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According to the reports by International Agency for Research on Cancer (IARC) of WHO, the incidence and prevalence rates of NHL is higher in the developed countries than the developing countries such as Asia and South Africa[final epi]. The contribution of NHL among all the cancers in the United States is about 4%[final epi]. With 54,370 estimated new cases and around 19,410 deaths in the year 2004 in United states, NHL stands as the fifth commonly diagnosed disease[final epi]. According to SEER (Data from Surveillance, Epidemiology and End Results) programme conducted by the National Cancer Institute (NCI) during the period of 1970's and 1980's, there was an increase in NHL by 3% to 4% (age-adjusted rate) annually[final epi].
Rituximab also known as Rituxan or MabThera is a chimeric murine or human monoclonal glycosylated IgG1-kappa antibody which binds to a hydrophobic transmembrane protein CD20 present on B-lymphocytes in order to destroy B-cells through various mechanisms[IB]. It is a highly purified 1328-amino acid antibody and is produced in a suspension culture by a Chinese hamster ovary (CHO) in a nutrient media of gentamicin antibiotic[IB]. During the purification process gentamicin is removed along with other impuities.
2. Mechanism of action:
Rituxan recognizes and binds to the CD20 receptor and ultimately starts destroying or eliminating its target cells. Although in-vivo mechanism of action is not fully understood, studies from in-vitro suggest that Rituxan depletes or destroys the B-cells by three different mechanisms[IB]:-
Complement-Dependent Cytotoxicity (CDC)
Complement, a large group of plasma proteins destroys invading pathogens or malignant cells. The complement system is activated by Ab-Ag (Rituxan-CD20) complexes. The C1q protein component of the complement recognizes and binds to the Fc domain of Rituxan starting a series of reactions. The products of these reactions penetrate the B-cells thereby eventually killing them.
Antibody-Dependent Cellular Cytotoxicity (ADCC)
After Rituxan binds to the CD20 receptor the effector cells recognizes the Fc domain Rituxan and binds to it. The effector cells now release cytotoxic granules which penetrates the B-cells to kill the ligated cells or phagocytose them.
Apoptosis also called as programmed cell death is a natural mechanism which eliminates undesired cells from the body. When Rituxan binds to the CD20 cells the antibody-receptor complex signals the cell to initiate the degeneration process. The cell changes its shape as its cytoskeleton collapses, nucleus condenses and enzymes fragment DNA into small pieces. Eventually the B-cells destroy themselves.
3. Drug History:
Rituximab was developed by IDEC pharmaceutical company formed in 1986. US FDA approved Rituximab in 1997 while in Europe it was approved in 1998 as the standard treatment of B-cell Non-Hodgkin's lymphoma[CD20 TAREGET].
Currently in US, Rituximab is co-marketed by Biogen Idec and Genentech whereas it is marketed under Roche in the European countries and Canada[IB]. Rituximab is also available as Reditux in Dr. Reddy's laboratory from 2007. It is marketed in Japan by Chugai Pharmaceuticals and Zenyaku kogyo.
4. Patent Status:
Rituxan was launched in 1997 in United States[final patent]:
patent number 5,677,180 will expire by 2014,
patent number 5,736,137 will expire by 2015,
and patent number 7,381,560 will expire by 2016.
5. Market Share:
Rituximab was the first monoclonal antibody to be approved by any regulatory authority. The effectiveness of the drug gave it a humongous response in terms of capita. In the year 2006, the sales revenue in US alone was $2,252 million and in 2007 it went up to $2,515.
C. DRUG DISCOVERY PROCESS:
1. Target Identification:
The target CD20 antigen or Bp35 antigen was discovered during the invention of the first monoclonal anti-CD20 antibody. A new antibody known as anti-B1 was formed which recognized the CD20 antigen after Balb/c mice were immunized with Burkitt's lymphoma cells[CD20 TARGET]. Once the target is identified it is then validated.
2. Target Validation:
The exact cellular mechanism of CD20 protein in not clearly understood. Due to greater number of abnormal B-cells in NHL a desirable finding is produced[TMA]. Till date no natural ligand is discovered. On the basis of ligation with other antibodies to CD20, little information has been gathered regarding the functioning of CD20[CD 20]. The target acts as a B-cell activating or proliferating molecule. Ligation of CD20 antigen with type-1 monoclonal CD20 antibody escorts the formation of signalling platforms or lipid rafts. This ultimately causes activation of capase-3 and calcium flux[CD20 TARGET]. The downstream signalling cascade and the formation of signalling platforms perhaps is in conjunction with the signalling potential of the B-cell receptor[CD20 TARGET].
3. Compound Screening, Lead Identification and Optimisation
STEP 1- Murine anti-CD20 monoclonal antibody production.
Balb/c mice were immunized with human lymphoblastoid cell line SB. Mice spleens were extracted with high serum titers of anti-CD 20 antibodies and then spleenocytes were fused with mouse myeloma. Radio-immunoassay was used to record all assays CD20 reactivity.
After purified anti-CD20 B1 were radiolabeled with I, the hybridomas were screened by co-incubation with I-B1in 1% bovine serum albumin (BSA), 100,000 SB cells and phosphate buffered saline for an hour at room temperature. Using a 96-well filter plate the cells were harvested. Those wells which showed more than 50% inhibitions were cloned and antibody with highest activity was derived named as B28.
STEP 2- Engineering anti-CD20 Ig DNA expression.
STEP 3- C2B8 was created by growing Chinese hamster ovary (CHO) cells using electroporation device. Protein-A affinity chromatography was used to purify chimeric monoclonal antibody from supernatant.
STEP 4- High Throughput Screening
Immunoreactivity and specificity of C2B8 was observed. The purified antibodies produced by CHO, radio-labelled were tested by binding to different target cells (CD20) and detected by flow cytometry using a FAC scan analyzer.
STEP 5- Evaluation of in-vitro effector function of C2B8 to detect that it binds to the human C1q component of the complementary system and initiate complement dependent cytotoxicity as well as a marked reactivity was observed in the assays of ADCC.
From the data of Human tissue cross reactivity, the anti-CD20 monoclonal antibody recognized the target CD20 antigen without destroying any other tissues except the B-cells[IB].
After the C2B8 was identified as a lead it was then optimized by using Structural Activity Relationship. Potency and toxicity assays were performed in order to confirm and optimize the lead, C2B8.
4. Preclinical Evaluation:
Drug metabolism- The route of administration is intravenous so the drug reaches the blood directly and onset of action is quick. The metabolism of Rituximab/ Rituxan is supposed to be same as that of IgG1 antibody as the entire structure of Rituximab is based on IgG1's structure.
Pharmacokinetics- Distribution of antibody (rituximab) in different parts of the body as well as the malignant lymph nodes and lymphoma may affect the sensitivity of the drug. As depletion of CD20 takes place either in lymphatic tissues or in blood Rituximab is distributed outside the vascular space[IB]. Tissue penetration of the drug may or may not be dose dependent but it depends on the target density, antibody affinity and diffusion characteristics of the antibody[IB,4]. In contrast to IgG1 molecules, Rituximab crosses the placental barrier and is excreted into milk[IB]. The free circulating antibodies enters the metabolic pathway of IgG while on the other hand the bound ones will be destroyed and eliminated from the body[IB].
In-vivo studies suggests that Rituximab has a long half-life (61-311hours)[IB]. The clearance of the drug mainly depends on the CD20 B-cells as binding to CD20 free B-cell will decrease the amount of free Rituximab[IB].
Toxicology- Single-dose toxicology in monkeys shows that Rituximab at maximum dose of 100mg/kg no toxicity as well as repeat-dose toxicology at maximum dose of 20mg/kg weekly is non-toxic. Although monkebservedys developed an anti-rituximab response when subjected to repeat-dose toxicology for 4 or 8 weekly doses of 20mg/kg. In the reproductive toxic studies, no toxic effects were observed in the pregnant monkeys at doses up to 100mg/kg during organogenesis[IB].
Invitro pharmacology- Various invitro studies show that Rituximab has high specificity for CD20 antigen present on B-lymphocytes and it binds to the CD20 receptor with an affinity of 5.2-11nM. Exact mechanism of Rituxan is not clear but based on evidences from invitro studies it acts via three different processes. These are CDC, ADCC or apoptosis induction[IB].
From Human tissue cross reactivity studies, Rituximab has a very restricted pattern of distribution as it recognizes CD20 antigen. Immunoreactivity was seen in large number of cells including bone marrow, spleen, lymph node, peripheral blood and lymphoid follicles leaving the stem cells and the plasma cells[IB]. The drug does not cause any harm to other normal tissues[IB].
Invivo pharmacology- The onset of action of CD20 B-cell depletion was immediate in the peripheral blood flow after single and repeat-dose of Rituximab was observed from the invivo experiments[IB]. More than 95% of peripheral B-cell depletion took place in almost all the cynomologous monkeys. Within a short course of treatment B-cell depletion was observed in the lymphatic system (mandibular lymph nodes, spleen and submucosal lymphoid tissues). From the observation, the B-cell depletion in the lymphoid tissues and peripheral blood appeared to be dose and time depedent. Recovery of the B-cells varied differently and did not show any dependability on the doses. Rituximab has high specificity to bind with CD20 B-cells and B-cells, so only these cells were destroyed and depleted. The T-cells, the plasma cells and the stem cells were unaffected by the drug[IB].
5. Clinical Evaluation (non-hodgkin's lymphoma[IB]):
The mean AUC as well as the mean maximum concentration were directly proportional to the increase in dose. There were no significant effects of age or sex on the pharmacokinetics of rituximab. The volume of distribution was small. The pharmacokinetics was almost the same between the Japanese and non-Japanese patients. The mean serum half-life of rituximab was short after first administration when compared after subsequent administrations. The mean clearance was greater after first infusion than after subsequent infusions. Moderate to high variability for AUC was observed.
In 83% of patients with indolent NHL, a significant decline in the peripheral blood cell (B cell) counts was observed within the first three infusions along with sustained depletion for about 6-9 months.
Recovery of the B-cell was seen after 6 months from the completion of the treatment.
Rituximab when given as monotherapy or combination therapy in different stages of Non-Hodgkin's produces varied effects.
EFFICACY IN INDOLENT NHL[MAB THER]:
From a six year follow-up study (rituximab along with Cyclophosphamide, Doxorubicin, Vincristine and prednisone) showed that 55% were in remission between 4 an seven years. ORR in patients who completed the treatment was around 100%.
D. SUMMARY AND CONCLUSIONS:
Targeted anti cancer therapy using anti bodies have become one of the top level strategies in the field of oncology. The credit goes to the ability of the therapeutic anti bodies to bind to the cancerous cells. This in turn creates high specificity and affinity of the anti bodies to target specific antigens. Rituximab was the first therapeutic monoclonal anti body approved by USFDA in the year 1997, has proved to be beneficial in the treatment of Non-Hodkin's Lymphoma, Rheumatoid Arthritis, Chronic Lymphocytic Leukemia and some other auto immune diseases[Targeted 2004]. This monoclonal antibody targets the CD20 antigen present on the B-Cell Lymphocytes. In spite of the positive outcomes of the CD20 targeted anti cancerous therapy with rituximab in B-Cell Lymphomas there is an issue of resistance to the drug. This resistance usually results in non responsiveness and early relapse of the Lymphoma. It is very important to identify the importance of resistance which will help to overcome the non responsiveness. Many investigations in this field have led to the development of second and third generation anti bodies [CD20 Target].