein/vaccine or drug) is not directly coupled to
VB.sub.12 or its analogue. Instead, VB.sub.12 or its analogues are
covalently coupled to micro/nano particle surface in which
bioactive is loaded. The micro and nano particles are made up of
biodegradable and pharmaceutically acceptable polymers.
DETAILED OF DESCRIPTION OF THE INVENTION
[0025] The conjugation of various peptides and proteins to the
Vitamin B.sub.12 molecule has been shown to facilitate the in-vitro
and in-vivo transport of these moieties across the epithelial cells
of the intestine. However, pharmaceutically relevant oral delivery
of many vitamin B12-pharmaceutical conjugates does not occur with
many of these bioactives due to the limited uptake capacity of the
VB.sub.12 transport system, loss of bioactivity of native protein
during conjugation to VB.sub.12, loss of intrinsic factor (IF)
affinity of the conjugates and finally due to the liability of the
bioactives to GI degradation. In order to overcome these
shortcomings we have endeavoured to develop vitamin
B.sub.12-particulate (both microspheres and nanospheres) systems
that could be taken up by the natural uptake mechanism for
VB.sub.12. Different biodegradable particulate systems
(microspheres and nanospheres) were prepared. These particles were
surface modified with vitamin B.sub.12 and these preformed sphere
conjugates were loaded with therapeutic proteins (insulin,
hepatitis `B` vaccine). In some cases the loading step was altered.
In-vitro studies showed that insulin was protected from degradation
by gastric enzymes. These systems, of different sizes, were fed to
diabetic rats, whose blood glucose levels were monitored over time.
The pharmacological bioavailability of these systems was 5-25%,
which showed not only oral bioactivity but also evidence of
controlled release of these biopharmaceuticals. These systems
showed interesting results with vaccine loaded VB.sub.2
microspheres and nanospheres. Thus the above carrier system and
various strategies mentioned herein, upon further testing and
optimization, may make per oral protein delivery a reality.
[0026] The present invention relates to the field of pharmaceutical
preparation of traditional injectable drugs given parenterally,
peptide and protein pharmaceuticals including vaccines,
particularly in the field of such pharmaceutical preparations,
which are suitable for oral delivery.
[0027] In accordance with the present invention there is provided a
drug delivery system comprising a protein drug (insulin and
hepatitis `B` vaccine) incorporated within the biodegradable
polymeric particulates, which are coated with VB.sub.12 on their
surface as shown in FIG. 1, wherein D is the injectable drug,
SP--is the long chain spacer to retain IF affinity of Vitamin
B.sub.12 and/or for ease of coupling, BDC is the biodegradable
carrier particle (microsphere/nano particle).
[0028] For the purpose of successful oral drug delivery, VB.sub.2
acts as targeting ligand for initial transport across the
intestinal epithelium the circulation or for site-specific delivery
of the vaccines. The VB.sub.12 carries the particulate spheres
(micro and nano) along with it until the desired site reached.
[0029] In the embodiment of the present invention,
VB.sub.12-microsphere and VB.sub.12-nanoparticle systems deliver
the traditional parenteral drugs, peptides/proteins including
vaccines by oral route. Various biodegradable microspheres and
nanoparticles are prepared with existing natural, semi synthetic
and synthetic polymers. These particulates systems (micro and nano)
can be surface modified to provide groups suitable for VB.sub.12
conjugation. Vitamin B.sub.12 derivatives or their various
derivatives with numerous spacers (Russell Jones et al 1995a and
1995b) were coupled to particulate carriers. Different linkages,
both biodegradable (cleaved by systemic enzymes) and
non-biodegradable covalent linkages are used to link the VB.sub.12
to the particulate. The coupling also involves ionic, coordinate
covalent and strong physical adsorptive bounds.
[0030] In an embodiment, the drugs to be delivered are the
traditional drugs administered exclusively by parenteral routes,
therapeutic peptides/proteins, other biopharmaceuticals such as
heparin and vaccines for immunization.
[0031] In another embodiment of the present invention, the drugs to
be delivered are the traditional drugs administered exclusively by
parenteral routes selected from gentamycin and Amikacin,
therapeutic peptides/proteins selected from insulin, EPO, G-CSF,
GM-CSF, Factor VIII, LHRH analogues and Interferons, other
biopharmaceuticals such as heparin and vaccines selected from
Hepatitis `B` surface antigen, typhoid and cholera vaccine for
immunization.
[0032] In yet another embodiment, the carrier system is a
pharmaceutically acceptable carrier comprising VB.sub.12 or its
analogs or their derivatives to be coupled to biodegradable
polymeric particulate carriers, which are, loaded with
drugs/vaccines given parentally.
[0033] In yet another embodiment, the carrier system is
transcytosed after receptor mediated endocytosis at or around ileum
by the natural uptake VB.sub.12.
[0034] In yet another embodiment, VB.sub.12 is the targeting ligand
for the intestine to systemic and/or lymphatic destiny.
[0035] In yet another embodiment, VB.sub.12 is native
cynocobalamine (VB.sub.12) or various analogs of VB.sub.12 viz.,
aquocobalimin, adenosylcobalamin, methylcobalamin, hydroxycobalamin
and/or their derivatives or alkylcobalamines in which alkyl chain
is linked to cobalt of VB.sub.12 or cynocobolamin with chloro,
sulphate, nitro, thio or their analide, ethylamide, propionamide,
monocarboxylic and dicarboxylic acid derivatives of VB.sub.12 and
its analogues or monocarboxylic, dicarboxylic and tricarboxylic
acid derivatives and prominamide derivatives of `e` isomer of
monocarboxy VB.sub.12 and analogues of VB.sub.12 in which cobalt is
replaced by other metals (zinc or nickel etc) and various spacers
attached to these derivatives or any derivatives which retains IF
affinity after coupling to biodegradable particulate carriers.
[0036] In yet another embodiment, the derivatives of VB.sub.12 also
include derivatization at primary 5'-hydroxyl and 2'-hydroxyl
groups of ribose moiety of VB.sub.12 and various spacers attached
at this site useful for coupling with particulate carriers.
[0037] In yet another embodiment, R'--R"-the spacers and/or agents
for derivatization of VB12 to provide either NH.sub.2 or COOH or SH
groups and combination thereof are selected from diacids or alkyl
diacids (COOH--COOH, (COO(CH.sub.2)nCOO)) alkyl diamines
(NH.sub.2(CH.sub.2)n-NH.- sub.2) or alkyl diamides (NHCO (CH)n
CONH.sub.2) or hydrazides (NH.sub.2NH.sub.2) or alkyl dihydrazides
(NH.sub.2NHCO(CH.sub.2)n CONHNH.sub.2 or substitution SH group
containing agents (N-succinymidyl3-(2-pyridyldithio) propionate or
its long chain alkyl derivatives or acid anhydrides [(CH)n CO CO
O)] or acid halide spacers (R(CH.sub.2)n COCl) or anhydroxy
activated ester functional groups (NHS) which are used for the
peptide bonds or surfactant derivatives or polymeric spacers, and
in all cases `n` is an integer from 1,2,3. . .infinite.
[0038] In yet another embodiment, the microspheres or nanoparticles
are made up of biodegradable and pharmaceutically acceptable
polymers.
[0039] In yet another embodiment, the drug loaded polymeric
particulates degrade in-vivo to release and deliver a protein
pharmaceutical/vaccine for its bioactive response.
[0040] In yet another embodiment, the particulate carriers are
biodegradable, hydrophobic or hydrophilic polymeric
microspheres/nanoparticles containing surface COOH, anhydride,
NH.sub.2, SH groups and combination thereof.
[0041] In yet another embodiment, the particulate carriers systems
are of both monolithic (matrix type, cross-linked) and/or reservoir
type (microcapsule and nanocapsule or multi particulate type
(particles within the particles) or particles formed by co-addition
the ligand VB.sub.12 to the polymer i.e., conjugate polymer
particulates.
[0042] In yet another embodiment, the particulate carriers include
polysaccharide polymers viz., starch and their derivatives, pectin,
Amylose, guar gum and their derivatives, dextran of different
molecular weights and their derivatives, chitosan and their
derivatives, chondriotan sulphate and their derivatives and finally
other natural and semi synthetic derivatives of polysaccharides and
such polymers thereof.
[0043] In yet another embodiment, the cross linking agents for
polysaccharides include epichlorohydrin, POCl.sub.3, borax (guar
gum), aldehydes (e.g., glutaraldehyde) and other cross linking
agents thereof which cross links polysaccharide polymers to give
particulate carriers.
[0044] In yet another embodiment, less hydrophilic to hydrophobic
particulate carriers include biodegradable polymers of
poly(methylmethacrylate), poly(hydroxybutyrate),
polylactide(coglycolic acid), poly(anhydride) microspheres of
(fumaric acid: sebacic acid and poly fumaric acid) and poly
(lactide co-glycolide), fatty acylated particulates (hydrophilic
core surrounded by fatty acyl layer), LDL carriers,
multiparticulate systems (polymeric particles encapsulated in
polymers of different hydrophilicity or hydrophobicity) and finally
different compositions of these polymers.
[0045] In yet another embodiment, the particulate carriers also
include natural protein polymers such as albumin, gelatin, semi
synthetic or peptide based synthetic polymers and derivatives
thereof.
[0046] In yet another embodiment, the cross-linking agents such as
terephthloyl chloride, glutaraldehyde and such cross linkers which
cross-links protein polymers to give particulate carriers.
[0047] In yet another embodiment, the particulates, which are
formed by polymerization of monomers such as glutaraldehyde to poly
(glutaraldehyde), and such particles formed by polymerization of
monomers and their coupling to VB.sub.12.
[0048] In yet another embodiment, the size of the system for the
systemic delivery of drugs, proteins and vaccines are ranging from
few nanometers to 10 .mu.m or more.
[0049] In yet another embodiment, these particulate systems which
are modified or activated on the surface to suit VB.sub.12 linkage
and/or complexation.
[0050] In yet another embodiment, the particulate system is surface
modified with VB.sub.12, and includes coupling of VB.sub.12 to
biodegradable particulate carriers.
[0051] In yet another embodiment, coupling of VB.sub.12 derivative
and particulates include both biodegradable and non biodegradable
bonds with and without spacers in between.
[0052] In yet another embodiment, coupling between VB.sub.12
derivative and particulate carriers or surface modified/activated
particulates is by amide bond with carbodiimides such as 1-ethyl
3-3dimethyl amino propyl carbodiimide (EDAC) or 1,1' carbonyl
diimidazole (CDI) or N, N' diisopropyl carbodiimide (DIPC) and
other peptide coupling agents or N-hydroxy succinimide activated
coupling (NHS) or periodate coupling or glutaraldehyde activated
coupling or CNBR mediated coupling or acid halide induced amide
coupling or by the use of hydrophilic spacer ethylene glycol bis
(succinimidyl succinate) (EGS) or hydrophobic spacers
disuccinimidyl suberate (DSS) or via a thiol cleavable spacer with
N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and any such
conjugates which retains IF affinity for VB.sub.12.
[0053] In yet another embodiment, the coupling involves physical
adsorptive type or complexation.
[0054] In yet another embodiment, the drugs, therapeutic
peptides/proteins and vaccines are entrapped within the highly
dense VB.sub.2 coupled biodegradable particulate carriers.
[0055] In yet another embodiment, the loading step of the bioactive
agent in question is in the preformed particulate carrier conjugate
or within the carrier during the preparation of the particulate
spheres or during the coupling between the particles and VB.sub.12
derivative.
[0056] In yet another embodiment, the bioactives to be delivered
are 1) physically entrapped with in the particles before/after/or
during the conjugation or 2) adsorbed or 3) covalently coupled or
4) by ionic interaction or 5) complexation and any other methods
there off to load VB.sub.12 particulate systems.
[0057] In yet another embodiment, the use of surfactants,
aggregation minimizers, protease inhibitors and permeation
enhancers as adjuvants in the formulations.
[0058] In yet another embodiment, the delivery systems are
co-administered with exogenous intrinsic factor (IF) of VB.sub.12,
especially for IF deficient species.
[0059] In yet another embodiment, the said delivery systems are
formulated into dosage forms suitable for oral delivery.
[0060] In yet another embodiment, the oral dosage forms include
solutions, suspensions, gels, pastes, elixirs, viscous colloidal
dispersions, tablets, capsules and/or oral control release types
and finally any such delivery forms for oral route.
[0061] Oral delivery of peptide and proteins has proven an elusive
target for the pharmaceutical industry over the past few decades.
In the present invention a model therapeutic protein, insulin and
vaccine hepatitis `B` vaccine were selected as model protein. The
presence of various groups on VB.sub.12 derivatives were assigned
by comparing IR spectra with that of native VB.sub.12. The `e`
carboxylate of VB.sub.2, which has highest affinity for IF
affinity, was separated and covalently linked to various spacers to
give various VB.sub.12 derivatives (FIG. 2). Various VB.sub.12
particulate systems of different sizes were prepared and their
surface was modified by various means to suit VB.sub.12 conjugation
(sizes were illustrated in the text). These VB.sub.12 coupled
particulate systems (FIG. 1) were loaded with insulin and hepatitis
`B` vaccine. It is assumed, even if some of the carrier systems
delivers sub therapeutic doses of protein (insulin), its relevance
would be adequate to trigger immune response. Consequently the
problem is far less complicated for traditional drugs that are
exclusively given parenterally (hence model drug was not chosen)
and will be loaded at a later stage. For these studies permeation
enhancers were not taken along with delivery systems, as the main
objective is to study uptake mechanism of this carrier system. It
is assumed that the above additives may probably potentiate the
efficacy of the carrier system by allowing absorption through other
mechanisms. In-vitro enzymatic study showed that insulin was
protected against digestive action of intestinal enzymes when
entrapped within the spheres, whereas free insulin solutions were
completely degraded under the same experimental conditions (FIG.
3). Various nanoparticles VB.sub.12 conjugates in the size range of
160-250 nm showed interesting results with model protein insulin.
Antidiabetic activities of some representative insulin delivery
systems are shown in FIGS. 4-8. These systems showed maximum blood
glucose reduction of 48.4.+-.4.1 (% initial) at 5 hours. The onset
of action was observed 2 hours after administration for all the
delivery systems. Maximum % decrease in glucose level (tmax) was
found at the 5th hour for all the systems microparticulate
conjugates, which was maximal at the 6th hour. The above results
show similar uptake kinetics to that observed for VB.sub.12
absorption in the rat, and suggest that the uptake be due to
specific VB.sub.12 mediated absorption, which takes several hours.
During preliminary assessment of the different systems the duration
of action was studied for 7 hours for all the systems (n=3
animals), but in later studies the uptake was continued for up to
12 hours (n=5) (FIG. 9). The Antidiabetic activity of plain insulin
was estimated following intravenous (IV) and subcutaneous
administration (FIG. 10). The pharmacological availability was
assessed by the above results. The AUC.sub.0-7 values were
significantly higher (p<0.01) for nanoparticle conjugates and
higher size nanoparticle conjugates (p<0.05) when compared to
AUC.sub.0-7 value for i.v. route. With microparticulate conjugates
there was little difference in AUC's when compared to i.v. route
but there was reduction in blood glucose levels at 3rd, 4th, 5th
and 6th hours whereas i.v. injection of insulin showed no activity
at these times. The pharmacological availability of various
particulate conjugates was from 5% to as much as 23%. It is
expected that microparticle conjugates may give good results with
vaccines for mucosal immunity, as dose to trigger immune response
will not be more than therapeutic proteins (work with hepatitis `B`
is under progress. Nanoparticle conjugates containing 10 I.U./kg
showed best results. Uptake studies showed that the absorption is a
VB.sub.12 specific carrier-mediated type (FIG. 9). This carrier
system and the various strategies described in the present
invention have potential to replace the current need for injectable
protein drugs/vaccines and other traditional drugs that are given
exclusively by parenteral route. These preliminary findings provide
a sound platform for further testing and optimizing a delivery
technology for peroral delivery of injectable drugs.
[0062] For ease of understanding, the distinction between the
instant invention and the prior art is schematically represented in
FIG. 11. As shown therein, FIG. 11(A) represents the prior art
wherein VB.sub.12, a bioactive covalent complex is formulated as
solutions or as dispersion or as paste or used as tablets, in
appropriate dosage forms, FIG. 11(B) represents the instant
invention wherein VB.sub.12 or its analogues are covalently coupled
to microspherelnanoparticle surface in which bioactive ingredient
is loaded and formulate dosage forms.
[0063] For easier understanding of this present invention,
schematic representation of earlier patents and the present
invention is shown in FIG. 11 wherein the Vitamin B.sub.12 is
covalently linked to the bioactive materials and administered as a
solution, dispersion, paste and tablet forms. In the present
invention, bioactive (protein/vaccine or drug) is not directly
coupled to Vitamin B.sub.12 or its analogues, instead Vitamin
B.sub.12 or its analogues are covalently coupled to
microsphere/nanoparticle surface in which bioactive ingredient is
loaded and formulate dosage forms. The micro and nano particles are
made up of biodegradable and pharmaceutically acceptable
polymers.
[0064] As shown in FIG. 11, D is bioactive material, SP is long
chain spacer to retain IF affinity of Vitamin B.sub.12 and/or for
case of coupling BDC is biodegradable carrier particle
(microsphere/nanoparticles- ) and VB.sub.12 is Vitamin
B.sub.12.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] FIG. 1 shows VB12 coupled particulate systems.
[0066] FIG. 2 shows structure of VB.sub.12 and sites of linkage for
IF affinity.
[0067] FIG. 3 graphical representation which shows effect of pH and
proteolysis on plain insulin and insulin containing
VB.sub.12-microsphere conjugates.
[0068] FIG. 4 graphical representation which shows hypoglycemic
effect of oral administration of insulin loaded VB.sub.12-sphere
conjugates in streptozotocine induced diabetic rats.
[0069] FIG. 5 graphical representation which shows hypoglycemic
effect of oral administration of insulin-loaded VB.sub.12 coated
microsphere/nanoparticle conjugates in streptozoticin induced
diabetic rats (n=3).
[0070] FIG. 6 graphical representation which shows hypoglycemic
effect of oral administration of insulin-loaded VB.sub.12 coated
microsphere/nanoparticle conjugates in streptozoticin induced
diabetic rats (n=3).
[0071] FIG. 7 graphical representation which shows hypoglycemic
effect of oral administration of insulin-loaded VB.sub.12 coated
microsphere/nanoparticle conjugates in streptozoticin induced
diabetic rats (n=3).
[0072] FIG. 8 graphical representation which shows hypoglycemic
effect of oral administration of insulin-loaded VB.sub.12 coated
microsphere conjugates in streptozoticin induced diabetic rats
(n=3).
[0073] FIG. 9 graphical representation which shows dose response
relation of effective delivery system (DDN1 CO--NH `e' VB.sub.12)
and its uptake studies.
[0074] FIG. 10 graphical representation which shows hypoglycemic
effect of plain conjugate and insulin given by I.V., S.C. and oral
routes in streptozotocine induced diabetic rats.
[0075] FIG. 11 shows schematic representation of present invention
in comparison with earlier inventions.
[0076] Various VB.sub.12 sphere conjugates were prepared as
follows. The following are given by the way of illustration of the
present invention and therefore should not be construed to limit
the scope of the invention.
SECTION I
[0077] 1.0 Synthesis of VB.sub.12 analogues suitable for
conjugation:
[0078] Native VB.sub.12 (FIG. 2, la) was subjected to mild acid
hydrolysis (0.4 ,M, HCl ; 72h, RT) to produce a monoacid derivative
from one of the three propionamide side chains (b, d and e) of the
corrin ring. The `e` monocarboxylate of VB.sub.12 (1b), which has
the highest affinity for IF (Kolhouse and Allen, 1977) was
separated from b and d isomeric mixture by a combination of DOWEX
AG 1.times.2 (Biorad) chromatography and semi-preparative
C.sub.18--RP--HPLC (with a gradient of 5-100% acetonitrile in 0.1%
trifluoroacetic acid) (Anton et al., 1980).
[0079] 1.1 Synthesis of amino derivatives of `e` VB.sub.12 (Russell
Jones et al., 1995):
[0080] Amino derivatives of `e` VB.sub.12 were prepared by reacting
the `e` isomer of VB.sub.12 (1b) with various spacers such as 1,2
diaminoethane, 1,6-diaminohexane, 1,12 diaminododecane,
1,3-diamino-2-hydroxypropane, and 1,6-diamino-3, 4-dithiahexane
(a.k.a cystamine) to produce amino `e` VB.sub.12 derivatives of 1c,
1d, 1e, 1f and 1g respectively. These reactions were carried out at
pH 6.5 using 20 fold molar excess of diamine and 1-ethyl-3-
[3-(dimethyl amino) propyl] carbodiimide (EDAC) respectively.
[0081] In a separate reaction 1,12 diaminododecane was coupled to
native VB.sub.12 (1a) to produce amino-dodecyl-5'O--VB.sub.12 (1h)
following activation of VB.sub.12 with 1,1'-carbonyl diimidazole in
dry DMF containing diisopropyl ethyl amine. Other amine derivatives
can also be prepared at this site (data not shown).
[0082] All amino derivatives of VB.sub.12 were purified by RP--HPLC
(semi-preparative C4 column) with a gradient of 5-100% acetonitrile
in 0.1% TFA. Eluted material was further purified by adsorption to
S-Sepharose and elution with 0.1 M HCl. The amino derivatives were
further purified by extraction into phenol and back extraction into
water after the addition of methylene chloride to the phenol phase.
Finally each of the products was lyophilized and recovered as a
red-powder.
[0083] 1.2 Synthesis of dithiopyridyl (DTP) amino VB.sub.12
derivatives:
[0084] Three dithiopyridyl (DTP) amino-eVB.sub.12 derivatives were
prepared by reacting succinimidyl 3-(2-pyridyldithio) propionate
(SPDP) with 2-aminoethyl-eVB.sub.12 (1c) 6-aminohexyl-eVB.sub.12
(1d) and 12-aminododecyl-5'O VB.sub.12 (1h) to give the respective
derivatives of (1i), (1j) and (1k). Typically the reaction was
carried out by dissolving the amino-VB.sub.12 derivative (50 mg/ml)
in 0.1 M PO4 buffer, pH 7.5 containing 0.1 M NaCl, followed by the
addition of 800 .mu.l of solution of SPDP (50 mg/ml in acetone) to
the amino-eVB.sub.12 derivative. After overnight reaction at room
temperature the DTP-amino-eVB.sub.12 product was purified by
RP--HPLC on a semi-preparative C4 column and then lyophilized.
[0085] 1.3 Synthesis of long chain dithiopyridyl (DTP)
amino-VB.sub.12 derivatives:
[0086] These derivatives were prepared from the 6-aminohexyl-e
VB.sub.12 (1d) by sequential reaction with disuccinimidyl suberate
(DSS) (to give [[(monosuccinimidyl) suberyl] hexyl]-e VB.sub.12)
and 1,12-diaminododecane to give
[[(12-aminododecyl)suberyl]hexyl]-e VB.sub.12 (1 l). This product
obtained was derivatized at the terminal amino group with SPDP,
purified on RP--HPLC, and lyophilized to give derivative (1 m).
[0087] 1.4 Synthesis of hydrazidyl derivatives of e-VB.sub.12
carboxylate:
[0088] These derivatives were prepared to couple to COOH
particulates. Hydrazido-e VB.sub.12 (1 n) was prepared by a
two-step synthesis involving the coupling of tertiary-butyl
carbazate to eVB.sub.12 carboxylate and subsequent removal of the
t-Boc group to generate the free hydrazide.
Cys-hydrazido-eVB.sub.12 was synthesized from eVB.sub.12 cystamine
(1 g). The conversion of this material to eVB.sub.12 Cys-hydrazide
(1 o) proceeded by succinylation of the e VB.sub.12 cystamine and
subsequent conversion of the resultant terminal carboxyl group to a
hydrazide by the procedure outlined above for eVB.sub.12 hydrazide.
The (adipylhydrazido)-eVB.sub.12 reagent (1 p) was readily prepared
in one step from eVB.sub.12 carboxylate and a 20-fold excess of
adipylhydrazide by the addition of EDAC.
[0089] 1.5 Anilido derivative of eVB.sub.12:
[0090] eVB.sub.12 carboxylate (1b) was activated with NHS and EDAC
and than coupled to para-aminopheyl acetic acid to give the
VB.sub.12 anilide (1q).
[0091] 1.6 Formation of VB.sub.12 derivatives containing hindered
thiol groups:
[0092] Amino ethyl eVB.sub.12 (1c) was coupled with
4-[(succinimidyl oxy)- carbonyl] -.varies.-methyl-.varies.-(2
pyridyldithio) toluene using standard literature conditions
(Blackey et al., 1987). The product.varies.-methyl-.varies.-(2
pyridyldithio) tolyl]-hexyl] e-VB.sub.12 (1r) was purified by
RP--HPLC.
[0093] 1.7 Formation of Iodoacetamido derivative of VB.sub.12:
[0094] Amino ethyl eVB.sub.12 was converted to iodoacetamido
derivative by reaction with NHS ester of iodoacetic acid. The
product (1 s) was purified by RP--HPLC.
[0095] 1.8 Formation of tetra peptide spacer derivative of
eVB.sub.12:
[0096] eVB.sub.12 carboxylate (1b) was activated with EDAC and NHS,
as previously described, was added to solution of tetrapeptide
GGEA-OMe in bicarbonate buffer, pH 9.5. The product
VB.sub.12-GGEA-OMe (1t) was purified by RP--HPLC.
[0097] Structure of VB.sub.12 and sites of linkage for IF affinity
as shown in FIG. 2, wherein,
[0098] 1a R'.dbd.NH2; R".dbd.H
[0099] 1b R'.dbd.OH ; R".dbd.H
[0100] 1c R'.dbd.NH (CH.sub.2).sub.2 NH.sub.2: R".dbd.H
[0101] 1d R'.dbd.NH(CH.sub.2).sub.6 NH.sub.2: R".dbd.H
[0102] 1e R'.dbd.NH (CH.sub.2).sub.12 NH.sub.2: R".dbd.H
[0103] 1f R'.dbd.NHCH.sub.2 CH(OH)CH.sub.2 NH.sub.2: R".dbd.H
[0104] 1g R'.dbd.NH(CH.sub.2).sub.2 SS (CH.sub.2).sub.2 NH.sub.2:
R".dbd.H
[0105] 1h R'.dbd.NH.sub.2: R".dbd.NH (CH.sub.2).sub.12 NH.sub.2
[0106] 1I R'.dbd.NH (CH.sub.2).sub.2 NHCO (CH.sub.2).sub.2 SS-Py:
R".dbd.H
[0107] 1j R'.dbd.NH (CH.sub.2).sub.6 NHCO (CH.sub.2).sub.2 SS-PY:
R".dbd.H
[0108] 1k R'.dbd.H:R'.dbd.NH (CH.sub.2).sub.12 NHCO
(CH.sub.2).sub.2 SS-Py
[0109] 1l R'.dbd.NH (CH.sub.2).sub.6 NHCO (CH.sub.2).sub.6 CONH
(CH.sub.2).sub.12 NH.sub.2: R".dbd.H
[0110] 1m R'.dbd.NH (CH.sub.2).sub.6 NHCO (CH.sub.2).sub.6 CONH
(CH.sub.2).sub.12 NHCO (CH.sub.2) SSPY:R".dbd.H
[0111] 1n R'.dbd.NH NH.sub.2: R"--H
[0112] 1o R'.dbd.NH (CH.sub.2).sub.2 SS (CH.sub.2).sub.2 NHCO
(CH.sub.2).sub.2 NHNH.sub.2: R".dbd.H
[0113] 1p R'.dbd.NHNH CO(CH.sub.2).sub.4 CO NHNH.sub.2:
R".dbd.H
[0114] 1q R'.dbd.NH (CH.sub.2).sub.2 NH COph CH.sub.2 COOH:
R".dbd.H
[0115] 1r R'.dbd.NH (CH.sub.2).sub.2 NH COph CH (CH.sub.3) Sspy:
R".dbd.H
[0116] 1s R'.dbd.NH (CH.sub.2).sub.2 NH CO CH.sub.2 I: R".dbd.H
[0117] 1t R'.dbd.NH-Gly-Gly-Glu-Ala-Ome: R".dbd.H
SECTION II
[0118] Particulate carriers for drug loading are prepared by using
natural, semi-synthetic and synthetic polymers of biodegradable
nature. The polymeric particulate carriers prepared by various
techniques are elaborated in this section.
[0119] 2.0 Preparation of cross-linked polysaccharide cores
[0120] Polysaccharide (PC) microparticles/nanoparticles of starch
(DSM/DSN) and dextran (DDM/DDN were prepared by both emulsion and
gel methodologies.
[0121] 2.1.1 Preparation of PC cores by sonication of the
cross-linked polymer gel:
[0122] PC polymer cores were prepared according to the method of
Samain et al (1989) with some modifications. 2 gm of polymer starch
(Potato, soluble)/dextran (Mol. wt. 66,000 and 10,000) was
dispersed (hydrolyzed) homogeneously in 1 M NaOH by vigorous
shaking. Epichlorohydrin (0.18-0.4 ml) was then added under
constant stirring for cross-linking. Shaking was stopped and the
reaction was left to proceed at 70-80.degree. C. until (usually 2-6
hrs) until a soft gel (white to pale yellow) was obtained. The
resulting gel was diluted with water, mechanically ground and
subjected to sonication with a probe sonicator (M/s Branson
ultrasonified). The particulates so obtained were fractioned into
different sizes by centrifugation and the precipitate (aggregates
and larger particulates) was discarded.
[0123] Finally, the chosen fractions were dried by lyophilization
(DDM1, DDM2 and DDM3) or spray dried (DSM1 and DSM2) [mini spray
dryer: Inlet temperature 200.degree. C., out let temperature
140.degree. C.; pump control 3, aspirator control 19, heating
control 12, flow indicator 700] to get fme powder particulates.
[0124] 2.1.2 Cross-linking hydrolyzed starch with POCl.sub.3:
[0125] This was carried out according to method of Ignacio De
Miguel et al., 1995 with some modifications. In this procedure, 5
gm of soluble starch (potato) was solubilized by homogenization in
2 M NaOH (10-12 ml). The temperature was adjusted to 4.degree. C.
by immersing in an ice bath then POCl.sub.3 (2.5 gm 0.33 M) was
added drop-wise together with a solution of 7.5 ml of 10 M NaoH
under stirring. After addition of cross-linking agent it was
further stirred at level 3 for 15 minutes (homogenizer). Then pH of
the preparation was adjusted 7 with dil HCI. The resulting gel was
diluted with water and subjected to sonication, centrifugation
followed by spray drying (conditions same as above) to get fine
powder (DSN1 and DSN2).
[0126] 2.2 Interfacial cross-linking by emulsion method:
[0127] 2.2.1 Cross-linking with epichlorohydrin:
[0128] Some of the PC cores prepared by the above method showed
some irregularities in shape, and so fresh microparticles were
prepared by an emulsion method. Typically polysaccharide (2 gm
Dextran mol. wt. 10,000 or soluble starch 5 gm) was solubilized in
1 M NaOH (5-10 ml). The polymer aqueous phase was emulsified in
liquid paraffin (20-30 ml) containing span 80 (2%) by stirring at
15,000-22,000 rpm for 10 to 15 minutes to disperse the polymer
droplets to a fine state. Then epichlorohydrin (0.5-1.0 ml) was
added under agitation at 1000-5000 rpm with constant stirring at
70-80.degree. C. for 2-3 hrs followed by addition of water (2-5 ml)
and stirred for further 2-3 hrs with intermittent addition of
water. The stirring was stopped until the white emulsion turned
pale yellow with visible separation of colloidal particulate system
(when agitation is stopped). The resultant particles were washed
thrice with equal volume of petroleum ether/hexane and sufficient
water. The organic phase was separated to remove oil phase from the
aqueous particulate system. The aqueous particulate system was
freeze dried/spray dried as described previously to get DDN1/DDN2
and DDN3/DSN3.
[0129] 2.2.2 Cross-linking with POCl.sub.3 by emulsion method:
[0130] 5 gm of starch (potato soluble) was solubilized in 2 M NaOH
(12.5 ml) and emulsified in liquid paraffin (40 ml) containing 2%
span 80 by stirring at 22,000 rpm for 15 minutes. The temperature
of dispersed aqueous polymer was brought to 4.degree. C. (ice bath
immersion), and cross-linking was carried out by adding POCl.sub.3
(0.66 M 2.5 gm) drop wise together with a solution of 10 M NaOH
(7.5 ml). This step was carried out under stirring at low rpm. The
oil phase removed by washing three times with equal volumes of
hexane/petroleum ether (separating funnel). The aqueous phase was
diluted sufficiently, pH to 7.0 and finally spray dried as above to
get fine powder DSN4.
[0131] 2.3 Preparation of cross-linked guar gum (GG) and chitosan
hydrogels: (Kabir et al., 1998)
[0132] 5 gm of chitosan solubilized in 0.5 M acetic acid solution
(300 ml). Similarly 4 gm of guar gum was dispersed for 2 hrs at
45.degree. C. in 800 ml water. Cross-linking was carried out by
addition of 25% w/v glutaraldehyde. The reaction mixtures were
homogenized for 30 at 22,000 rpm by the addition of 0.5 M
concentrated H.sub.2SO.sub.4 followed by 25% w/v glutaraldehyde for
chitosan emulsions and guar gum emulsions under the stirring at the
same rpm. The reaction mixture was stirred for an additional 30
minutes at 15,000 rpm and kept in sealed container (separating
funnel) for 48 hrs. The oil phase was removed by washing repeatedly
with equal volumes of hexane. The aqueous phases were mixed with 5%
w/v NaHC0.sub.3 by stirring for 3-4 hrs and then rinsed with I
litre of water until no traces of aldehyde were found. Then the
particulate systems were spray dried to get chitosan products (DCN1
and DCN2) and guar gum products (DGM1 and DGM2) respectively.
[0133] 2.4 Preparation of nanoparticles by solvent evaporation
method:
[0134] Procedure of Ando et al., 1999 (with some modification)
[0135] Microspheres and nanoparticles of poly (methylmethacrylate)
(PMAM/PMAN), poly (hydroxybutyrate) (PHBM/PHBN), poly (D,L lactide)
(PLAM/PLAN) and poly (DL lactide Co-glycolic acid) ( 85:15) of
different ratios (PLGAM/PLGAN) were prepared by water-oil-water
double emulsion solvent evaporation process.
[0136] Due to the fragility of the protein bioactives encapsulation
was carried out through cryopreparation (cold temperature) and
carbohydrate (lyophilization and osmotic balance-in emulsion). The
typical procedure is out-lined for the PLGA polymer. 500 .mu.l (500
.mu.g) of hepatitis `B` surface antigen or 100-200 .mu.l 100IU
insulin containing 80-100 mM trehalose was emulsified in 9-10 ml
dichloromethane containing 100 mg PLGA by sonication for 10 seconds
at 4-6.degree. C. The primary emulsion temperature was than lowered
below the freezing point of inner aqueous phase (dry ice/acetone)
and then emulsified with 50-60 ml of 5% PVA (88% hydrolyzed)
solution containing 100 mM trehalose (4-7.degree. C.) at 6000-9000
rpm for 30-50 sec. The resulting secondary emulsion was diluted in
100 ml of 1% PVA and the system was stirred magnetically for 6-9
hrs to allow for evaporation of organic phase. The colloidal
particles were finally collected by centrifuigation and washing
thrice with water to remove excess of PVA as well as protein
bioactive. Particle size was controlled by changing level of
sonication/agitation and also by centrifugation step to yield
microspheres and nanoparticles and finally dried by
lyophilization.
[0137] 2.5 Preparation of Albumin microspheres:
[0138] 2.5.1 Terephthaloyl chloride cross-linking:
[0139] 3-4 ml solutions of (20-25% w/v) human serum albumin (HSA)
in carbonate buffer (pH 9.8) were emulsified separately in
chloroform: cyclohexane (1:4 w v/v) mixtures (15 ml) containing
4-5% v/v sorbitan trioleate. Then 20-35% solutions of terephthaloyl
chloride were added (0.5, 1, 1.25 and 2.5% w/v) respectively. After
5-30 minutes of reaction time, the microspheres were washed,
centrifuged, resuspended in PBS 7.4 and finally freeze-dried.
Microspheres made in the conditions 1) pH 9.8, TC 2.5%, time 5 min
and 2) pH 9.0, TC 2.5% and time 30 minutes were taken for
conjugation.
[0140] 2.5.2 Glutaraldehyde cross-linking using an emulsion
method:
[0141] 0.5 ml of 100 mg HSA was homogenized in 100 ml of cottonseed
oil by slowly adding aqueous polymer phase drop wise to get w/o
emulsion. 0.1 ml of an aqueous solution of glutaraldehyde (25% w/v)
was added to the stabilized emulsion and stirring was continued for
1 hr, then 50 ml of acetone was added. After 1 minute microspheres
were collected by centrifugation, washed with water, quenched with
10% w/v glycine solution (to remove reactive aldehyde groups),
centrifuged appropriately, washed with water and lyophilized. Two
centrifugation fractions were chosen HSAM and HSAN.
[0142] 2.6 Preparation of gelatin nanoparticles:
[0143] These were prepared according to a modification of the
procedure of Lig et al., 1997. 1 ml of aqueous gelatin solution 7%
w/v was added to 90 ml of cotton seed oil at 40.degree. C. This
biphasic system was homogenized at a speed of 22,000 rpm for 7-9
minutes to form w/o emulsion. The emulsion was cooled to a
temperature of 40.degree. C. (gelling point of gelatin) by keeping
in a refrigerator when the emulsion turned to a suspension with the
formation of gelatin nanoparticles, the oil phase was washed with
hexane/petroleum ether. The aqueous particulate phase was then
lyophilized to get DGN1.
[0144] Where ever cross-linking is involved, drug loading is
carried out at different stages i.e. either after preparation of
particulate cargo or after coupling of VB.sub.12 derivative.
[0145] 2.7 Preparation of poly (anhydride) nanoparticles: Phage
inversion nano-encapsulation (PIN) method.
[0146] Poly(anhydride) WP were prepared according to the procedure
of Mathiowitz et al (1997). Protein (100 IU/mg, insulin/HBsAg 500
.mu.g/mg) to be encapsulated (0.5-1 ml containing appropriate
quantities to be encapsulated) was sonicated with 2-4% w/v of
dichloromethane containing anhydride polymers of poly fumaric acid
and sebacic acid and poly (FA:SA, 20:80) and poly fumaric acid:
polylactide-co-glycolic acid [poly (FA:PLGA)50:50] for 10-20 sec at
4-6.degree. C. The resulting dispersion was then poured rapidly
into an unstirred bath of non-solvent solvent such as petroleum
ether (polymer solvent-non solvent ratio 1:100) to get polymeric
nanoparticles. Desired particle size can be tailored by changing
initial polymer %, solvent-non-solvent ratio and selection of
non-solvent. The optimized formulations chosen were poly [FA:SA]
80:20 .RTM.AFSN and poly [FA:PLGA] 50:50 .RTM.AFPLGAN.
[0147] 2.8 Poly glutaraldehyde nanoparticles:
[0148] Margel, et al., (1979) processes with some modifications. 10
N NaOH was added drop wise to solution of to aqueous glutaraldehyde
(3-25%) solution 100 ml containing surfactants (Guar C13 (1% and
poly ethylene oxide mol wt. 10, 000 (s)/or Aerosol 604 (1%)) until
alkaline pH was reached (7-14, preferably 9-11). The mixture was
de-aerated with N.sub.2 gas in a tightly closed container and
agitated on a mechanical shaker for 24 hrs at ambient temperature.
On hourly intervals for 4-6 hrs the pH was adjusted to 11. Finally
the system was dialyzed extensively with water and centrifuged to
get PGL spheres (nanoparticles), particles of varying sizes that
can be tailored by varying the concentration of monomer, surfactant
or pH.
[0149] Two preparations of various optimized conditions PGLN1 (200
nm) (COOH) and PGLN2 (400 nm) (CHO) were taken for coupling.
SECTION III
[0150] 3.0 Particulate sphere surface derivitazation suitable for
VB.sub.12 coupling:
[0151] Different methods of surface activation of the particulate
carriers (microspheres/nanospheres) are illustrated as follows:
[0152] 3.1.1 Activation by periodate oxidation:
[0153] The particulate fractions used for periodate oxidation were
DSM1, DSM2, DSN1, DSN2, DSN3, DSN4, DCN1, DGN1 and DGN2.
[0154] The weighed quantity (250-500 mg) of microparticles was
suspended in distilled water (5-10 ml) and was agitated by magnetic
stirrer. Then sodium periodate (equal weight to particles) was
added and mixing was continued for 45-50 minutes in the dark. The
oxidized particulate system was thoroughly washed either by
dialysis or centrifugation and finally lyophilized.
[0155] 3.1.2 Coupling of VB.sub.12 with oxidized particles.
[0156] The oxidized particulate system (100-150mg) was then reacted
with amino VB.sub.12 derivative (20-30 mg) for 18-24 hrs at
4.degree. C. in the dark. The conjugate was reduced by sodium
borohydride (4 mg) for 2 h at 4.degree. C., dialyzed and finally
lyophilized as a powdered conjugate.
[0157] The VB.sub.12 derivatives and particulates used were:
[0158] (Schematic representation of the reaction as mentioned in
the following table.)
[0159] Conj. No. VB.sub.12
derivative+Particle.fwdarw.Conjugate-(Size)
1 VB12 derivative Conj + No Particle Conjugate Size 1 1O DSN1 DSN1
- NH NH--(CH.sub.2).sub.2CON- H(CH.sub.2).sub.2--S--S-- 207 .+-. 11
nm (CH.sub.2).sub.2--NHeVB- .sub.12 2 1d DCN1
DCN1-NH(CH.sub.2).sub.6--NHeVB.sub.12 243 .+-. 18 nm 3 1g DGN1
DGN1-NH(CH.sub.2).sub.2 S--S--(CH.sub.2).sub.2NH-eVB.- sub.12 261
.+-. 6 nm 4 1f DSN2 DSN2 NH--CH.sub.2(OH)--CH--CH.sub.2--
-NHeVB.sub.12 410 .+-. 23 nm 5 1n DSM3 DSN3 NHNHeVB.sub.12 792 .+-.
38 nm 6 1e DGN2 DGN2 NH(CH.sub.2).sub.12NHeVB.sub.12 914 .+-. 47 nm
7 1c DSN4 DSN4 NH(CH.sub.2).sub.2NHeVB.sub.12 1.2 .+-. 0.05 nm 8 1d
DSM1 DSM1 NH(CH.sub.2).sub.6NHeVB.sub.12 4.1 .+-. 0.3 nm 9 1g DSM2
DSM2 NH(CH.sub.2).sub.2SS(CH.sub.2).sub.2NHeVB.sub.12 6.6 .+-. 0.7
nm
[0160] 3.2.1 Surface derivatization of the particulates to provide
COOH groups suitable for upling with VB.sub.12.
[0161] Surface derivatization of spheres to provide a functional
group for VB.sub.12 linkage: Particulates were succinylated using
the method reported by Peyrot et al, (1994) to provide a functional
group for linkage to amino VB.sub.12 derivatives. The
polysaccharide cores were first completely dried and then dispersed
in dichloromethane (100 mg/ml) containing 50 mg/ml triethylamine.
Succinic anhydride (30% w/w was added and the reaction was carried
out under anhydrous conditions at (35.+-.2.degree. C.) for 20 hrs.
Finally the chosen fractions DDM3, DDM2, DSM1, DDN1, DDN2 and DDN3,
were dialyzed against distilled water and lyophilized.
[0162] 3.2.2 Coupling of succinylated particles with VB.sub.12
derivatives: VB.sub.12-sphere conjugates were prepared by a direct
amide linkage of the succinylated cores DDM/DDN) with
amino-derivatives of VB.sub.12. In general, coupling was carried
out by dispersing succinylated dextran cores in 0.15M NaCl solution
(pH 4.5) and mixing it thoroughly with NH.sub.2-Spacer VB.sub.12
and 1-ethyl-3[(dimethylamino)pr- opyl] carbodoiimide (EDAC) in a
total of 3 ml. The pH was then adjusted to 7.5 with NaHCO.sub.3.
The reaction mixture was stirred at 30.+-.2.degree. C. for 2 hours
and incubated at 4.degree. C. for 20 hours and finally freeze-dried
as dark pink to red powders. Typically 100 mg of succinylated cores
were coupled with molar excess of (with respect to COOH) equal
quantities of amino VB12 and EDAC.
[0163] 3.2.3 Alternative method for formation of VB.sub.12 spheres
using aminohexyl-5'OVB.sub.12:
[0164] 1. Succinylated dextran cores were dispersed in solution (pH
4.5) at 100 mg/ml and mixed thoroughly with an equal volume of 50
mg/ml N-hydroxysuccinimide (50 mg/ml in DMF).
[0165] 2. 1-ethyl-3{-3(dimethylamino)propyl} carbodiimide (EDAC)
(equal weight to spheres) was added at 100 mg/ml, and allowed to
react for 15 minutes.
[0166] 3. An equal weight of Aminohexyl-5'OVB.sub.12, (to spheres)
dissolved at 100 mg/ml in 0.025 M K2CO3 was added and the reaction
mixture was stirred at RT for 20 hours and finally dialyzed
extensively against, before being lyophilized.
2 Conjugate VB.sub.12 No. derivative +particle .fwdarw. Conjugate
(Size) 10) 1 h +DDM3 .fwdarw. DDM3 (CH.sub.2).sub.12-5'OVB.sub.12
(6.1 .+-. 0.9 nm) 11) 1 h +DDM2 .fwdarw. DDM2
(CH.sub.2).sub.12-5'OVB.sub.12 (3.6 .+-. 0.39 nm) 12) 1 h +DDM1
.fwdarw. DDM1 (CH.sub.2).sub.12-5'OVB.sub.12 (1.7 .+-. 0.32 nm) 13)
1 h +DDN3 .fwdarw. DDN3 (CH.sub.2).sub.12-5'VB.sub.12 (875 .+-. 52
nm) 14) 1 h +DDN2 .fwdarw. DDN2 (CH.sub.2).sub.12-5'OVB.su- b.12
(436 .+-. 38 nm) 15) 1 c +DDN1 .fwdarw. DDN1
(CH.sub.2).sub.2-eVB.sub.12 (164 .+-. 13 nm)
[0167] 3.3.1 CNBR mediated coupling:
[0168] 2 gm of CNBR was dissolved in 25 ml of PBS, pH 11.5 in a
fume cup board. Then CNBR solution was added to particulate
suspension under vigorous agitation for 10-15 minutes. In the above
reactions pH was maintained by addition of NaOH. Particulates were
centrifuged and thoroughly washed with ice-cold distilled water and
sodium bicarbonate.
[0169] 3.3.2 Coupling
[0170] 25 mg of amino VB.sub.12 was added to CNBR activated
particles (100 mg) suspended in 3 ml bicarbonate buffer pH 7.5 and
mixed by rotation for 72 hours at 4.degree. C. and finally washed
by centrifugation and extensive dialysis against buffer.
3 VB.sub.12 S. No. derivative +Particle .fwdarw. Conjugate (Size)
16) 1e +DSM2 .fwdarw. DSM2NH (CH.sub.2)12eVB.sub.12 (6.6 .+-. 0.7
.mu.m)
[0171] 3.4.1 Derivitization of polysaccharide particulates to
provide amino groups:
[0172] One of the amino groups of the diamine (1,2 diamino ethane)
was protected by adding slowly 50% v/v benzyl oxychloride/acetic
anhydride and purified by dowex chromatography. The mono protected
diamine was treated with periodate activated particles/succinylated
particulates. The protective group on the NH.sub.2 group was
removed to produce amino group of the particles on the surface.
[0173] 3.4.2 Surface modification to provide thiol groups:
[0174] The amino groups modified particulate system was dispersed
in phosphate buffer, pH 7.5 (50 mg/ml). SPDP was dissolved in
acetone (50 mg/ml). Both the phases were mixed thoroughly and left
to proceed at room temperature over night and excess of the reagent
removed by dialysis. A free thiol was introduced by reduction with
.beta.-mercaptoethanol and finally lyophilized.
[0175] 3.4.3 Formation of VB.sub.12-pqlysaccharide conjugates by
disulfide linkage:
[0176] SPDP treated particles (25 mg/ml; 50 mg/ml) were suspended
in sodium acetate buffer, pH 4.5. Then 2.5 ml of DTP amino ethyl
derivative (10 mg/ml) dissolved in acetate buffer, pH 4.5. Both
phases were mixed and the reaction allowed to proceed for 48 hours
at 4.degree. C. The conjugates were dialyzed and finally
lyophilized. The various derivatives used and their conjugates from
the above reaction are given below:
4 VB.sub.12 S. No. derivative +Particle .fwdarw.Conjugate (Size)
17) 1K +DCN1 .fwdarw.DCN1 - SS-(CH).sub.12 eVB.sub.12 (243 .+-. 13
nm) 18) 1m +DGN1 .fwdarw.DGN1-SS-(CONH)n (CH.sub.2)6 (CH).sub.12
eVB.sub.12 (261 .+-. 16 nm) 19) 1r +DDN2
.fwdarw.DDN2-SS-(CH.sub.2).sub.12- eVB.sub.12 (436 .+-. 38) 20) 1j
+DSN3 .fwdarw.DSN3-SS-(CH.sub.2).sub.6-eVB.su- b.12 (792 .+-. 45
nm) 21) 1i +DSM1 .fwdarw.DSML-SS-(CH.sub.2).sub.2- -eVB.sub.12 (4.1
.+-. 0.41 .mu.m) 22) Ij +DDM3
.fwdarw.DDM3-SS-(CH.sub.2).sub.6-eVB.sub.12 (6.1 .+-. 0.9
.mu.m)
[0177] 3.5.0 Formation of noncleavable VB.sub.12-polysaccharide
core conjugates:
[0178] 1) Micro particles containing NH.sub.2 groups
(DGN1-previously modified with monoprotected diamine) on the
surface were treated with 1.5 fold molar excess of ethylene glycol
bis (succinimidyl succinate) [EGS] for 20 minute at room
temperature. Amino eVB.sub.12 (1c) was added and the coupling
carried out over night. The product was dialyzed/centrifuged and
lyophilized.
[0179] 2) Similarly, a particulate system (DDN2-previously modified
with mono-protected diamine) was treated with disuccinymidyl
suberate (DSS). The reaction intermediate was treated with amino
VB.sub.12 (1d) and the conjugate washed and lyophilized.
[0180] 3) To SPDP modified particles (DSM1), a solution of
iodoacetemido hexyl eVB.sub.12 (1s) in diisopropyl
ethylamide/diethyl formamide (1:20 v/v) was added under vigorous
stirring (at inert atmosphere) for 30 minutes at room temperature
and the thioether conjugate was washed and lyophilized.
[0181] 4) The tetrapeptide eVB.sub.12 (1t) was conjugated to amino
group containing particles ((DCN1-previously modified with
mono-protected diamine, 3.4.1) in the presence of EDAC/NHS in
bicarbonate buffer, pH 9.5 for 30 minutes at room temperature and
was mixed with 2% acetic acid washed and lyophilized.
[0182] 5) The anilido derivative of eVB.sub.12 (1q) was coupled
with amino group containing particles (DSN3-previously modified
with mono-protected diamine, 3.4.1) in the presence of EDAC/NHS in
bicarbonate buffer, pH 9.5 for 30 minutes at room temperature and
diluted with 2% acetic acid, washed and lyophilized.
5 VB.sub.12 S. No. Derivative +particle .fwdarw. Conjugate (Size)
23. 1C +DGN1 .fwdarw. DGN1 - [EGS]- eVB.sub.12 (261 .+-. nm)
(Activity less) 24. 1d +DDN2 .fwdarw. DDN2 - [DSS]-eVB.sub.12 (436
.+-. 38 nm) 25. 1s +DSM1 .fwdarw. DSM1-CH2-CONH (CH2)2
NH-eVB.sub.12 (4.1 .+-. 0.41 .mu.m) 26. 1t +DCN1 .fwdarw.
DCN1-(GGEA) - eVB.sub.12 (243 .+-. 13 nm) 27. 1q +DSN3 .fwdarw.
DSN3 - NHCO-CH2-Ph-NH-eVB.sub.12 (792 .+-. 45 nm)
[0183] 3.6 Amide conjugates of amino VB.sub.12/hydrazide and micro
particles prepared by solvent evaporation by EDAC:
[0184] 20 fold molar excess of amino VB.sub.12/hydrazide VB.sub.12
and EDAC were used to couple to particles containing COOH in
bicarbonate buffer, pH 7.5 and conjugation was carried out
overnight at 4.degree. C., washed and lyophilized. Particulates
used and their VB.sub.12 derivatives are given below:
6 VB.sub.12 S. No. Derivative +Particle .fwdarw. Conjugate (Size)
28) 1p +PLGAN1 .fwdarw. PLGAN1 CONHNH - spa- eVB.sub.12 (120 .+-.
6.0 nm) 29) 1c +PLGAN2 .fwdarw. PLGAN2 CONH - eVB.sub.12 (135 .+-.
7.0 nm) 30) 1d +PLGAM3 .fwdarw. PLGAM3 CONH - spa - eVB.sub.12 (1.5
.+-. 0.09 .mu.m) 31) 1o +PHBN1 .fwdarw. PHBN1 CONH - SS-spa-
eVB.sub.12 (173 .+-. 6.0 nm) 32) 1n +PHBN2 .fwdarw. PHBN2
CONHNH-eVB.sub.12 (932 .+-. 30.0 nm) 33) 1e +PLAN1 .fwdarw. PLAN1
CONH-spa-eVB.sub.12 (190 .+-. 9.0 nm) 34) 1g +PLAM2 .fwdarw. PLAM2
CONH-SS-eVB.sub.12 (3.4 .+-. 0.3 .mu.m)
[0185] 3.6.1 VB.sub.12 particulate systems coupled by disulfide
linkages:
[0186] The nanoparticles PLGAN1, PLGAN2, PHBN1 and PLAN1 were
surface modified with monoprotected diamine and treated with SPDP
as described previously (section 3.2.1 and 3.2.2) to give SH
containing particles.
[0187] 3.6.2 Derivatization of particulates to provide amino
groups:
[0188] One of the amino groups of the diamine (1,2 diaminoethane)
was protected by adding slowly 50% v/v benzyl oxychloride/acetic
anhydride and purified by DOWEX chromatography. The mono protected
diamine was treated with particulate systems, and the protective
group on the NH.sub.2 group was removed to produce amino group of
the particles on the surface.
[0189] 3.6.3 Surface modification to provide thiol groups:
[0190] The amino groups modified particulate system was dispersed
in phosphate buffer, pH 7.5 (50 mg/ml). SPDP was dissolved in
acetone (50 mg/ml). Both the phases were mixed thoroughly and left
to proceed at room temperature over night and excess of the reagent
removed by dialysis. A free thiol was introduced by reduction with
B-mercaptoethanol and finally lyophilized.
[0191] 3.6.4 Coupling:
[0192] SPDP treated particles (25 mg/ml, 50 mg/ml) were suspended
in sodium acetate buffer, pH4.5. Then 2.5 ml of DTP amino ethyl
derivative (10 mg/ml) dissolved in acetate buffer, 4.5. Both phases
were mixed and the reaction allowed to proceed for 48 hours at
4.degree. C. The conjugates were dialyzed and finally
lyophilized.
[0193] 6.6.5 Co-incorporation of surface functional groups for
linkage into nanospheres (PLGA):
[0194] Surface functional groups suitable for VB.sub.12 linkage are
co-administered to the nano sphere matrix during preparation.
Nanospheres containing (L-alpha-phosphatidyl ethanolamine) were
prepared by mixing a chloroform solution of lipid to a polymer
solution in dichloromethane (lipid to polymer ratio 1:5).
Emulsification was then carried out as above (PLGA solvent
evaporation process) PGLAN2. The formed particles were modified
with SPDP to make them suitable for coupling.
[0195] These systems were designed for conjugation to dithiopyridyl
derivate of VB.sub.12.
7 Cong. VB.sub.12 No. derivative +Particle .fwdarw. Conjugate
(Size) 35) ij +PLGAN1 .fwdarw. PLAGN1-SS- eVB.sub.12 (120 .+-. 6.0
nm) 36) 1k +PLGAN2 .fwdarw. PLGAN2-SS-eVB.sub.12 (135 .+-. 7.0 nm)
37) 1m +PHBN1 .fwdarw. PHBN1-SS-eVB.sub.12 (170 .+-. 6.0 nm) 38) 1i
+PLAN1 .fwdarw. PLAN1-SS-eVB.sub.12 (190 .+-. 9.0 nm)
[0196] 3.6.6 Adsorption of VB.sub.12 derivative copolymers on
colloidal particulates:
[0197] This was done by the method of Neal et al., with some
modifications by VB.sub.12 coupling on poloxamers Neal et al.,
1998.
[0198] STEP 1:
[0199] The terminal hydroxyl groups in poloxamer 407 and
poloxamine-908 were substituted with amino groups followed by
coupling of the amino copolymers with VB.sub.12.
[0200] 1) e-carboxylate of VB.sub.12 (1b) in presence of EDAC as
above. The reactions were slightly modified to include an organic
solvent, tetrahydrofuran instead of aqueous solvents as used
previously.
[0201] 2) In another reactions the amino copolymers were modified
with SPDP, followed by linkage with dithiopyridyl derivatives of
eVB.sub.12 (1i) as described previously.
[0202] The procedure for amino derivatization of copolymers is
out-lined briefly (Ref. Neal, J. C. et al., 1998). 20% w/v solution
of copolymer in CH.sub.2C.sub.12 was reacted with a 2-fold molar
excess of p-toluenesulfonyl chloride and pyridine at room
temperature for 24 h. The p-toluenesulfonate ester product was
extracted by first washing with 3 M HCl, followed by washing the
organic layer with NaHCO.sub.3, with rotary evaporation used to
obtain the copolymer. In the second step, the p-toluenesulfonate
ester product was reacted with 25% w/v NH.sub.3 in H2O for 6 h at
120.degree. C. in a pressurized reaction vessel, to produce the
ainiated copolymer. The reaction products were cooled to room
temperature and extracted with CH.sub.2C.sub.12 to separate the
ammonium toluenesulfonate salt from the aminated copolymer. The
product was then washed with base (NaOH/H.sub.2O) to produce the
free amino product.
[0203] STEP 2:
[0204] The aminated copolymers of VB.sub.12 were adsorbed onto
nanoparticle surface. Briefly VB.sub.2 derivatized copolymer 0.5%
w/v solutions were incubated for 12 hr with equal volume and
concentration of microparticle suspension.
8 Conjugates adsorbed on nanoparticles. Cong. eVB.sub.12 No.
derivative +particle .fwdarw. Conjugate (Size) 39)
P-407NHCO-eVB.sub.12 +PLGAN1 .fwdarw. PLGAN1 - P407- eVB.sub.12
(120 .+-. 6.0 nm). (eVB.sub.12-polo-eVB.sub.12) 40) P-908
NHCOeVB.sub.12 +PGBN1 .fwdarw. PHBN1-P908-eVB.sub.12 (173 .+-. 6.0
nm) 1 41) P407-SS-eVB.sub.12 +PHBN1 .fwdarw.
PHBN1-P407-SS-eVB.sub.12 (173 .+-. 6.0 nm) 42) P-908-SS-eVB.sub.12
+PLGAN1 .fwdarw. PLGAN1-P908-SS-eVB.sub.12 (120 .+-. 6.0)
[0205] Surface adsorption
[0206] Particulate systems containing surfacial COOH groups are
anionic in nature, mixing them with cationic agents gives their
adsorption by ionic bond on the nanoparticle surface. The required
quantity of the surface-modifying agent can be adsorbed. Typically
for particles with 5-10% DMAD, 5-10 mg of this agent dissolved in
10-20 ml water containing 95-90 mg of preformed particles, was
suspended by sonication on an ice bath and dried by lyophilization.
2
[0207] Epoxy activation of nanoparticles: Vinod et al., 1998
[0208] A sample of (80) of pre-formed nanoparticles was suspended
in 10 ml of borate buffer (50 MM, pH5) over in an ice bath. Then,
zinc tetrafluroborate hydrate (catalyst 24 mg) and Denacol solution
(24 mg in 4-ml borate buffer) were added while stirring at
37.+-.1.degree. C. for 30-50 minutes. The nanoparticles were
separated by centrifugation and washed extensively with H.sub.2O to
remove unreacted impurities.
9 Particle COOH + DeOH - - - Particle COO-De-OH Amino derivatives
VB.sub.12 are coupled by 1,1'carbonyl diimidazole activation.
[0209] 3.6.7 Conjugation of anhydride particles prepared by PIN
method:
[0210] Weighed quantities of polyanhydride nanoparticles [ASSN1,
AFPLGAN2; 50 mg each] were suspended in borate buffer [0.20 M, pH
9.0, 10ml] then a hydrazide derivative of eVB.sub.12 was added in
molar excess over anhydride groups of nanoparticles. After 30
minutes acetate buffer [0.2 M, pH 5.0, 25 ml] was added. The
conjugated nanoparticles were washed with 2 M NaCl, distilled water
and lyophilized.
10 Cong. eVB.sub.12 No. derivative +particle .fwdarw. Conjugate
(Size) 45) 1o +AFSN1 .fwdarw. AFSN1-CONHNH-SP-eVB.sub.12 (500 .+-.
22 nm) 46) 1p +AFPLGAN1 .fwdarw. AFPLGAN1CONHNH-SP-eVB.sub.12 (200
.+-. 9.0 nm)
[0211] 3.6.8 Disulfide conjugates of anhydride particles and
eVB.sub.12:
[0212] The particles (AFSN1, AFPLGAN2) were surface modified with
mono-protected diamine and the protecting agent cleaved as
described previously (Section 3.6.2).
[0213] In another reaction nanoparticles (AFSN1) were surfaced
modified with arginamide Ref. (Gao, J. et al., 1998). Briefly 50 mg
of nanoparticle were suspended in a solution containing 12 mM
arginamide in borate buffer (0.20 M, pH 9.0, 3 ml at R.T.). After
15 minutes the suspension was acidified by adding acetate buffer
(0.20 M, pH 5.0, 9 ml). The particles were washed with 2M NaCl,
distilled water and lyophilized.
[0214] The above amino derivatized particles were coupled to SPDP
to give particles containing SH groups as described previously
(section 3.6.3).
[0215] The coupling is carried out as described in 3.6.4.
11 Cong. eVB.sub.12 No. derivative +particle .fwdarw. Conjugate
(Size) 47. 1k +AFSN1 .fwdarw. AFSN1-SS-eVB.sub.12 (500 .+-. 22 nm)
(Amino .fwdarw. SH) 48. 1j +AFPLGAN2 .fwdarw.
AFPLGAN2-SS-eVB.sub.12 (200 .+-. 22 nm) (Amino-.fwdarw. SH) 49. 1i
+AFSN1 .fwdarw. AFSN1 -SS-eVB.sub.12 (500 .+-. 22 nm)
(Arginamide-.fwdarw. SH)
[0216] 3.6.9 Conjugation of protein polymer based
micro/nanoparticles to VB.sub.12:
[0217] Different fractions of microparticles and their conjugates
are given below:
[0218] In this reaction HSAM particles (100 mg) were activated with
0.25-0.5 ml, 25% w/v glutaraldehyde in borate buffer that forms
Schiff's base and gives CHO groups for conjugation. The disulfide
coupling between particulates and VB.sub.12 derivative is carried
out as described in 3.6.2., 3.6.3 and 3.6.4.
12 Cong. eVB.sub.12 (Surface .fwdarw. Conjugate No. derivative
+particle nature) .fwdarw. HOC (Size) 50. 1p +HSAM1 (anhydride
& .fwdarw. HSAM1-CONHNH-spa-eVB.sub.12 (3.7 .+-. 0.18.mu.m)
EDAC 51. 1e +HSAM2 (anhydride & .fwdarw.
HSAM2-CONH-(CH).sub.12-eVB.sub.12 (6.4 .+-. 0.3 .mu.m) NH2)
reduction 52. 1k +HSAM2+SPDP.fwdarw. (SH groups) .fwdarw.
HSAM2-SS-spa-eVB.sub.12 (6.4 .+-. 0.3 .mu.m) 53. 1o +DAN1 (COOH)
.fwdarw. EDAC DAN1-Co-NHNH-spa-eVB.sub.12 (534 .+-. 23 .mu.m) 54.
1d +DAM2 .fwdarw. COOH .fwdarw. DAM2CONH-(CH.sub.2).sub.6 -
eVB.sub.12 (2.3 .+-. 0.9 .mu.m) reduction 55. 1l + DGN1 + SPDP
.fwdarw. SH .fwdarw. DGN1-SS-eVB.sub.12 (700 .+-. 31 m) 56. 1b
+DGN2 (NH.sub.2) .fwdarw. DGN1-NHCO-eVB.sub.12 (914 .+-. 47 nm)
[0219] 3.7 Coupling of poly glutaraldehyde nanoparticles to
eVB.sub.12:
[0220] An aqueous particulate suspension (PGLN2, 100 mg/ml; 5 ml)
was diluted with PBS (pH 7.4, 15 ml). Then hydrazide VB.sub.12
derivative (10 ml, 15-20 mg) solution was added under stirring for
2 hrs at 4.degree. C. then glycine (200 mg) was added to remove the
reactive aldehyde groups and agitation was continued for another 1
hr at R.T. The particles were dialyzed/centrifuged and finally
lyophilized. PGLN1 was coupled with amino hexyl eVB.sub.12 (1d) by
carbodiimide chemistry.
13 Cong. eVB.sub.12 No. derivative +particle .fwdarw. Conjugate
(Size) 57. 1d +PGLN1 .fwdarw. PGLN1-CONH (CH.sub.2).sub.6eVB.sub.12
(200 .+-. 12 nm) Reduction 58. 1o +PGLN2 .fwdarw.
PGLN2-CH.sub.2-NHNH-spa (SS)-eVB.sub.12 (400 .+-. 27 nm)
[0221] 3.8 Degradable hydrogel by covalent coupling of VB.sub.12
derivatives:
[0222] Ref: Zhao, X. and Harris, J. (1998)
[0223] Two step hydrogel preparation-A two-Step Gel made from
difinctional double-Ester PEG-fifty milligrams of difunctional
PEG-CM-HBA-N-hydroxysuc- cinimide (NHS) 2000 (vide infra) was
dissolved in 0.25 mL of deionized water. To the solution was added
0.5 mL of a buffered solution with or without FITC-BSA (10 mg/mL)
and 0.25 niL of 8-arm PEG-amine solution (250 mg/mL). After
vigorous shaking, the solution was allowed to sit. The gel formed
in a few minutes. A suitable pH range was 5.5 to 8.
[0224] A two-step gel made from difunctional PEG-succinimidyl
carbonate containing an ester in the fifty milligrams of
difunctional ester containing PEG-succinimidyl carbonate 6800 (vide
infra) was dissolved in 0.3 mL of deionized water. To the solution
was added 0.3 niL of 8-arm PEG amine (250 mg/mL). After vigorous
shaking, the solution was allowed to sit. The gel formed in a few
minutes. A suitable pH range was 5.5-8.
[0225] Conjugates 59 and 60: Amino and hydrazide derivatives are
coupled during the preparation.
[0226] Section-IV
[0227] Protein loading (insulin) into the preformed conjugate:
[0228] Typically 100 I.U. of plain bovine insulin solution (40
I.U./ml) or a solution of 500 .mu.g hepatitis `B` vaccine was mixed
with preformed conjugates containing 100 mg of polysaccharide
cores. The gel was freeze-dried and a dark pink to pale red powder
and stored at 4-8.degree. C. until use.
[0229] Wherever, cross-linking is involved drug loading is carried
out at different stages i.e. either after preparation of
particulate cargo or after coupling of VB.sub.12 derivative.
[0230] Specific advantages:
[0231] The delivery system described in this invention has
advantages over the other reported VB.sub.12 based conjugated
systems (1) The pharmaceutical to be delivered is loaded in the
biodegradable polymeric carriers. (2) Particulate carriers prepared
in this invention are made up of biodegradable and pharmaceutically
acceptable carriers and hence degrade in-vivo to release and
deliver the bioactive for its pharmacological response, which is a
prerequisite for drug delivery. (3) Peptide/protein pharmaceuticals
or vaccines or drugs given exclusively by parental groups can
easily be loaded within the biodegradable polymers for
pharmaceutical purpose. (4) Polymeric particles afford protection
against harsh environment of the intestine and gut enzymes. (5)
Uptake is amplified many times as compared to VB.sub.12 protein
conjugated system. (6) The pharmaceutical to be delivered is not
chemically modified and hence retains its full bioactivity.
[0232] In-vivo performance:
[0233] Various insulin loaded drug delivery systems prepared were
tested for antidiabetic activity in Streptozotocin induced diabetic
rats and the results are briefed in summary section. These insulin
drug delivery systems are orally administered to diabetic rats
(n=number of animals). At various time intervals after feeding the
rats were bled and their blood glucose levels determined.
* * * * *