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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5640_Библиотеки_им_академика_М_И_Перельмана.pdf
Drug Delivery in Rare Diseases: Orphan Drugs and Therapies 409
Table 2
Various routes of orphan drug administration/delivery
Drug Route Disease Developer
Soliris Intravenous infusion Paroxysmal noctur nal hemoglobinuria Alexion Pharmaceuticals
Vimizim Intravenous infusion Morquio A syndrome BioMarin Pharmaceutical
Orkambi Oral Cystic fibrosis Vertex pharmaceuticals
Translarna Oral Duchenne muscular dystrophy PTC therapeutics
Galafold Oral Fabry disease Amicus therapeutics
Ocaliva Oral Primary biliary cholangitis Intercept pharmaceuticals
Xuriden Oral Hereditary orotic aciduria Wellstat therapeutics
Ravicti Oral Urea cycle disorders Horizon therapeutics
Vitrakvi Oral NTRK fusion-positive tumors Bayer
Mepsevii Intravenous infusion Mucopolysaccharidosis VII Ultragenyx pharmaceutical
Lumizyme Intravenous infusion Pompe disease Sanofi Genzyme
Kanuma Intravenous infusion Lysosomal acid lipase deficiency Alexion Pharmaceuticals
Myozyme Intravenous infusion Pompe disease Genzyme
defects, offering a potential cure for inherited rare diseases such as
spinal muscular atrophy and certain types of hemophilia
16, 17]. Advanced drug delivery techniques also include
[
antibody-drug conjugates, which link potent drugs to antibodies
that specifically target disease-causing cells, thus minimizing systemic side effects and improving the precision of rare disease thera-
18, 19]. One of the most innovative approaches involves the
pies [
use of nanotechnology, which enhances drug solubility, stability,
and bioavailability, thereby improving therapeutic outcomes
[
dr
toxicity, which
[
1
20, 2
ugs
in
22, 23].
].
Liposomal delivery systems, for instance, encapsulate
lipid
bilayers,
is
par
allowing
ticularly
targeted
for
beneficial
delivery and
treating
for
rare
reduced
cancers
Nanoparticles serve as an advanced drug delivery system with
the merits of targeted dr ug deliver y and low toxicity due to their
minute size and consequently larger surface-to-volume ratio,
enabling long-term controlled release of bioactive ions with fewer
side effects [
24]. For example, liposomal nanoparticles have been
utilized to deliver migalastat for the treatment of Fabry disease,
improving its pharmacokinetic profile and reducing the frequency
of dosing [
25]. Furthermore, polymeric nanoparticles have been
employed to deliver enzyme replacement therapies in lysosomal
storage disorders, ensuring a sustained release and targeted delivery
to affected tissues [
26]. Polymeric nanoparticles have also been
410 Anil Kumar et al.
used for the delivery of asfotase alfa in hypophosphatasia, a rare
metabolic disorder, providing a controlled release and enhanced
stability of the drug [
Dendrimers, another type of nanoparticle, have shown potential in the targeted delivery of drugs for rare diseases, such as cancer
and genetic disorders, by loading drug molecules both in their
interior and on surface groups, thus allowing controlled drug
release [
to deliver bone morphogenetic proteins for the treatment of rare
bone diseases, offering sustained release and improved bone regeneration [
particles is their use as drug-releasing support for neural diseases,
capable of negotiating the blood-brain barrier. This barrier is a
severe limitation for the delivery of potentially useful drugs; however, it has been demonstrated that different drugs bound to nanoparticles can be transported across the blood-brain barrier and
achieve pharmacological effects in the brain, such as in brain
tumor treatment [
been employed to deliver RNA therapeutics for the treatment of
glioblastoma, showing significant potential in improving drug
delivery efficiency and reducing tumor growth [
the delivery of siRNA in transthyretin amyloidosis, where nanoparticles have enhanced the stability and delivery efficiency of siRNA,
thus improving therapeutic outcomes [
delivery systems have demonstrated substantial potential in improving the pharmacokinetic profiles and therapeutic indices of orphan
drugs, thereby addressing the unique challenges associated with
treating rare diseases.
29, 30]. Mesoporous silica nanoparticles have been used
31, 32]. One of the most promising applications of nano-
Nanotechnology-based deliver
27, 28].
33, 34]. For instance, gold nanoparticles have
35, 36].
y systems
have been utilized for
37, 38]. Nanoparticle-based
2 Liposomes
Liposomes are spherical vesicles consisting of one or more phospholipid bilayers, which closely resemble the structure of cell membranes. They can encapsulate both hydrophilic and hydrophobic
molecules, making them versatile carriers for various substances.
These structures can vary in size from very small unilamellar vesicles
(SUVs) with diameters less than 100 nm to large multilamellar
vesicles (MLVs) that can exceed 1 μm in diameter [
liposomes consist of a single lipid bilayer, while multilamellar liposomes have multiple concentric bilayers [
lipids used in liposome formulation include phosphatidylcholine
(PC), phosphatidylethanolamine (PE), and phosphatidylserine
[
40]. Cholesterol is often included to modulate membrane fluidity
and stability, preventing leakage of encapsulated drugs [
surface charge of liposomes can be neutral, positively charged, or
negatively charged, depending on the composition of the lipids
39].
38]. Unilamellar
Common phospho-
40]. The
Drug Delivery in Rare Diseases: Orphan Drugs and Therapies 411
used. Charged liposomes can be prepared by incorporating cationic
or anionic lipids into the bilayer [
41]. The charge influences lipo-
some stability, interaction with biological membranes, and the
encapsulation efficiency of charged molecules [41].
Liposomes have been extensively studied and utilized for a
variety of purposes due to their biocompatibility, ability to encapsulate diverse molecules, and capacity to deliver drugs to specific
sites within the body. These bodies are widely used as drug delivery
vehicles for both hydrophilic and hydrophobic drugs, improving
the solubility, stability, and bioavailability of therapeutic agents
41]. They can provide targeted delivery to specific tissues, reduce
[
systemic toxicity, and enhance therapeutic efficacy [
42]. Liposomal
formulations of chemotherapeutic agents, such as liposomal doxorubicin (Fig. 1a, b) and liposomal daunorubicin (DaunoXome),
have been developed to reduce cardiotoxicity and enhance drug
accumulation in tumor tissues through the enhanced permeability
and retention (EPR) effect [
22].
Liposomal drug delivery systems have gained attention for
their capability to encapsulate both hydrophilic and hydrophobic
drugs, protecting them from degradation and enhancing their
therapeutic index [
41]. A notable example is the use of liposomal
amphotericin B, which has significantly reduced the toxicity associated with conventional formulations in the treatment of fungal
infections in immunocompromised patients [
43]. Moreover, lipo-
somal formulations have been successfully applied in delivering
drugs for rare genetic disorders, such as liposomal ciprofloxacin
21]
for cystic fibrosis-related lung infections [
A significant
challenge in developing many specialized applica-
.
tions of liposomes is the difficulty in directing them to tissues where
they typically do not accumulate [
42]. As a result, extensive
research has been dedicated to creating liposomes with targeting
vectors attached to their bilayer surface [41]. These vectors include
ligands such as oligosaccharides, peptides, proteins, and vitamins
[44]. Most research has focused on antibody conjugates, as methods for producing highly specific monoclonal antibodies (MAbs)
are well established [
any cell type if the cells are accessible to the carrier [
Ideally, liposomes could be delivered to
45].
46]. However,
this is complicated by issues such as tissue access, competition, and
rapid clearance [
47]. Antibodies can become immunogenic when
coupled to liposomes, though immunogenicity can be reduced by
formulating the liposomes with the cytotoxic drug doxorubicin
48]. These challenges indicate that developing antibody-targeted
[
liposomes for in vivo applications will be difficult [
41]. Moreover, it
is essential to understand the rationale behind attaching a targeted
ligand. The basic concept is to crosslink liposomes, which react to
form a permanent covalent bond when activated [
49]. The most
common method involves the reaction of sulfhydryl groups with
maleimide groups, which is clean, fast, and efficient, and has been
412 Anil Kumar et al.
Fig. 1 (a) Liposome as a drug carrier. (b) Liposomes delivering drugs to the cancerous cells
adapted to modify all antibody functional groups in liposome conjugates [
50]. The choice of chemistry and modification site should
be based on compatibility with the specific antibody [
antibodies may react differently to various procedures, so multiple
protocols might need to be tested [
41]. The recommended general
procedure involves thiolating antibodies with succinimidyl 3(2-pyridyldithio) proprionate (SPDP), followed by deprotection
with DTT (dithiothreitol (DDT) and conjugation to liposomes
42]. Different
Drug Delivery in Rare Diseases: Orphan Drugs and Therapies 413
containing maleimide-derivatized 1,2-distearoyl-sn-glycerol-3phosphoethanolamine (DSPE) or 1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine (DPPE) [
ifying IgG antibodies to attach them to liposomes. This process
enhances the functionality of liposomes for targeted drug delivery
and other therapeutic applications [
3 Preparation of Liposomes
49]. It involves chemically mod-
51].
3.1 Reagents
3.2 Hydration and Liposome Extrusion
1. Lipid mixture (appropriate composition).
2. Chloroform.
3. Methanol (if needed).
4. Nitrogen gas.
5. Buf fer (PBS or HBS).
6. Liquid nitrogen.
7. Polycarbonate filters (100 nm).
8. Extruder (e.g., Lipex Biomembranes).
1. Dissolve the lipid mixture in chloroform (~1 mL per 50–100
μmole of lipid) in a glass tube.
2. Add a small amount of methanol if required.
3. Dry the lipid to a thin film using a nitrogen gas stream.
4. Use a warm water reservoir to facilitate solvent evaporation and
prevent lipid crystallization.
5. Dry the lipid overnight on a lyophilizer.
6. Add an appropriate buffer to the dried lipid film.
7. Vortex the mixture until the lipid film is fully dispersed.
8. If necessary, warm the sample above the lipid’s phase transition
temperature to facilitate dispersion.
9. Transfer the hydrated lipid suspension to a cryovial.
10. Freeze the suspension in liquid nitrogen for 5 min.
11. Thaw the suspension in a water bath set above the lipid’s phase
transition temperature.
12. Repeat this freeze-thaw cycle four more times to ensure
uniform liposome formation.
13. Use an extr uder to pass the suspension through a stacked pair
of 100-nm polycarbonate filters.
14. Allow the suspension to equilibrate for 5 min inside the
extruder before applying pressure for the first pass.
15. Repeat the
extrusion process nine more times to ensure consis-
tent liposome size and homogeneity.
414 Anil Kumar et al.
3.3 Amine
Modification
3.4 Conjugation
It involves chemically modifying IgG antibodies to attach them to
liposomes, enhancing the functionality of liposomes for targeted
drug delivery [51].
1. Prepare liposomes incorporating 1% N-(4-(p-maleimidophenyl)butyryl)-(1,2-distearoyl-sn-glycerol-3-phosphoethanolamine) (MPB-DSPE) using the hydration and extrusion
methods described above.
2. Prepare a 1 mM SPDP solution in 2,4-hydroxethyl-1-piperazineethanesulfonic acid (HEPES)-buffered saline (HBS).
3. Add SPDP, (succinimidyl 3-(2-pyridyldithio)propionate) a
hetero-bifunctional crosslinker, (5 mol equivalents) to the
IgG solution and stir at room temperature for 20 min.
4. Pass the solution through a Sephadex G-50 column equilibrated in SAS (pH 4.4) and collect fractions with an absorbance
>1.0 at 280 nm.
5. Add DTT (reducing agent, cleaves disulfide bonds) to the
solution and stir at room temperature for 20 min.
6. Pass the solution through another Sephadex G-50 column
equilibrated in HBS (pH 7.4) and collect fractions with an
absorbance >1.0 at 280 nm.
1. Determine the IgG concentration from the absorbance at
280 nm.
2. Add the IgG solution to the liposome solution (75 μg protein
per μmol of lipid) and stir at room temperature for 16 h.
3. Pass the mixture through a Sepharose CL-4B column and
collect liposome-containing fractions.
4. Determine coupling efficiency using protein and lipid assays.
5. Determine the size of the conjugates using a particle sizer.
3.5 Carbohydrate
Modification
It involves altering the carbohydrate moieties of IgG antibodies to
facilitate their attachment to liposomes. This method is useful for
creating targeted delivery systems in various therapeutic
applications [
51].
1. Prepare a sodium metaperiodate solution (1 mg/mL), add to
the antibody solution, and stir at room temperature for 1 h.
2. Pass the
solution through a Sephadex G-50 column equilibrated in SAS (pH 4.4) and collect fractions with an absorbance
>1.0 at 280 nm.
Drug Delivery in Rare Diseases: Orphan Drugs and Therapies 415
3.6 PDPH (N-(4-(pMaleimidophenyl)
Butyryl) Hydrazide)
Coupling
3.7 Disulfide
Modification
3.8 PEGylation
1. Add PDPH suspension (40 μL PDPH stock per mL of IgG
solution) and stir at room temperature for 5 h.
2. Pass the solution through a Sephadex G-50 column equilibrated in SAS (pH 4.4) and collect fractions with an absorbance
>1.0 at 280 nm.
3. Add DTT to the solution and centrifuge at 3000 rpm for
20 min.
4. Pass the supernatant through a Sephadex G-50 column equilibrated in HBS (pH 7.4) and collect fractions with an absorbance >1.0 at 280 nm.
It creates disulfide bonds between the IgG antibodies and liposomes to enhance their stability and functionality for targeted
applications [51].
PEGylation, the process of attaching polyethylene glycol (PEG)
chains to drug molecules, has revolutionized the delivery of orphan
drugs by enhancing their solubility and prolonging their half-life in
circulation [
52]. This technique has been effectively applied in the
delivery of pegloticase for the treatment of chronic gout, significantly improving its pharmacokinetic properties and reducing
immunogenicity [
53]. It has been instrumental in the development
of therapies for hemophilia, such as PEGylated recombinant factor
VIII, which offers extended protection against bleeding
episodes [
54].
3.8.1 Materials Required 1. Pre-formed liposomes (e.g., made of phosphatidylcholine
(PC) and cholesterol).
2. PEGylated lipid (e.g., DSPE-PEG2000).
3. Organic solvent (e.g., ethanol).
4. Phosphate-buffered saline (PBS), pH 7.4.
5. Equipment: Magnetic stirrer, water bath, dialysis tubing,
UV-Vis spectrophotometer.
3.8.2 Procedure 1. Dissolve the PEGylated lipid (DSPE-PEG2000) in a small
volume of ethanol.
2. The concentration should be prepared such that when added to
the liposome suspension, the final desired PEGylation percentage is achieved (e.g., 5% molar ratio of DSPE-PEG2000 to
total lipids).
3. Add the PEGylated lipid solution to the pre-formed liposome
suspension slowly while stirring.
4. The volume
of ethanol should not exceed 5% of the total
volume to avoid disrupting the liposomes.
416 Anil Kumar et al.
5. Incubate the mixture at a temperature suitable for the lipid
phase transition (e.g., 37 °C) while stirring for 1–2 hours to
ensure the insertion of PEGylated lipids into the liposome
bilayer.
6. Ensure constant stirring to facilitate uniform distribution of the
PEGylated lipids.
7. After the incubation, remove free PEGylated lipids and ethanol
by dialyzing the liposome suspension against PBS (pH 7.4)
using dialysis tubing with an appropriate molecular weight
cut-off (e.g., 10,000 Da).
8. Dialyze for 24 h with frequent changes of the PBS to ensure
complete removal of free PEGylated lipids and ethanol.
9. Measure the particle size and polydispersity index (PDI) using
dynamic light scattering (DLS) to confirm the integrity and
uniformity of PEGylated liposomes.
10. Assess the surface charge (zeta potential) to confirm successful
PEGylation. PEGylated liposomes usually exhibit a slightly less
negative or neutral zeta potential due to the shielding effect of
PEG chains.
11. Verify the presence of PEG on the liposome surface by measuring the absorbance at specific wavelengths (e.g., 220 nm) using
a UV-Vis spectrophotometer.
3.9 Liposomal Doxorubicin (LD)
LD is used in the treatment of rare cancers such as Kaposi’s sarcoma. The encapsulation of doxorubicin in liposomes enhances
drug delivery by improving targeting to tumor tissues while reducing systemic exposure and associated toxicities [
55]. Liposomes
preferentially accumulate in tumor tissues due to the enhanced
permeability and retention (EPR) effect [41]. The use of LD
significantly reduces cardiotoxicity, a common adverse effect associated with conventional doxorubicin therapy [55]. The liposomal
encapsulation alters the pharmacokinetics of doxorubicin, resulting
in prolonged circulation time and reduced peak plasma concentrations, thereby minimizing damage to cardiac tissues [
41]. LD is
administered intravenously, allowing the liposomes to circulate
through the bloodstream and gradually release the encapsulated
doxorubicin at the tumor site [
22]. This targeted delivery system
enhances the therapeutic efficacy of doxorubicin while minimizing
adverse effects on healthy tissues [
Clinical s
tudies h
ave demonstrated the effectiveness of LD in
55].
treating Kaposi’s sarcoma, with improved response rates and
reduced toxicity compared to conventional doxorubicin
56]. Patients treated with LD have shown significant tumor
[
regression and better tolerability [
Doxorubicin
is typically loaded into liposomes using a pH
56].
gradient method. This active loading method achieves high encapsulation efficiency and stable drug retention within the liposomes
Drug Delivery in Rare Diseases: Orphan Drugs and Therapies 417
[22]. To improve the circulation time of LD in the bloodstream,
the surface of the liposomes is modified with polyethylene glycol
(PEG). This process, known as pegylation, helps the liposomes
evade the immune system and reduces clearance by the mononuclear phagocyte system (MPS) [
41].
3.10 Marqibo (Vincristine Sulfate)
This liposomal formulation of vincristine sulfate is used to treat
acute lymphoblastic leukemia (ALL). It uses sphingomyelin/cholesterol liposomes to encapsulate vincristine. The drug is loaded
into liposomes via active gradient loading. It enhances the pharmacokinetics and reduces toxicity compared to conventional
57].
3.11 DepoCyt (Cytarabine)
vincristine [
DepoCyt is a liposomal formulation of cytarabine, used for the
treatment of lymphomatous meningitis. DepoCyt utilizes multilamellar liposomes to encapsulate cytarabine. The liposomes are
prepared by reverse-phase evaporation method, followed by extrusion to control the size. This formulation allows for sustained
release of cytarabine into the cerebrospinal fluid [
4 Poly(Lactic-co-Glycolic Acid, PLGA) Nanoparticles
Makadia and Siegel [59] reviewed the use of PLGA-based nanoparticles for controlled drug delivery. These nanoparticles degrade
slowly, providing a sustained release of the encapsulated drug. This
approach has been utilized in the delivery of various therapeutics,
including those for rare diseases, due to PLGA’s biocompatibility
and FDA approval [
glycolic acid. It degrades into lactic acid and glycolic acid, which are
metabolized by the body, making PLGA suitable for biomedical
applications [
60]. One of the most common methods for preparing
PLGA nanoparticles is the emulsion solvent evaporation technique
(Fig. 2). This involves dissolving PLGA and the drug in an organic
solvent (e.g., dichloromethane), forming an emulsion with an
aqueous phase containing a surfactant (e.g., polyvinyl alcohol,
PVA), and then evaporating the solvent to form nanoparticles [
Another method
in a water-miscible organic solvent (e.g., acetone) and then added
to an aqueous phase, leading to the formation of nanoparticles as
the solvent diffuses and evaporates [
depends on factors such as the polymer-to-drug ratio, the type of
solvent, and the method used. High encapsulation efficiency can be
achieved by optimizing these parameters [
PLGA nanoparticles can be modified with targeting ligands (e.g.,
antibodies, peptides) to enhance specificity. PEGylation (attachment of polyethylene glycol) is also used to improve circulation
time and reduce immune recognition [
59]. PLGA is a copolymer of lactic acid and
is nanoprecipitation, where PLGA is dissolved
58].
61].
62]. Encapsulation efficiency
63]. The surface of
64].
418 Anil Kumar et al.
Fig. 2 Preparations of polycaprolactone-based nanoparticles
PLGA nanoparticles are generally stable in aqueous suspension
but can aggregate over time. To enhance stability, nanoparticles are
often lyophilized with cryoprotectants like trehalose or sucrose
59]. While PLGA nanoparticles are less pH-sensitive compared
[
to chitosan nanoparticles, the degradation rate of PLGA can be
influenced by pH. Typically, neutral pH conditions are used for
storage to minimize hydrolytic degradation [
4.1 Drug Release
Profile
PLGA nanoparticles provide controlled and sustained release of
encapsulated drugs. The release profile can be tailored by adjusting
the polymer composition (ratio of lactic acid to glycolic acid),
molecular weight, and the presence of any surface modifications
[
63]. The release mechanism generally involves an initial burst
release followed by a sustained release phase. This can be modulated by altering the preparation conditions and particle size
61]. PLGA is FDA-approved for use in various biomedical
[
applications.
60] (Table 3).