Introduction
Avastin (Bevacizumab solution for injection; Genentech, San Francisco, Canada) is a humanized (93% human, 7% murine sequence) IgG1 monoclonal antibody prepared by recombinant DNA engineering. It is a Vascular Endothelial Growth Factor (VEGF)-specific angiogenesis inhibitor and belongs to the category of drugs considered the fourth mode for cancer treatment. In 2004, it became the first angiogenesis inhibitor drug approved for cancer chemotherapy, first for the treatment of colorectal cancer, and subsequently approved for other malignant conditions (1, 43).
Chemistry/Composition
Bevacizumab is typically a monomer with a molecular weight of 149 kDa. It has three major fragments: Fv (Fragment variable of murine origin), Fab (Fragment antigen-binding), and FC (Fragment crystallizable of human origin) which performs effector function. Each V (variable) domain contains three short stretches of peptide and hypervariable sequences (HV1, HV2, and HV3) known as the complementarity-determining regions (CDR)-antigen-binding region (30).
The heavy chains show C-terminal heterogeneity (lysine discrepancies) and also contain one N-linked glycosylation on asparagine at position 303. Two inter-chain covalent disulfide bonds couple the two heavy chains. Each light chain is covalently joined through a disulfide bond at cysteine 214 to a heavy chain at cysteine 226 (6).
Avastin comes as a lyophilized solid for reconstitution prior to use. The solid is a clear to slightly iridescent, colorless to brown, sterile; pH 6.2 solution given by intravenous infusion. It is supplied in 100 mg and 400 mg preservative-free single-use vials to deliver 4 mL or 16 mL of Avastin (25 mg/mL).
The 100 mg product is formulated in 240 mg α, α-trehalose dihydrate (acceptable non-compendial specifications), 23.2 mg Na phosphate (monobasic, monohydrate), 4.8 mg Na phosphate (dibasic, anhydrous), 1.6 mg polysorbate 20, and water for injection USP. Its 400 mg product contains the same amount of ingredients in the order of four folds. It can be stored up to 24 months at 2-8°C and given IV, mostly on days 0, 28, 35, and 42 or every 14 days (6).
Pharmacology
Tumor Angiogenesis
Angiogenesis is the formation of new blood vessels shooting from the preexistent vasculature. It occurs in normal and pathological conditions, including those associated with malignant neoplastic disease (2, 9). The association of angiogenesis and malignant neoplastic disease was ab initio discovered about 50 decades ago (11-13).
Folkman et al., in 1971, first proved that angiogenesis was one of the major steps in tumour patterned advance and metastasis (4, 14). Gullino showed that cells in pre-malignant tissues acquired angiogenic capacity, which is dominant when malignancy is attained (15), which was confirmed through genetic studies, that acquisition of an angiogenic phenotype was one of the trademarks of malignant neoplastic disease (3, 16-18).
VEGF A is the major angiogenic factor and regulator of tumour neovascularisation in humans. It enhances endothelial cell proliferation and blood vessel formation. Over-expression of VEGF in most tumours worsens the forecast (8, 20). AvastinA® explores the most efficacious of the three possible ways of blocking VEGF’s activity (neutralise VEGF); others either block production (Iressa) or block receptors for VEGF and other angiogenic stimulators – Sutent (7).
Fig. 2. Tumour angiogenesis. Angiogenesis is initiated by the production of angiogenic factors from tumor cells, such as vascular endothelial growing factor (VEGF). Upon adhering to its blood relation receptors on endothelial cells, VEGF triggers endothelial cell proliferation and migration. Degradation and invasion of extracellular matrix (ECM) then follow. Endothelial cells assemble into a tubular structure. The process is completed by loop formation and vessel wall ripening. (EC = endothelial cell) – Ref- [7].
Pharmacodynamics:
Bevacizumab binds with high affinity to all human VEGF-A with its Fab fragment, which interacts selectively with ligand VEGF (complement arrested development), prior to VEGF’s connection to the natural endothelial receptor, which leads to antibody-dependent cellular cytotoxicity (ADCC). Normal ligand-receptor interaction is blocked; besides, receptor phosphorylation and downstream tracts are activated.
Therefore, vascularization is regressed; tumour vasculature formation and tumour growth are also inhibited (23). It binds and inhibits the biological activity of human VEGF during in vitro and in vivo assay systems (64). Preclinical in vivo tumour growth studies of Bevacizumab disposal to xenograft and metastatic models of malignant neoplastic disease in mice showed a decrease in microvascular tumour growth and suppression of metastatic disease patterned advance (24, 25).
Pharmacokinetics:
In contrast to small molecule drugs, the typical metabolic enzymes and transporter proteins such as cytochrome P450 and multidrug resistance efflux pumps are not involved in the clearance of monoclonal antibodies (mAbs) (62). Pharmacokinetic studies of Bevacizumab were conducted in mice, rats, and rabbits given by intraperitoneal injection; absorption was complete but slower for subcutaneous injection. Its distribution was assessed in rabbit models because recombinant mAb (rhuMAb VEGF) was found to bind to rabbit VEGF but not mouse or rat VEGF recombinant MAb (24).
This model showed that most of rhuMAb was retained in the plasma, with more distribution to the heart, testes, bladder, and kidney compared with other organs (which justifies its use in cancer treatment of these organs); suggesting that endogenous antibodies and Avastin are similarly cleared and regulated by Brambell receptors (54). Its pharmacokinetics was therefore characterized as a two-compartment model from previous studies.
Patients who received doses ranging from 0.1 to 10mg/kg intravenously over 4-24 weeks had an average clearance of 239mL/day, with an average Vd (volume of distribution) of 3260mL in the central compartment (6). The mechanism of metabolism and elimination has not yet been described in published information. However, results from a phase II trial showed that human clearance and half-life for Avastin were about 2.79 mL/kg/day and 21 days, respectively (6).
Clinical Applications:
It is used for the management of metastatic colorectal cancer, with intravenous interferon alfa or 5-fluorouracil-based chemotherapy for first- or second-line treatment (31); non-squamous non-small cell lung cancer, with carboplatin and paclitaxel for first-line treatment of unresectable, locally advanced, recurrent, or metastatic disease (32, 34); first-line therapy for metastatic HER2-negative breast cancer (33, 35); spongioblastoma, as a single agent (intra-arterially/intracranially) for patients with progressive disease following previous therapy; and macular degeneration (48, 56).
Side Effects / Adverse Effects:
Commonly reported effects are high blood pressure, albuminuria, and bleeding. Less frequent adverse events include arterial and venous thromboembolic events (ATE, VTE), congestive heart failure, wound healing complications, and GI perforations. Generally, these adverse events are not dose-dependent in any indication (with the exception of high blood pressure and grade 1 albuminuria).
These adverse events require regular monitoring (high blood pressure, albuminuria), avoiding overdosage (high blood pressure, VTE), temporary dose interruption (high blood pressure, albuminuria, VTEs, wound healing), and absolute discontinuation of the regimen (for all life-threatening events) – 38, 40-43.
Development / Formulation:
The first approved mAbs for therapy, Orthoclone (OKT3), which was of murine origin, stimulated the formation of neutralizing human anti-mouse antibodies that reduced the drug’s activity via immunogenicity. Murine MAbs were engineered to the chimeric type (mouse CDR, human Fc) but still had the same restrictions typified by rituximab; nevertheless, they had a longer in vivo half-life (57,62). Bevacizumab development (humanized MAbs) was meant to cut down the xenogenic part, thereby restricting immunogenicity (23). Two methods of accomplishing absolute biocompatibility of mAbs with humans (fully humanized mAbs) are phage display library (Adalimumab/Humira) and the usage of transgenic XenoMouseA® – Panitumumab/Vectibix (63).
Bevacizumab is produced by recombinant DNA engineering in Chinese hamster ovary (CHO) cells, with Garamycin (antibiotic) as a part of the expression system. Its industry is based on a CHO master and working cell bank system, which has been exhaustively characterized and tested to exclude harmful contaminations and endogenous viruses in conformity to ICH guidelines. The industry consists of a series of steps, which include agitation, crop, and purification. Chromatographic and viral inactivation/removal processes were combined to purify the product (45).
Structurally, it is large, complex, lipophilic, and prone to degradation by acids, enzymes, as well as temperature extremes and solvents (58,69). The development of the product was intended to achieve a stable liquid intravenous preparation (as extract), the most outstanding means of delivery of antibody therapeutics, which is able to reach diffuse and inaccessible tumor sites (49,64). Besides, liquid preparations are cheaper, easy to develop, and easier to prepare for disposal.
Its presentation as a liquid preparation was limited by aggregation and deamidation reaction (water-accessible parts) which could affect the product’s integrity/efficacy (65,68,75) by inducing charge heterogeneity noticeable by isoelectric focusing or high-performance cation-exchange chromatography (66,67).
At low concentration, it adheres to container walls and exhibits aggregation at high concentrations. The concentration allowed will not be sufficient for clinical response in chronic therapy. Besides, it is difficult for concentrations >50mg/ml to pass through the needle gauge due to its viscosity – solution dimerization (70,71). This justifies its disposal by extraction. Other conditions that encouraged this situation were pH 6.5-7.5, IM NaCl (72,73).
This challenge was partially managed by post-translational modification, i.e., N-linked glycosylation at asparagine 303 during the production of Avastin (45, 55, 71). However, the antigen specificity of some therapeutics could be affected by replacing some amino acids of the Fv fragment (59-61). Also, the solution was prepared by lyophilization to reduce the impact of H2O (deamidation, attachment/collection, and loss of authority) on Bevacizumab because it is stable to stop dead drying in the absence of excipients that act as cryoprotectants (76-78).
Its freeze-drying with residual H2O content (1-8%) allows for optimum stabilization in the dry province and upon reconstitution, as confirmed by a recent study (22). Avastin was formulated with a buffer (sodium phosphate-glandular fever & dibasic), sugar (trehalose dihydrate), and detergent (polysorbate 20) after freeze-drying, to achieve a favorable pH for the product and reduce its collection rate (45, 74, 44). Alpha, alpha-trehalose dihydrate is a better anti-aggregation agent than sucrose (which can induce acute renal failure), with a higher Tg-glass passage temperature (39, 46).
The use of polymeric systems to improve the stability of some products has shown an inflammatory response (47). An exception is the case of intra-vitreal injection of Bevacizumab loaded PLGA microspheres for macular degeneration, which provides sustained drug effects with fewer inclinations to immunogenicity (48).
The Fab fragment of Bevacizumab for therapy may avoid the immunological reactions associated with the whole antibody while improving the pharmacokinetics of the drug when conjugated with polyethylene glycol (PEG). Although it has not been developed for clinical usage, results from in vitro site-specific mono-PEGylation of Fab of Avastin showed that PEG-Fab (10 kDa) bound in a similar mode as the native Fab (as analyzed by Biacore).
However, more studies would still be done to improve the binding affinity of executable PEGylated Fab. Besides, in vitro and in vivo studies on a manufactured Avastin tissue tablet for subconjunctival nidation showed that such preparation would prolong drug release and improve results of lesion healing after glaucoma filtration surgery (37a, B).
Clinical Information
Avastin has consistently been involved in several clinical tests that showed encouraging results. It is often combined in many stage II solid tumor tests, with erlotinib HCl, a tyrosine kinase inhibitor for synergy (5, 19, 21, 47, 50-52). Unusually, a result of a stage II test, multicenter, open-label, noncomparative survey was recently published, where the efficacy of Bevacizumab, as a single agent and in combination with irinotecan, in recurrent spongioblastoma was evaluated, and results shown in Table 1. A predetermined dosage of the drug was given to 167 patients, and the primary endpoints were the clinical response rate and 6-month progression-free survival, while safety and overall survival were the secondary endpoints. It was inferred from the results obtained that Bevacizumab, as a single agent or in combination with irinotecan, was moderately safe and effective in recurrent spongioblastoma (53).
Conclusions
Bevacizumab’s versatility for the management of a variety of malignancies is associated with the expression of VEGF in almost all tumor types (10, 15). Intravenous route /local bringing (intracranial/intravitreal) are the lone executable manner of bringing as other paths compromise drug efficacy/safety (79,80) .
It is hoped that specificity of its Fab-PEG conjugate for the VEGF ligand in future clinical surveies will cut down the majority of Avastin for bringing whilst optimizing therapy. Besides, It has been proven that antitumor activity of AvastinA® can be improved by its combination with chemotherapy (26), radiation (27, 28) and other antiangiogenic agents (29).
Avastin-resistant tumours have been managed by aiming other angiogenesis signalling tracts such as platelet-derived growing factor-C (36).
