A Preclinical Evaluation Of Rituximabs Effectiveness

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Non-Hodgkin's Lymphoma is defined as large group of cancers of lymphocytes (white blood cells) affecting the lymphatic system of the body. Non-Hodgkin lymphomas can occur at any age and symptoms are lymph nodes that are larger than normal, fever, and weight loss etc.

There are many different types of non-Hodgkin lymphoma. These types can be classified into aggressive (ie fast-growing) and indolent (ie slow-growing) types, and they can be formed from either B-cells or T-cells.

Lymphomas that arise after bone marrow or stem cell transplantation are generally B-cell non-Hodgkin lymphomas. Prognosis and treatment depends on the stage and type of disease.

Around 10,300 people are diagnosed with non-Hodgkin lymphoma in UK annually. 65,540 new cases and 20,210 deaths from non-Hodgkin lymphoma are estimated in the United States as of 2010.

2) Rheumatoid Arthritis:

It can be defined as an autoimmune disease causing chronic inflammation of the joint.

It can also cause inflammation of the tissue around the joints, as well as in other organs in the body.

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Autoimmune diseases occur when the body's tissues are mistakenly attacked by their own immune system.

Every year there are approximately 20,000 new cases of rheumatoid arthritis in the UK. There are around 400,000 adults in the UK with rheumatoid arthritis as of 2010. Prevalence is more common in women than men by a ratio of 3:1.

3) Chronic Lymphocytic Leukemia:

Chronic Lymphocytic Leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder marked by a progressive accumulation of functionally incompetent lymphocytes

In the majority of patients the cells of origin with chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) are clonal B cells arrested in the B-cell differentiation pathway, intermediate between pre-B cells and mature B cells. Morphologically in the peripheral blood, these cells resemble mature lymphocytes.

According to FDA, the disease mostly affects people of age 50 and above . It is

diagnosed in some 16,000 people each year, causing about 4,400 deaths

http://www.cancer.gov/images/spacer.gif 

TARGET HISTORY:

Target or receptor of a drug is defined as any part of the cell, usually a large protein molecule, on the cell surface or in the cytoplasm with which a drug molecule interacts to generate a response or effect. Fundamentally any bond could be involved in the drug-receptor interaction.

An agonist is a drug which produces a stimulation type response. The agonist is a very close mimic and "fits" with the receptor site and therefore initiates a response.

An antagonist drug interacts with the receptor site and blocks or depresses the normal response for that receptor as it only partially fits the receptor site and cannot produce desired effect.

The target for Rituximab is CD20 antigen expressed on B lymphocytes. CD20 is a CD marker - a molecule on the cell surface that can be used to identify and type a particular cell in the body.

CD20 is a marker for B-cells, a type of white blood cell (WBC) which defends the body from infections. There are two types of WBCs - B-cells and T-cells. CD20 is present only on the surface of B-cells but not T-cells.

Testing for CD20 is done using a special technique called immunohistochemistry. It is a routine method to determine if an abnormal cancerous WBC is a B-cell or T-cell.

Treatment and prognosis for B-cell and T-cell lymphomas are different. New drugs called monoclonal antibodies have been developed that target cells with only specific CD markers on their surface. Many of them have proven advantage in the treatment of B-cell lymphomas that have CD20 on the surface of the cancer cells. Thus testing for CD20 is a very important part of lymphoma diagnosis.

The Fab domain of rituximab binds to the CD20 antigen on B lymphocytes and the Fc domain can take on immune effector functions to mediate lysis of B-cell. Possible mechanisms of effector-mediated cell lysis include complement-dependent cytotoxicity resulting from C1q binding, and antibody-dependent cellular cytotoxicity . Rituximab binding to CD20 antigen has been established to induce cell death via apoptosis.

DRUG BACKGROUND:

Rituximab (chimeric anti-CD20 monoclonal antibody; Ritixan or MabThera) is a chimeric murine/human monoclonal antibody that binds to CD20, a hydrophobic transmembrane protein that is present on B-lymphocytes, and eliminates these cells potentially via a number of different mechanisms.

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It is approved for the treatment of patients with relapsed or chemo resistant, low- grade or follicular, CD20+ , B-cell non-Hodgkin's lymphoma.

MECHANISM OF ACTION:

The pharmacokinetics of rituximab is dose dependent after a single infusion, exposure and area under curve increases with increasing dose (dose range: 50-500 mg/kg). The prime target of rituximab is CD20 antigen on the B-lymphocyte which results in the CD20 mediated clearance of drug after the first infusion. The Fab domain of rituximab binds to the CD20 antigen and the Fc domain can recruit immune effector functions to mediate B-cell lysis.

The mechanisms for the cell lysis are complement-dependent cytotoxicity resulting from C1q binding and the antibody-dependent cellular cytotoxicity mediated by one or more of the Fcγ receptors on the surface of granulocytes, macrophages , and natural killer cells.

Rituximab binding to CD20 antigen on the lymphocytes causes cell death via apoptosis. After depletion of B cells, this clearance pathway no longer exists. Also depletion of circulating B cells may trigger greater recirculation of IgG through

FcRn receptors and less elimination through Fcγ receptors as a means to maintain IgG homeostasis.

DRUG HISTORY:

It was approved in June 1998 by the EC, although launched previously in the US

( as Rituxan ), rituximab (formerly IDEC-C2B8) was the first anticancer monoclonal antibody, heralded an influx of the molecules into treatment regimens for several types of tumours.

It was originally discovered by IDEC (now Biogen Idec) and jointly developed along with Genentech, Roche and Zenyaku Kogyo, the MAb targets the CD20 protein that is expressed on over 95 per cent of B-cell lymphomas.

It was the first monoclonal antibody therapy to exhibit a clinically-significant anti-tumour response, which finally provided proof of principle that these molecules could be efficacious, as well as specific and less toxic. These attributes has seen rituximab to have significant revenue, with sales of US$2,252 million and US$2,515 million in 2006 and 2007 respectively.

In 2001, RITUXAN received approval in the United States with a package insert expanded to include information on the administration weekly for 8 doses, for the treatment of bulky disease, and for 4 weekly doses as retreatment.

In 2006(February), it was approved for previously untreated DLBCL (diffuse, large B-cell lymphoma) in combination with CHOP or other anthracycline-based chemotherapy regimens.

In September 2006, RITUXAN received approval for the treatment of non-progressing (including stable disease), low-grade, CD20+ B-cell NHL, as a single agent, after first-line CVP chemotherapy. Also it got approved for previously untreated follicular, CD20+, B-cell NHL in combination with CVP chemotherapy

In February 2010, RITUXAN received approval for the treatment of patients with previously untreated and previously treated CD20-positive CLL in combination with fludarabine and cyclophosphamide.

PATENT STATUS:

Approved in June 1998 by the EC and launched earlier in the US, rituximab (formerly IDEC-C2B8) was the first anticancer MAb as the treatment regimens for multiple tumour types.

Sales of rituximab look set to remain at a steady level for the next five years.  It has been announced by Dr Reddy's to market Reditux (brand of rituximab) in other markets, also in US, in 2015 when the patent period of the product gets over.

MARKET SHARE:

Co-marketing of Rituxan is done by Genentech and Biogen Idec in the United States, and Roche markets MabThera in other countries, except Japan, as Rituxan is co-marketed by Chugai and Zenyaku Kogyo Co. Ltd.

40% or $156 million is Biogen's share of Rituxan's U.S. profits in the third quarter. These companies are working to get Rituxan approved to treat lupus and MS, which could offer a boost to their sales.

However under the agreement between the companies, Biogen's portion of Rituxan profits will minimize by a quarter, to 30%, as soon as the companies launch a successor drug - this might take place in 2010 or 2011.Biogen's share of the new drug's profits would also be 30%.

According to recent news, Biogen Idec's share of the co-promotion profits of RITUXAN will decrease from 40% to 35% due to developmental cost incurred for GA101 drug.

DRUG DISCOVERY PROCESS:

(no information was available for this section)

TARGET IDENTIFICATION:

It is the process by which various methods are used by the scientists to identify and isolate the targets associated with the disease and learn about its functions and how these can affect disease. Based on this method various compounds are identified that may have interaction with these targets and helpful in treatment of a specific disease.

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The identification for tumor-specific antigens that could serve as targets for antibody-mediated attack on cancer cells has been mainly inadequate till date, although genomic and proteomic approaches give the promise of the future identification of such tumorspecific proteins

It was observed that CD20 is a transmembrane surface antigen that appears to be involved in the regulation of B-cell growth and differentiation, possibly by functioning as a calcium channel (Janas et al 2005; Li et al 2003). The CD20 antigen has a number of properties that make it an attractive target for monoclonal antibody therapy in NHL.

TARGET VALIDATION:

This procedure is carried on to select the most useful target by analyzing and comparing each drug target to others based on their association with a specific disease and their ability to regulate biological and chemical process/molecules in the body.

These studies generate data to identify compounds that have an effect on the target selected.

B-cell-specific markers (CD20) could be targeted and would not involve individualized development. Rituximab targets CD20, a transmembrane protein present on virtually all B cells from the stage at which they become committed to B-cell development until it is down regulated when they differentiate into antibody-secreting plasma cell. Thus, CD20 was considered a pan-B-cell antigenic marker.

LEAD IDENTIFICATION:

A lead molecule is the one that has potential to treat a disease. A standard drug is compared with the new molecules to confirm their successful effect on the drug target. Evaluation is also carried on each of these molecules.

Out of many compounds, rituximab was observed to have a specific binding to the CD20 antigen with high affinity and inducing depletion and death of these cells via several mechanisms.

LEAD OPTIMIZATION:

In this step, various lead compounds are assessed to choose the one which

has the greatest potential to get developed into safe and effective

medicines.

Therefore CD20 antigen and anti-CD20 monoclonal antibodies produced were isolated and characterized. Out of them the murine IDEC-2B8 was chosen for process of chimerization (to develop a therapeutic antibody that can be safely injected into humans).The chimeric monoclonal antibody was preferred in order to prevent the immunogenicity of the murine parent monoclonal antibody to utilize a human framework which allowed for complement and effector cell binding and to selectively result into β-cell depletion.

The technology that was necessary for humanization was not yet fully developed at that time and thus was not followed. A vector was so engineered that facilitated high Mab yields when inserted in CHO (Chinese hamster ovarian) cells and used in a high volume fermentation process.(Sufficient information was not available for this section)

PRECLINICAL EVALUATION:

PHARMACOKINETICS OF RITUXIMAB-

The distribution and metabolism of rituximab is believed to be similar as the endogenous IgG1 molecules with exception that it has specific distribution to CD20 antigen-bearing cells.

Invivo pharmacology studies described that rituximab distributes outside the vascular space as depletion of CD20+ cells occurs in the lymphatic tissues and the blood. It is expected that rituximab is transported through the placental barrier and excreted into milk in which physiologically low levels of IgG can be measured.

Free circulating rituximab enters the metabolic pathway of endogenous soluble IgG and rituximab bound to CD20+ lymphocytes will be phagocytosed, together with destroyed B cells, by infiltrating macrophages and granulocytes.

The clearance pathway of rituximab is minimal after the depletion of circulating cells. Pharmacokinetics for single and two IV doses in cynomolgus monkeys are shown in the table below:

PK

Single doses(10,30, & 100mg/kg)

Two doses (0.05,0.2,0.5,2 &10 mg/kg)

Clearance

-

21.9+/-5.86mL/day for 10 mg/kg

Volume of distribution

239-281 µg/ml

3970+/-1220mL for lowest dose

Half-life

61-311 hours

154+/-43.9 hours

There exist regulatory mechanisms that may contribute to the longer half-life of IgG 1 antibodies which results from recycling through the FcRn receptor and depletion of B-cells maintains IgG homeostasis.

Overall it can be summarized that the disposition of rituximab in monkeys is dependent on antibody- specific and non-specific processes. Also clearance is partially dependent on circulating b cells.

NON-CLINICAL TOXICOLOGY:

A Good Laboratory Practice single-dose, repeat-dose and reproductive studies were conducted in monkeys by administering rituximab intravenously at various doses. In single dose toxicity, there were no treatment related effects on mortality, body weight, and food consumption or body temperature. There was a transient and mild decrease in platelet count, lymphocyte ratio and increase in neutrophil ratio all animals 1 day after the administration with mild hematologic changes. Therefore rituximab was non-toxic at the maximum dose of 100mg/kg in single -dose toxicology study.

In repeat-dose study, dose range was 0-20 mg/kg with vehicle control group of 1 male and 1 female per group and treatment groups of 3 males and 3 females per group. There were no treatment related effects but incidence of vomiting in 3 of treated females. The expected pharmacologic effects of rituximab were consistent with decreased B-cells. Some monkeys developed an anti-rituximab response for 4 or 8 weekly doses of 20mg/kg.

Dose range were 20, 40, 100 mg/kg given weekly for reproductive toxicity studies to pregnant female monkeys. Rituximab was well tolerated not resulting into embryotoxicity or teratogenicity.

NON-CLINICAL PHARMACODYNAMICS:

It was evident in the in-vitro studies that rituximab binds to CD20 antigen expressed on primate b lymphoma cell lined and primary malignant and non-malignant B-cells, with high specificity and affinity of 5.2-11nM.This results in CD20+ B-cell elimination by mechanisms of antibody-dependent cellular cytotoxicity , complement-dependent cytotoxicity and direct effects. However individual contribution to apoptosis is not known.

Human tissue cross-reactivity studies shows that rituximab is specific to CD20 antigen and immune-reactivity is seen in subset of cells in bone marrow, peripheral blood etc.

Rituximab does not recognize or promote destruction of any normal human tissues.

In-vivo studied in cynomolgus monkeys demonstrated that there is immediate onset of CD20+ B-cell depletion (>95%) in peripheral blood. This depletion is both time and dose dependent and T-cells numbers are unaffected by rituximab. After stopping the treatment, a normal B-cell pool can be reconstituted from precursor cells.

CLINICAL EVALUATION:

The mainstreams of pharmacokinetic and pharmacodynamic studies have been performed in patients with B-cell lymphoma. In 9 patients given 375 mg/m2 as an IV infusion for four doses, the mean serum half-life was 59.8 hour(range 11.1 -104.6 hours) after the first infusion and 174 h(range 26 - 442 h) after the fourth infusion.

The serum concentration of rituximab was inversely associated with tumor burden directly associated with response.

The wide range of half-lives therefore be a sign of the inconsistent tumor burden among patients and the changes in CD20+ (normal and malignant) B-cell populations upon repeated administrations.

Rituximab can be detected in the serum for many months after the dose of drug. This persistence of rituximab in the serum has inferences for cross-match and tissue typing analyses. Since rituximab is cytotoxic when complements are present, sera that contain rituximab would produce a positive B-cell cytotoxic-positive cross match.

The human portion of the IgG1 would provide a target for the anti-human Ig fluorochromes used in flow cytometry. Flow and cytotoxic cross-matches and

PRA (panel reactive antibody) determinations, however, can be successfully done after either elimination of the cell surface CD20 by pronase treatment of the cells or removal of the circulating rituximab by immune-magnetic bead absorption.

The main toxicities reported with rituximab as monotherapy and in combination with chemotherapy are infusion- related reactions and associated with first infusion. Some of them are hypotension, fever, chills, rigors etc.

SUMMARY :

Rituximab (chimeric anti-monoclonal antibody) is a chimeric mirine/human monoclonal antibody that binds to CD20 antigen on B lymphocytes and eliminates these cells potentially via a number of different mechanisms. It was discovered by IDEC (now Biogen Idec) and jointly developed along with Genentech, Roche and Zenyaku Kogyo. Rituximab is mainly indicated for the cases of lymphoma, chronic lymphocytic leukemia and rheumatoid arthritis however further clinical studies have been conducted for use of this drug in many other oncology indications and autoimmune diseases, example- rheumatoid arthritis

It is used in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) and CVP (cyclophosphamide, vincristine, and prednisolone).In the European Union and other countries, it is also indicated as maintenance therapy for relapsed/refractory follicular lymphoma.

Limited data are available for use of rituximab in other malignancies such as multiple myeloma or post-transplant lymphoproliferative disorder and for non-malignant hematologic disorders such as cold hemagglutinin disease or autoimmune hemolytic anemia.

It is evident from the non-clinical studies that rituximab binds specifically to CD20 antigens on B-cells. In-vitro, binding of rituximab promotes complement-mediated lysis, antibody-dependent cell-mediated killing of CD20+ cells and apoptosis. In human tissue cross-reactivity studies, rituximab did not recognize or promote destruction of any other normal human tissues. Rituximab was well tolerated in cynomolgus monkeys, and no significant toxicologic effects were observed.

CONCLUSION:

From all the data available about rituximab, it can be concluded that rituximab is no less than a boon for humankind and a significant merge to our therapeutic armamentarium as it is one of the potential monoclonal antibody that is successful in combating life-threatening diseases. Also an improved humanized version of rituximab that can decrease the chances of development of neutralizing human antibodies against the chimeric murine portion of the molecule (HACA) and allow chronic dosing is currently in clinical trials for rheumatoid arthritis. Thus deep interest in B cells and antibody in transplant is essential for application of carefully designed clinical trials. Such trials promoting potential life-saving drugs are really required.