430 |
R. Baid et al. |
Encapsulation of CNTF in an implant filled with RPE cells may be an effective method to deliver sufficient amounts of protein over a long period of time to treat retinal neurodegenerative diseases.
Other growth Factors: Retinal diseases such as RP and AMD are caused by apoptotic cell death (Travis 1998; Dunaief et al. 2002). Impediment of apoptosis (i.e., retinal cell death) is one of the promising fields, which can have a major impact on blindness. A variety of neurotrophic growth factors have shown great potential in inhibiting retinal degeneration in several animal models (Wenzel et al. 2005). Basic fibroblast growth factor (bFGF) was shown to delay photoreceptor degeneration in Royal College of Surgeons (RCS) rat (Faktorovich et al. 1990). Brain-derived neurotrophic factor (BDNF) (LaVail et al. 1998), CNTF (LaVail et al. 1998; Thanos et al. 2004), glial-derived neurotrophic factor (GDNF) (AndrieuSoler et al. 2005; Buch et al. 2006), lens epithelium derived growth factor (LEDGF) (Machida et al. 2001), and rod derived cone viability factor (RdCVF) (Leveillard et al. 2004) have been shown to inhibit retinal degeneration in various animal models. Pigment epithelium derived factor (PEDF) is a neuroprotective factor preventing neovascularization by protecting the retina and retinal pigmented epithelium and by inhibiting angiogenesis (Steele et al. 1993; Cayouette et al. 1999; Mori et al. 2002). Adenoviral vector delivery of complimentary DNA encoding human PEDF (AdPEDF.11;GenVec, Gaithersburg, MD, USA) has successfully inhibited ocular neovascularization (Mori et al. 2002). A phase I trial has shown that there are no adverse events or dose-dependent toxicities associated with PEDF in patients with NVAMD (Campochiaro et al. 2006).
Table 17.2 summarizes clinical trials of several macromolecule drugs in the eye. Table 17.3 summarizes promising protein drugs useful in treating diseases of the back of the eye.
17.6 Ophthalmic Protein Formulation Development
Ophthalmic protein formulation development is a complicated process as proteins are sensitive and easily perturbed by changes in their surroundings. Conformational stability of proteins, which is maintained by weak physical interactions and disulfide linkages, can be compromised by changes in pH and ionic strength (Saishin et al. 2003).
The three-dimensional structure of proteins can also be disrupted by a number of variables that are encountered during the development of suitable formulations. One of the major concerns while formulating proteins is the humidity of the surroundings. A “low humidity” environment in most manufacturing units will be around 20% relative humidity. However, this can be too high for proteins, which have an inherent nature to absorb large amounts of water leading to degradation during storage or distribution. Changes in protein structure can not only negatively impact its therapeutic effect, but can also trigger adverse immune reactions in the body (Hermeling et al. 2004).
Table 17.2 Clinical trials of various ophthalmic macromolecule therapies
Therapeutic agent |
Target disease |
Clinical trial |
Observation |
Clinical level |
|
|
|
|
|
Pegaptanib |
CNV |
VISION (Chakravarthy et al. 2006) |
Risk of ³3 lines vision loss reduced to 67% |
FDA approved |
|
|
|
in 1 year |
|
|
DME |
MDRS-phase II (Cunningham et al. 2005) |
In 1 year, a gain of 18% vision |
Phase-III ongoing |
Ranibizumab |
CNV |
ANCHOR MARINA, PIER, PrONTO |
In 2 years, there was a gain of 6.6 letters |
FDA approved |
|
|
(Takeda et al. 2007; Regillo et al. 2008) |
|
|
|
DME |
READ-2-phase II (Hayashi et al. 2009) |
Reasonable safety profile |
Phase IIIfor DME-ongoing |
Bevacizumab |
CNV |
Case series (Avery et al. 2006; Bashshur |
Visual improvement of 15–30 letters |
Phase-III ongoing |
|
|
et al. 2006; Rich et al. 2006; Spaide |
|
|
|
|
et al. 2006) |
|
|
|
DME |
Case series (Haritoglou et al. 2006; Arevalo |
In 1 year, visual improvement of 7 letters |
Phase-III ongoing |
|
|
et al. 2007) |
|
|
VEGF-trap |
CNV |
CLEAR IT-1 (Nguyen et al. 2006) |
In 6 weeks, visual improvement of 4.8 |
Phase-II ongoing |
|
|
|
letters |
|
|
DME |
DAVINCI – phase II (Ferrara et al. 2006) |
Visual acuity gain of 8.6–11.4 letters |
Phase II completed |
|
|
|
depending on dose |
|
|
Wet AMD |
Phase II |
5.3 mean letter gain in visual acuity in 52 |
Phase-III- VIEW-1 and |
|
|
|
weeks |
VIEW-2 ongoing |
CNTF (NT-501) |
RP |
Phase I (Einmahl et al. 2002) |
Reasonable safety profile |
Phase I completed |
|
Dry AMD |
Phase II (Ehrlich et al. 2008) |
Stabilized best corrected visual acuity |
Phase II completed |
|
|
|
(BCVA) in 12 months |
|
|
RP |
Phase II/III (Feher et al. 2009) |
Not available |
Phase II/III ongoing |
CNV choroidal neovascularization; DME diabetic macular edema; AMD age related macular degeneration; RP retinitis pigmentosa; ANCHOR anti-VEGF antibody for the treatment of predominantly classic choroidal neovascularization in age-related macular degeneration; CLEAR clinical evaluation of antiangiogenesis in the retina; DA VINCI DME and VEGF trap-eye: investigation of clinical impact; ETDRS early treatment for diabetic retinopathy study; FAIS fluocinolone acetonide implant study; MARINA minimally classic/occult trial of the anti-VEGF antibody ranibizumab in the treatment of neovascular age-related macular degeneration; MDRS Macugen diabetic retinopathy study; PIER phase I AMD, multi-center, randomized, double-masked, sham injection-controlled study of the efficacy and safety ranibizumab; PrONTO prospective optical coherence tomography imaging of patients with NAMD treated with intra-ocular ranibizumab (Lucentis); READ ranibizumab for edema of the macula in diabetes; VEGF vascular endothelial growth factor; VIEW VEGF trap-eye: investigation of efficacy and safety in wet age related macular degeneration; VISION VEGF inhibition study in ocular neovascularization
Development Formulation and Delivery Drug Protein 17
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432
Table 17.3 Growth factors for the treatment of retinal degenerative diseases
|
Growth factor |
Target disease |
Species tested |
Delivery approach |
Reference |
|
|
|
|
|
|
|
Basic fibroblast growth factor |
Retinal degeneration |
Rat |
Subretinal injection |
Faktorovich et al. (1990) |
|
(bFGF) |
|
|
|
|
Brain-derived neurotrophic |
Retinal degeneration slow |
Mouse |
Intravitreal injection |
LaVail et al. (1998) |
|
|
factor (BDNF) |
(RDS), nervous (NR), |
|
|
|
|
|
and Purkinje cell |
|
|
|
|
|
degeneration (PCD) |
|
|
|
Ciliary neurotrophic factor |
Retinal degeneration |
Rabbit |
Encapsulated cell therapy (ECT)-based |
Thanos et al. (2004) |
|
|
(CNTF) |
|
|
NT-501 device implant |
|
Glial-derived neurotrophic |
Retinal degeneration |
Mouse |
PLGA-microspheres, intravitreal |
Andrieu-Soler et al. (2005) |
|
|
factor (GDNF) |
|
|
injection |
|
|
|
Retinal degeneration |
Rat |
Mouse embryonic stem cells (mES) |
Gregory-Evans et al. (2009) |
|
|
Glaucoma |
Rat |
Biodegradable microspheres, intravitreal |
Jiang et al. (2007) |
|
|
|
|
injection |
|
Lens epithelium derived |
Retinal degeneration |
Rat |
Intravitreal injection |
Machida et al. (2001) |
|
|
growth factor (LEDGF) |
|
|
|
|
Pigment epithelium-derived |
Retinal degeneration, RDS |
Mice |
Intravitreal injection |
Cayouette et al. (1999) |
|
|
growth factor (PEDF) |
|
|
|
|
Rod derived cone viability |
Retinitis pigmentosa |
Mice |
Subretinal injection |
Leveillard et al. (2004) |
|
|
factor (RdCVF) |
|
|
|
|
|
|
|
|
|
|
.al et Baid .R
17 Protein Drug Delivery and Formulation Development |
433 |
Effective formulations must, therefore, safeguard a protein’s structural integrity, while achieving the desired therapeutic effect. In order to maintain the protein’s efficacy, the formulation developed must be resistant to both physical degradation, such as aggregation and denaturation, as well as chemical degradation, such as oxidation and deamination.
Table 17.4 lists four macromolecule formulations that were either approved (ranibizumab and pegaptanib) or used off-label (bevacizumab and infliximab) for administration to the vitreous humor of the eye. Of these, all formulations are protein based, except pegaptanib, which is an aptamer. While the off-label use of bevacizumab is widely undertaken with no known serious adverse events, off-label use of infliximab has been associated with retinal toxicity and immunogenicity (Giganti et al. 2010).
17.6.1 Protein Biosynthesis
The first step in protein formulation is to genetically engineer a cell to produce therapeutic protein. For instance, ranibizumab is produced in E. coli cells. The genetic information encoding the protein (DNA) provides the cell with the complete instructions to produce (generate) the protein. Typically the cells are engineered to express the protein in the cell and then depending on the nature of the protein it might either be secreted or retained within the cell. Genetically engineered cells are kept frozen as stock for future use in a manufacturing process. At the time of use, these cells are thawed and allowed to grow in a culture medium. The medium properties and growth parameters adopted during this step are crucial since they can drastically affect the cell growth and consequently the protein output. Once the cells have grown to a significant number, they are transferred to a larger tank (e.g., 1,000 L capacity), wherein their growth is continued. The cell medium is separated and if the protein is secretary in nature, the media is subjected to additional steps wherein any possible contaminants including cell debris, salts, or unwanted proteins are removed. When the protein is retained in the cell, the cell is disrupted either by sonication or lysis and the protein is separated from the cellular debris. Bioburden within the manufacturing room should be controlled during the processing. Also, bacterial endotoxins in the end product should be eliminated or minimized as per regulatory guidelines. A pure protein devoid of contaminants prepared as above is used in further development.
17.6.2 Preformulation Studies
Development of a stable protein formulation is one of the crucial steps in developing a protein as a therapeutic moiety. The first step in developing a formulation is the selection of a dosage form for the delivery of the protein. Most of the formulations
434
Table 17.4 Macromolecule formulations used for intravitreal administration in the clinic
|
Product brand name (generic name) |
Dose; route of administration |
pH of the formulation |
Excipients |
|
|
|
|
|
|
Lucentis® (Ranibizumab) |
0.05 mL of a 10-mg/mL solution; |
pH 5.5 |
10 mM histidine HCl, 10% a, a-trehalose |
|
|
intravitreal injection |
|
dihydrate, 0.01% polysorbate 20, q.s. water |
|
|
|
|
for injection |
Avastin® (Bevacizumab) 4 or 16 mL of a 25-mg/mL pH 6.2 solution; intravenous injection
1.25 mg/0.05 mL; intravitreal injection
Each 100 mL solution contains 240 mg a, a-trehalose dehydrate, 23.2 mg of sodium phosphate monobasic monohydrate, 4.8 mg of sodium phosphate dibasic anhydrous, 1.6 mg polysorbate 20, q.s. water for injection
Macugen® (Pegaptanib sodium) |
0.3 mg/90 mL; intravitreal |
pH 6–7 |
Each 90 mL contains 0.069 mg sodium phosphate |
|
injection |
|
monobasic monohydrate, 0.11 mg of sodium |
|
|
|
phosphate dibasic heptahydrate, 0.8 mg |
|
|
|
sodium chloride, q.s. water for injection |
Remicade® (Infliximab) |
100 mg/10 mL; intravenous |
pH 7.2 |
Each 10 mL contains 500 mg sucrose, 0.5 mg |
|
injection |
|
polysorbate 80, 2.2 mg monobasic sodium |
|
0.5 mg/0.05 mL; intravitreal |
|
phosphate monohydrate, 6.1 mg dibasic |
|
injection |
|
sodium phosphate dehydrate, q.s. water for |
|
|
|
injection |
|
|
|
|
.al et Baid .R