Oral Iron Therapy


Iron deficiency anaemia is treated by iron supplementation that may be administered orally or parenterally.  Oral iron absorption is inefficient, is interfered by food and is associated with high incidence of gastrointestinal adverse effects. About 17% of the world population is estimated to be suffering from iron deficiency. Iron deficiency is more common the poorer regions of the world. These regions have limited resources allocated for healthcare. Oral iron is inexpensive and convenient to administer making it the modality of choice for initiation of treatment of iron deficiency. The availability of safer parenteral iron preparations that can replete the iron deficit in one dose has reduced the threshold of switching a person intolerant to iron to parenteral iron.

Oral Iron Absorption

Dietary iron may be heme iron or non-heme iron. Heme iron is absorbed after oxidation to hematin. The absorption of heme iron is more efficient. Dietary non-heme iron is present in the ferric state. Ferric iron is insoluble and needs to be reduced to ferrous iron. Gastric acidity aids this conversion. Medicinal iron is most often administered in the ferrous state. Ferrous iron tends to oxidize to ferric iron at physiological pH. Gastric acid lowers the pH and retards oxidation. The absorption of non-heme iron is aided by ascorbate, animal proteins, human milk, keto sugars, organics, amino acids that form soluble chelates and retarded by phytates present in grains and vegetables, dietary fibre, polyphemols present in tea, coffee and wine, phosphates and phosphoproteins present in egg yolk, bovine milk, calcium and zinc. For a detailed discussion on iron absorption see intestinal iron absorption. Preparations containing iron in the ferric state have a 3-4 fold lower bioavailability than ferrous iron preparations. Ferric iron is insoluble in the alkaline medium of the duodenum and needs to be converted to ferrous iron (ScientificWorldJournal. 2012; 2012: 846824).

Oral Iron Preperations

Oral iron preparations may be heme or non-heme. Heme iron is available as heme iron polymer. Non-heme iron may be in the ferrous or ferric form. Carbonyl is pure iron prepared from the decomposition of iron pentacarbonyl. The preparations are listed in the table below.

 

Preparations Iron Content
Ferrous Sulfate, anhydrous 30%
Ferrous Fumarate 33%
Ferrous Sulfate 20%
Ferrous carbonate, anhydrous 48%
Ferrous Gluconate 12%
Ferric Ammonium Citrate 18%
Ferric bisglycinate 20%
Ferric pyrophosphate 12%
Carbonyl iron ~100%
Heme-iron peptide 100%
Polysaccharide iron complex 100%

Indications of Oral Iron Therapy

Oral iron is indicated for prevention and treatment of iron deficiency anaemia. The rate of iron delivery is insufficient to provide iron when erythropoiesis is stimulated by erythrocyte stimulating agents like erythropoietin and darbepoetin. Oral iron should not be used for such patients.

Trial of Oral Iron Therapy

Serum ferritin is an indicator of total body iron. It is also an acute phase reactant. Low ferritin indicates iron deficiency. The traditional cutoff is 12ng/mL. At this cutoff the sensitivity of ferritin for the diagnosis of iron deficiency anaemia is only 25%. The sensitivity can be increased to 92% with a positive predictive value of 83% if a cutoff of 30ng/mL is used. A trial of oral iron may be given if other causes of anaemia are excluded.

Contraindications to Oral Iron Therapy

  1. Primary hemachromatosis: Primary hemochromatosis is a is absolute contraindication
  2. Peptic ulcer, regional enteritis, or ulcerative colitis can be exacerbated by oral iron.
  3. β-Thalassaemia trait is a relative contraindication. Some patients may develop iron overload. Patients should be given iron only if iron deficiency is established by laboratory investigation.

Failure of Oral Iron Therapy

  1. Failure to take prescribed medication: Oral iron therapy causes gastrointestinal adverse effects in a large proportion patients that are severe enough in some to discontinue therapy. A detailed history must be taken to ascertain that the patient has taken the prescribed dose.
  2. Incorrect or incomplete diagnosis: Iron deficiency anaemia is hypochromic microcytic (see Evaluating Anaemia). Other diseases that result in hypochromic microcytic anaemia are thalassaemia and anaemia of chronic disease. Both are common and can both be confused with iron deficiency as well as co-exist with iron deficiency. Thalassemia trait affects about 1.5% of the world population. About 1.4% of the population is estimated to have anaemia due to infection, inflammation or chronic renal disease (https://dx.doi.org/10.1016%2FS0140-6736(15)60692-4). Incidental occurrence of iron deficiency with thalassaemia trait or anaemia of chronic disease will result in an incomplete response to iron supplementation. Some inflammatory conditions e.g. ulcerative colitis cause blood loss. Blood loss may be seen due to use of non-steroidal inflammatory agents in patients with autoimmune arthritis. Anaemia in these patients shows an incomplete response to iron supplementation because part of the anaemia results from chronic inflammation.
  3. Insufficient amount or inappropriately taken oral iron: The ideal dose is 200mg of elemental iron taken 2 hours before or 1 hour after food. Food interferes with iron absorption but also relieves gastrointestinal adverse effects. Gastrointestinal adverse effects are related to dose. Prescriptions of oral iron may have an insufficient dose or may be administered after food to reduce gastrointestinal adverse effects. This results in an inappropriate haemoglobin response.
  4. Iron demand exceeding intake: Iron demand may exceed supply if there is continued blood loss or in patients where erythropoiesis is stimulated with an erythropoiesis stimulating agent like erythropoietin or darbepoietin. In both these situations the rate of oral iron absorption limits iron availability. These patients need intravenous iron.
  5. Malabsorption of iron: Food interferes with oral iron absorption. Ferrous iron is rapidly oxidised to ferric iron at physiological pH. Gastric acid reduces ferric iron to ferrous iron. Proton pump inhibitors, H2 antagonists, antacids and gastrectomy reduce acidity and can interfere with iron absorption. Iron malabsorption may be seen as part of a malabsorption syndrome. Iron deficiency refractory to iron is rare. Iron refractory iron deficiency anaemia is a disorder resulting from mutations in the TMPRSS6. This mutations results in increase hepcidin production which is sensed by the body as an iron repeated state. Iron is not absorbed despite iron deficiency. The patients do not respond to oral iron and shows a partial response to parenteral iron.

 Interactions

  1. Interaction of iron with food: The absorption of non-heme iron is affected by food (See Intestinal Iron Absorption).
    1. Foods enhancing iron absorption: Ascorbate, animal proteins, human milk, keto sugars, organics, amino acids that form soluble chelates with iron enhance absorption of non-heme iron.
    2. Inhibiting iron absorption: `Inhibitors of absorption of non-heme iron include
      1. Phytates present in grains and vegetables
      2. Dietary fibre
      3. Polypohenols present in tea, coffee and wine,
      4. Phosphates and phosphoproteins present in egg yolk, bovine milk
      5. Calcium and zinc.
  2. Drug-Iron interactions
    1. Iron decreases the absorption of ACE inhibitors, bisphosphonates, levodopa, levothyroxine, penicillamine, quinolone and tetracyclines
    2. The absorption of iron is decreased by drugs that reduce gastric pH. These include H2 antagonists(Cimetidine, ranitidine, famotidine), proton pump inhibitors (omeprazole, pantoprqzole, esomeprazole, lansoprazole, rabeprazole, etc), antacids and cholesterol lowering agents (Cholestyramine and Colestipol).

Dose

  1. Children: 3–6 mg/kg daily in 3 divided doses.
  2. Adult: Usual therapeutic dosage: 50–100 mg 3 times daily but a dose of 200mg produces the maximal results. A smaller dosages (e.g. 60–120 mg daily) may be given if patients are intolerant of oral iron, but response in such patients takes a longer time.

Side Effects Of Oral Iron

  1. Gastrointestinal: The commonest adverse effects of iron are gastrointestinal symptoms, including heartburn, nausea, abdominal cramps, diarrhoea or constipation. These may be seen in up to a fifth of the patients. They can be reduced by decreasing the dose or taking iron after food. Taking iron with food can reduce the absorption by about 50%. Enteric coated preparation decrease the side effects by delaying the release of iron. Delaying release may bypass the duodenum that is the site of absorption of iron. The decrease in absorption is particularly marked in patients with aclorhydria as they can not dissolve the enteric coating. The stool of patients taking iron supplements may be discoloured black or green.
  2. Discolouration of teeth: Iron syrups may cause staining to teeth.
  3. Iron overload: Iron overload from oral iron therapy is rare. It has been described in patients with hemachromatosis and chronic haemolytic anaemias.
  4. Iron Poisoning: Iron poisoning usually occurs in children, particularly those younger than 5 years of age, because of accidental ingestion of medicinal iron. Children are likely to ingest these believing them to be candies.

Granulocyte Colony Stimulating Factor (G-CSF)


Neutropenia is a dose limiting toxicity of chemotherapy. It results in delay and dose reduction both of which adversely affect outcomes of treatment. Myeloid growth factors are biological agents that stimulate the production go granulocytes and offset the myelosupressive effect of chemotherapy. Two myeloid growth factors are available Granulocytic colony stimulating factor (G-CSF) and granulocytic monocytic colony simulating factor (GM-CSF). This article will discuss G-CSF as it is used more often than GM-CSF. Commertially available G-CSF is made by recombinant DNA technology and may be produced in E. coli (Filgrastim) or chinese hamster ovary cell lines (lenograstim). The half life of filgrastim can be increased by covalently linking it to polyethylene glycol (PEG) and converting it to pegfilgrastim.

Mechanism of Action of G-CSF

G-CSF is a 174 amino acid peptide the gene for which is on chromosome 17. It has a molecular weight of 18kDa. It is produced by monocytes, macrophages, fibroblasts, endothelial cells and keratinocytes in response to inflammatory cytokines and bacterial endotoxin.

G-CSF acts via the G-CSF receptor. G-CSF receptor is a transmembrane receptor that form a homodimer on binding G-CSF. Activation of G-CSF receptor results in activations of  JAK/STAT, SRC family of kinases, PI3/AKT and Ras/ERK 1/2. The details of the pathway are not completely understood.

G-CSF AAB.002

Activations of G-CSF has the following effects that lead to increased production of neutrophils

  1. Increased Proliferation of Neutrophilic precursors
  2. Shortened neutrophilic precursor bone marrow transit time
  3. Functions maturation of neutrophils – increased chemotaxis, phagocytosis and antibody dependent cytotoxicity

G-CSFs Available for Clinical Use

G-CSFs for clinical use is manufactured by recombinant DNA technology. Two molecules are available for clinical use. Filgrastim is produced using E. coli and lenograstim is obtained from Chinese hamster ovarian cells. Lenograstim is glycosylated (4% glycosylation).

Filgrastim on subcutaneous administration filgrastim has a half life of 2.5-5.8 hours. The drug is eliminated by uptake be G-CSF receptors on neutrophils and glomerular filtration. Pegylation, that involves attaching a 20kDa polyethylene glycol (PEG) molecule to the N terminal eliminates renal elimination prolonging the half life to 27-47 hours. The product, pegfilgrastim, is only eliminated by binding to neutrophil G-CSF receptors, patients with low neutrophil counts have a lower clearance. Prevention of glomerular filtration allows administration of pegfilgrastim only once in a chemotherapy cycle.

Indications for G-CSF

The discussion that follows applies to filgrastim and perfilgrastim as these drugs are used more commonly than lenograstim. The general principle apply to lenograstim but readers are advised to refer to information on lenograstim for details of use and adverse effects.

  1. Primary prevention of febrile neutropenia (FN) in patients with non-Myeloid malignancy on chemotherapy: Patients where on chemotherapy protocols that have a risk of febrile neutropenia equal to or greater than 20% should be administered G-CSF.
  2. Prevention of recurrence of febrile neutropenia: G-CFS may be used to prevent recurrence of febrile neutropenia in patients who have had an episode of infection in a previous chemotherapy cycle.
  3. Treatment of patients with febrile neutropenia: Initiating therapy with G-CSF after febrile neutropenia has set in has not been shown to decrease mortality of antibiotic use. It may however be used in patients who are at high risk of mortality.
  4. Mobilisation of stem cells for stem cell transplant
  5. Use in patients with myeloid malignancies: There is an apprehension that G-CSF may stimulate leukaemia cells and G-CSF is not used in induction. It may however be used after induction to reduct the duration of neutropenia.

Filgrastim is administered in a dose of 5μg/kg/day subcutaneously, by a short iv infusion or prolonged intravenous infection. therapy should be initiated at least 24 hours after the  chemotherapy. The adult dose of pegfilgrastim is 6mg. The paediatric dose depends on the weight of the child. Children less than 10 kg: 0.1 mg/kg, those between 10 to 20 kg be administered 1.5 mg, between 21 to 30 kg be administered 2.5 mg and between 31 to 44 kg administered 4 mg. Children weighing 45kg or more should be administered the adult dose of 6 mg. Pegfilgrastim should not be administered less than 14 days after a cycle of chemotherapy. It should be administered more than 24 hours after a cycle of chemotherapy.

Adverse Effects

  1. Bone Pain: Bone pain is the commonest side effect with about 20-30% of the patients suffering the side effect.
  2. Rare but serous side effects include splenic rupture, acute respiratory distress syndrome,  precipitation of sickle cell crisis and capillary leak syndrome

Chronic Myeloid Leukaemia



Chronic myeloid leukaemia (CML) is a myeloproliferative disorder characterised by anaemia, leucocytosis and splenomegaly. The natural history of CML is characterised by three phases chronic phase, accelerated phase (AP) and blast phase (BP). CML is characterised by the presence of fusion gene BCR-ABL1 that results from the t(9;22) translocation. This gene encodes for a constitutionally active tyrosine kinase that has been shown to drive the CML stem cells. The diagnosis of CML can not be made in the absence of this gene.  Onset of AP signals a change in the biological behaviour of disease. The disease follows a more agressive course that culminates in blast phase. The blast phase mresembles an acute leukaemia and is a terminal event in the natural history of CML. Inhibitors of BCR-ABL1 prevent the emergence of accelerated and blast phase and have dramatically improved the outcome of CML.

Etiology and Epidemiology

CML constitutes about 15% of all leukaemias. The incidence increase with age and the disease is slightly more common in males. The incidence of the disease does not show geographic variation. The only know etiological factor for CML is exposure to radiation.

Pathogenesis

CML is characterised by the presence of Philedelphia chromosome [t(9;22)(q34;q11)] which results from a reciprocal translocation between the long arms of chromosomes 9 and 22 (figure 1). The ABL1 proto-oncogene is located on chromosome 9 at q34. Chromosome 22 has the BCR gene at 22q11. The ABL1 gene translocates downstream to the BCR gene as a result of the t(9;22)(q34;q11) translocation. This results in the formation of the BCR-ABL1 fusion gene (see The BCR-ABL1 Gene).

Molecular Biology of CML

 

The expression of the ABL1 tyrosine kinase is tightly regulated. The t(9;22)(q34;q11) results in the N terminal segment of the ABL1 gene being replaced by that of the  BCR gene. This results in the ABL1 tyrosine kinase being constitutively expressed. The size of the N terminal amino acids contributed by BCR determine the length and the clinical properties of the fusion gene.

A model of pathogenesis of CML has to account for three features of the disease

  1. Uncontrolled proliferation of leucocytes accompanied sometimes with the proliferation of platelets
  2. Progression from a relatively stable phase of proliferation, the chronic phase, to a agressive  phase charecterized by increasing leucocytes counts, anaemia and falling platelet counts ultimately culminating in a acute leukaemia like picture, the blast phase. Unlike the chronic phase that shows myeloid and sometimes platelet proliferation, the blast phase may show a myeloid, lymphoid or rarely megakaryocytic lineage.
  3. Failure of tyrosine kinase inhibitors to errdicate the malignant clone despite pronounced and prolonged supression of the BCR-ABL1 positive clone.

The precise mechanism how BCR-ABL1 leads to chronic myeloid leukaemia is not known. Activation of phosphatidylinositol kinase (PI3K), RAS/Mitogen activated protein kinase (RAS/MAPK) and JAK/STAT pathway has been demonstrated in BCR-ABL1 positive cells. These pathways have been implicated in malignant transformation of cells and are believed to be responsible for the malignant phenotype of CML. Progression of CML from chronic phase to accelerated phase and eventually blast phase marks change in the disease that makes it progressively less responsive to drugs inhibiting BCR-ABL1 kinase. Cells with BCR-ABL1 fusion gene have an increase in the reactive oxygen species predisposing them to DNA damage. Progression from chronic phase to acclerated phase and eventually blast phase is associated with the cell accquiring additional mutations. This is known as clonal evolution. Reactive oxygen species induced DNA damage is believed to result in clonal evolution which results in progression to accelerated phase and blast phase resulting in treatment failure.

CML Pathogenesis-600px

Imatinib, an inhibiter of ABL1 tyrosine kinase has been in use for over a decade. Treatment of CML patients with Imatinib results in normalisation of blood counts, regression of splenomegaly and a decrease in the number of cells showing BCR-ABL1 fusion gene. The BCR-ABL1 kinase expressing cells are suppresses to undetectable levels in about 40% of the patients creating an impression that disease has been eradicated. If treatment is discontinued in such patients cells expressing BCR-ABL1 fusion reappear in 50-60% of such patients. These observations have been interpreted as suggesting that the CML stem cells have mechanisms that survive inhibition by Imatinib and other inhibitors of ABL1 tyrosine kinase. Studies to identify and target these pathways to erradicate CML stem cells with an goal to curing CML are undertaken. Some of the pathways that have shown a promise are Alox5pathway, the sonic hedgehog pathway (SHH), the Wnt/β-catenin pathway, the JAK/STAT pathway, the TGF-Beta/FOXO/BCL-6 pathway (Stem Cells International Volume 2013 (2013),  Article ID 724360, 12 pages)

Clinical Manifestations

Presentations

The manifestation of CML included

  1. Anaemia
  2. Fatigue, weight loss, fever, night sweats because of a hyper metabolic state induces by high leucocyte counts
  3. Bone pain
  4. Abdominal pain, early satiety and fullness because of splenomegaly. The pain is usually a tugging pain but sharp pain may indicate a splenic infarct. Splenic rupture though described is a rare event.
  5. Leucostasis because of very high white blood cell counts may present with neurological deficits, respiratory insufficiency or priapism.
  6. Incidental discovery for a haemogram performed for another reason is becoming a common presentation in populations that have a good healthcare system.

Splenomegaly is commonly seen. Historically CML is associated with massive splenomegaly (splenomegaly below the umbilicus). Today this may be seen only in communities with suboptimal health care and diagnosis is delayed. Hepatomegaly may be seen in some patients.

CML-Phases

Stages of Disease

CML has three stages, chronic Phase, accelerated phase and blast phase. Before the introduction of definitive therapy (Initially interferon, currently inhibitors of BCR-ABL1 kinase) every patient progressed from chronic phase to accelerated phase and finally to blast phase. The blast phase was terminal. The WHO definitions of the phases are listed in table 1. Acclerated phase definition is defined differently by different authors. Definitions may differ in the details but recognise the following

  1. Increasing WBC counts with appearance of new anomalies. These may be basophilia, eosinophilia or increase in the immature forms particularly blasts and promyelocytes. Progression of patients on BCR-ABL1 kinase inhibitors is accompanied by appearance mutations in BCR-ABL1.
  2. Increasing splenomegaly

The TKIs have dramatically reduced the rate of progression to accelerated phase or blast crisis. 4.6% for Dasatinib (DASSISION trial 5 year follow up), 3.5% for nilotinib (ENESTnd)and 8% imatinob (IRIS trial 8 Year follow up). Patients who do not achieve an early response to TKIs have a higher risk of progression.

Investigation

Haemogram

A complete haemogram should be performed in all patients. Patients of CML have leucocytosis with shift to the left and often have eosinophilia and/or basophilia. Platelet counts are often slightly increased but may be normal high or occasionally low. There is mild to moderate anaemia. The differential count of peripheral blood depends on the phase of disease. Patients with chronic phase have <10% blasts and ≤ 20% basophilis. Patients with accelerated phase have 10-19% blasts or >20% basophilic. Patients with blast crisis have ≥20% blasts.

Bone Marrow

Bone marrow studies are not needed for diagnosis of CML. This can be made by demonstration of BCR-ABL1 on peripheral blood. Bone marrow studies are essential for determining the phase of disease. The bone marrow of a patients of chronic phase of CML shows a high myeloid to erythroid ratio with a normal myeloid maturation. Dysplasia is not a feature of CML and suggests the diagnosis of myeloproliferative disease/mydlodysplasia overlap.  The chronic phase is characterised by <10% blasts. The megakaryocytes are smaller with reduced lobulation and may be increased in number. Bone marrow of patients from accelerated phase show myeloid hyperplasia with myelodysplasia and a blast percentage between 10-19%. Megakaryocytes may be seen in clusters or sheets. Bone marrow from patients with blast phase shows ≥20% blasts.

Risk Scores

Risk scores help in prognosticating the patients and should be performed at diagnosis. The three scores are listed in table 2. Hasford and Sokal scores are prognostic scores for predicting outcomes of CML patients. The  EUTOS score was developed to predict the outcome of patients receiving imatinib at 18 months of therapy and was reported to perform better than Sokal and Hasford scores. It has not been validated by other investigators.

CML Prognostic Scores

Table 2. CML Prognostic (Risk) Scores

 

Treatment

The approval of imatinib in May 2001 by the US FDA saw CML become the first disease to benefit from targeted therapy. The 8 year survival of patients in chronic phase has improved from 6% in 1975 to 42%-65% from 1983-2000 and 87% for patients diagnosed after 2001 (Blood 119:1981-87;2012). Before the introduction of imatinib the treatments used for patients of CML included (in chronological order) splenic radiation, arsenic, busulfan, hydrourea and a combination of interferon and cytarabine. With the exception of interferon and cytarabine none of the other therapies suppress the BCR-ABL1 positive clone.  The IRIS trial established imatinib, an inhibitor of BCR-ABL1 tyrosine kinase as the first line treatment for chronic myeloid leukaemia. DASSISION trial and the ENESTen trials established dasatinib and nilotinib as front line therapy.
 The aims of therapy is prevent progression of disease to accelerated phase and blast phase. This can be achieved by
  1. Inducing a haematological remission
  2. Inducing a molecular remission
  3. Eradicating the CML stem cell

Tyrosine kinase inhibitors (TKIs) are the first line of treatment in all patients of CML. The use of imatinib in pregnancy is associated with increase in malformations. These include malformations of the skeleton, respiratory system, kidney and gastrointestinal the gastrointestinal tract. The risk of exompholous is increased by almost 1000 times. Imatinib and other TKIs are contraindicated in pregnancy. The treatment of pregnant woman is challenging. Interferon may be used after the period of organogenesis. Women who have not completed their family are encouraged to do so early and should be switched to TKI. Women in the childbearing age on TKI should be made to understand that contraception is mandatory.

Tyrosine Kinase Inhibitors (TKI)

Many inhibitors of BCR-ABL1 tyrosine kinase have been developed. Out of these imatinib, dasatinib and nilotinib are approved for use in the first line setting. Dasatinib and nilotinib can be used for patients who relapse on imatinib. Busotinib and ponatinib are approved for use relapsed CML. The TKIs drugs used in CML are listed in the table below.

 Dose Indications Adverse Effects
Imatinib
  1. Chronic Phase CML: 400mg
  2. CML AP/BP: 400mg od the doses may be increased to 600mg-800mg/day. The 800mg dose is administered in two daily doses
  3. Ph+ALL: 600mg od
  4. PDGFRA mutated MDS/MPD: 400mg/day
  5. GIST: 400mg/day
  6. HES/CEL: 400mg od
  1.  CML First line therapy for chronic, accelerated and blast phase
  2. Ph+ve ALL
  3. Gastrointestinal stromal tumour
  4. MDS/MPD with PDGFA gene rearrangements
  5. Hypereosainophilic syndrome/chronic eosinophilic leukaemia
  1. Skin depigmentation
  2. Nausea, vomiting
  3. Periorbital oedema
  4. Fluid retention manifesting as effusions and oedema
  5. Myalgias
  6. Diarrhoea
  7. Insomnia, deprtesson
Dasatinib
  1. Chronic Phase: 100mg od
  2. Accelerated and blast phase: 140mg od
  1. CML first line and second line therapy
  2. Ph+ve ALL resistance or intolerance to prior tyrosine kinase therapy
  1. Myelosupression
  2. Bleeding
  3. Fliud retention manifestion as oedema and pleural effusion
  4. Diarrhoea, nauseas vomiting
  5. Fatigue
  6. Insomnia, depression
  7. Elevated transaminases
  8. Hypocalcaemia, hypophosphataemia
  9. QTc prolongation
Nilotinib
  1. CML Chronic Phase 300mg bd
  2. CML chronic phase resistant/intolerant to other TKIs: 400mg bd
  3. AP/BC: 400mg bd
CML first line and second line therapy

 

  1. Myelosupression
  2. Fatigue, asthenia, anorexia
  3. Prolonged QTc
  4. Electrolyte anomalies – hypophosphatemia, hypokalemia, hypocalcaemia, hyponatraemia
  5. Elevated transaminases
Bosutinib
  1. CML chronic phase: 500 mg orally once daily with food. Escalation to 600 mg daily in patients who do not respond.
  2. Dose in Hepatic impairment: Reduce 200 mg daily
CML failure or intolerance to firstling therapy
  1. Diarrhoea
  2. Nausea/vomiting
  3. Abdominal pain
  4. Myelosupression
  5. Skin rash
  6. Elevated Transaminases
  7. Fliud retention manifestion as oedema and pleural effusion
  8. Fatigue
Ponatinib
45mg reduce dose to 30mg in patients taking strong CYP3A inhibitors
  1. CML first line and second line therapy
  2. Ph+ve ALL resistance or intolerance to prior tyrosine kinase therapy
  3. The only TKI active against the T315I BCR-ABL1 mutation
  1. Arterterial thrombosis including myocardial infraction. Toxicity may be seen in up to 10% of the patients
  2. Cardiac toxicity – arrhythmias, pericardial effusions
  3. Elevated transaminases,  liver failure
  4. Pancrititis
  5. Hypertension
  6. Fluid retention,
  7. Gastrointestinal perforation
  8. Tumour lysis syndrome
Omacetaxine (Non TKI, inhibitor of protein synthesis)
 1.25mg/m2 SC bd for 14 consecutive days every 28 days till haematological response. Continue as 1.25mg/m2 sc bd for seven days every 28 days after as maintenance CML chronic or acclerated phase resistant to two more TKI
  1. Myelosupression – thrombocytopenia, neutropenia and anaemia
  2. Impaired glucose tolerance with hyperglycaemia in upto 11%
  3. Diarrhoea, nausea, vomiting abdominal pain
  4. Fatigue asthenia, arthralgia peripheral oedema
  5. Injection site reactions

A newly diagnosed patients should be initiated on treatment with Imatinib (400mg od), dasatinib (100mg od)  or nilotinib (300mg bd). Dasatinib and nilotinib. Busotinib and  ponatinib can be used in patients progressing on these the first line drugs. Ponatinib is indicated in the T315I mutations. Patients carrying this mutation do not respond to any of the other TKI.

Monitoring Therapy

Patients are monitored for response by clinical examination, haemogram and determining the levels of the BCR-ABL1 transcript. The BCR-ABL1 transcript is measured by RT-PCR. The response definitions are given in table below

Response Definitions

Complete Haematological Response
  1. WBC counts < 10 X 109/L
  2. Platelet Count < 450 X 109/L
  3. No immature myeloid cells on the peripheral blood differential count
  4. Basophils less than 5%
  5. no splenomegaly
Cytogenetic Response
  1. Complete (CCyR) 0% Ph+
  2. Partial Cytogenic Response  (PCyR) 1-35%
  3. Major Cyogenic response 36%-65%
  4. Minor Cytogenic Response 66%-95%
  5. No Cytogenic Response >95%
Molecular Response
  1. Early molecular response (EMR) – BCR-ABL1 transcripts ≤10% by QPCR (IS) at 3 and 6 months.
  2. Major molecular response (MMR) – BCR-ABL1 transcripts 0.1% by QPCR (IS) or ≥3-log reduction in BCR-ABL1 mRNA from the standardized baseline, if QPCR (IS) is not available.
  3. Complete molecular response (CMR) – no detectable BCR-ABL mRNA by QPCR (IS) using an assay with
  4. a sensitivity of at least 4.5 logs below the standardized baseline. CMR is variably described, and is best defined by the the assay’s level of sensitivity (eg. MR 4.5).
Relapse Resistance
  1. Any sign of loss of response (defined as hematologic or cytogenetic relapse)
  2. 1-log increase in BCR-ABL transcript levels with loss of MMR (two consecutive samples to be tested)
  3. loss of CCyR

The haematological and cytogenetic milestones in the treatment of CML have been described by the NCCN and ELN.  Secondary resistance to TKIs results from point mutations. Mutations analysis of the kinase domain of ABL should be performed in case of failure to achieve or loss of a milestone. Mutations that result in resistance to the first line TKIs are characterised and mutational analysis serves as a guide to choose second line therapy. The T315I mutation imparts resistance to all TKIs except ponatinib.

The TKI treatment needs to be continued lifelong. Attempts to withdraw treatment in patients who have achieved prolonged deep molecular response have shown that the disease returns in a majority of the patients on discontinuation of treatment.

Allogenic stem cell transplant was the mainstay of treatment of CML before the introduction of TKIs. The availability of many TKIs has diminished the role of allogenic stem cell transplant. Allogenic stem cell transplant is indicated only in patients who progress on TKIs.

Prognosis

Introduction of imatinib altered the prognosis of CML by converting it form a progressive ad fatal disease to a chronic disease. The 8 year survival of CML on imatinib is 85%. Those progressing on imatinib can be treated with other TKIs.

 

The BCR-ABL1 Gene


CML Pathogenesis-600pxBCR-ABL1 is a fusion gene formed as a result of the t(9;22)(q34;q11) chromosomal translocation, the translocation that results in the formation of the Philadelphia chromosome. The Abelson murine leukaemia viral oncogene homolog 1 (ABL1) gene from 9q34 is translocated downstream to a region at 22q11 known as breakpoint cluster (BCR). The fusion gene encodes for a constitutionally active tyrosine kinase that has been shown to drive the expression chronic myeloid leukaemia phenotype. BCR-ABL1 gene has also been on implicated in the pathogens is of acute lymphoblastic leukaemia and in rare cases of acute myeloid leukaemia. The gene has been targeted with unparalleled success by the first tyrosine kinase inhibitor approved in clinical practice, imatinib.

 Molecular Biology of BCR-ABL

The ABL1 proto-oncogene is located on chromosome 9 at q34. Chromosome 22 has the BCR gene at 22q11. The ABL1 gene translocated downstream to the BCR gene as a result of the t(9;22)(q34;q11) translocation. ABL1-BCR translocation also occurs and may express but is of no clinical significance.

 

Molecular Biology of CML

The BCR-ABL1 fusion gene and it’s variants

The breakpoint of the ABL1 gene may be upstream exon 1a, between exon 1a and 1b or downstream exon 1b but it is almost always upstream exon 2. With rare exceptions all transcripts of BCR1-ABL1 gene have exon 2-11 of the ABL1 gene. The BCR breakpoints are variable and determine the size as well as the pathogenic properties of the BCR-ABL1 gene.. The breakpoint on the BCR gene are clustered in three regions known major cluster, minor cluster and micro cluster (Table 1). Depending on the location of breakpoint  on the BCR gene three types of protein are synthesized. The p210 transcript is associated with CML and some patients with Philadelphia positive acute lymphoblastic leukaemia. The shorter p190 transcript is associated with philedelphia positive acute lymphoblastic leukaemia and some patients of chronic myeloid leukaemia. The CML that carry this mutation show monocytosis and have a more aggressive course. The p230 is the largest and the rarest of the BCR-ABL1 transcripts. It is associated with a more indolent course and is found in patients with the rare chronic neutrophilic leukaemia. Atypical transcripts e1a3, e13a3 and e6a2 have been described.

Table 1: The BCR-ABL1 fusion genes

 Major Cluster  Minor Cluster  Micro-Cluster
 Synonym M-Cluster m-cluster µ-cluster
Location exons 12-16 Between alternative exon 2, e2’ and e2 between exons e19 and e20
Protein p210 p190 p230
Associated Leukaemia  CML, e14a2 shown to have thrombocytosis in some studies., Ph+ ALL  Ph + ALL; CML that tends to have monocytosis and an agressive course  Chronic Neutrophilic Leukaemia, Small reports describing patients with a course resembling classical CML

Mutagenicity of BCR-ABL1

ABL1 is a nuclear kinase whose activity is tightly regulated by the cell. BCR-ABL1 translocation results loss of regulation and the kinase is  cosntitutively active. Sustitution of ABL1 at the N termnal by segments of the BCR gene result in the synthesis of a protein that has the capacity to dimerise. Dimerisation transphosphorylates and then aurtophsophorylates the the kinase fully activating it. The precise mechanism how BCR-ABL1 leads to chronic myeloid leukaemia is not known but activation of  phosphatidylinositol kinase, RAS/Mitogen activated protein kinase and JAK/STAT pathway has been demonstrated in BCR-ABL1 positive cells. These pathways are involved in cellular growth and differentiation. The BCR-ABL1 kinase also phosphorylates proteins involved in adhesion and migration and this may have a role in premature release of myeloid cells in circulation. CML cells have a two to sixfold increase in reactive oxygen species and have impaired DNA repair. Reactive oxygen species can induce DNA double strand breaks. The results is additional mutations and these are believed to be responsible for blast crisis and acclerates phase.

Tyrosine kinase inhibitors targeting the BCR-ABL1 protein induce a remission in most patients of CML. About half the patients who have achieved sustained complete molecular response relapse on discontinuation of the tyrosine kinase inhibitors. This suggests that the stemat least some CML stem cells are not BCR-ABL1 dependent for growth. Experimental observations support this hypothesis.

Targeting the BCR-ABL1 Gene

The BCR-ABL1 gene was the first gene to be targeted by a tyrosine kinase inhibitor, imatinib. Imatinib was followed by dasatinib, nilotinib, Busotinib and Panotinob. Imatanib, Dasatinib and Nilotinib are approved to first line use. Imatinib has resulted in a 85% 8 year survival. Dasatinib and nolitinib are active in imatinib resistant CML and are now approved for first line use. Drug resistance results from mutations in BCR-ABL1 kinase. The T315I mutation or the gatekeeper mutations impaires access of TKIs to the BCR-ABL1 kinase making most drugs inactive. Panotinib can inhibit the T315I mutation.

 

Further Reading

Barnes DJ Melo JV. Molecular Basis of Chronic Myleoid Leukaemia. In Chronic Myeloproliferative Disorders: Cytogenetic and Molecular Anomalies. Bain Barbra J (Ed) 2003.

Bendamustine


Bendamustine is a mechlorethamine derivative synthesised by Ozegowski and Krebs from the former East Germany in 1963. The  molecule has three parts,

  1. The alkylator mechloethamine,
  2. Benzimidazole ring that mimics purines
  3. Butyric acid side chain.

Each of these part has potential anti-malignacy action but only the alkylating actions are of clinical significance.

Mechanism of Action

Bendamustine acts as a classical chlorethyl alkylating agent. It causes inter and intrastrand DNA cross links that result in DNA strand breaks. The number of DNA strand breaks caused by bendamustine is grater than those caused by cyclophosphamide or melphalan. In addition the breaks are repaired slowly. This is probably because of the bulky structure of bendamustine. It has a partial cross sensitivity against other alkylating agents.

The benzimidazole side chain mimics purines and butyric acid side chain can react with membranes and proteins.  These actions do not contribute to the anti-tumour effect of bendamustine.

Mechanisms of Resistance

Resistance to bendaustime may develop because  of

Increased activity of DNA repair enzymes

Increased expression of sulfhydryl proteins, e.g. glutathione and glutathione-related enzymes

Bendamustine is partially cross-resistance with other alkylators.

Pharmacokinetics

  1. Absorption and Distribution: Bendamustine has a high oral bioavailability of 90% but no oral preparation is available. It is tightly (94%-96%) bound to human serum plasma proteins. Protein binding is not affected by hypoalbuminaemia.  It does not appear to displace other drugs or be displaced by other drugs. It is mainly distributed in the extracellular space and has a steady state volume of distribution of 25 L.
  2. Elimination: It is metabolised by hydrolysis. Active metabolites are formed as a result of drugs metabolism but theses have little clinical significance.  The half-life after a 30 minute infusion is 40 minutes with clearance of 700ml/min.
  3. Effect of Renal Impairment: Renal impairment with creatinine clearance unto 40 ml/min does not affect the elimination of bendamiustine. The effect of more severe renal failure is not studied.
  4. Effect of Hepatic Impairment: Mild Hepatic impairment does not have an effect on bendamustine elimination. The effect of moderate and severe liver impairment is unknown.

Toxicity

  1. Myelosupression: The main toxicity is myelosupression with about half the patients having grade 3 or 4 neutropenia. The incidence of febrile neutropenia is about 7%. Grade 3  to 4 thromboctopenia is about 24%. Anaemia is less common.
  2. Nausea and vomiting is usually mild to moderate
  3. Infusion reactions: infusion reaction is characterised by fevers, hypotension, back and muscle pain, chills, and rigours and may be seen within the 24 hours of infusion. It may be seen unto the third cycle. Steroids may help in patients getting infection reactions.
  4. Skin Toxicity: Skin rash and bullous exanthema
  5. Tumour lysis syndrome
  6. Carcinogenicity: Bendamustine has been associated with myelodysplastic syndrome and acute leukaemia.

Indications

  1. Chronic Lymphocytic leukaemia
  2. Low grade non-Hodgkin lymphoma
  3. Other disease wherebendamustine is used but the role is not established:
    1. Multiple Myeloma
    2. Acute Leukaemia
    3. Solid Tumours – breast cancer, small cell lung cancer, germ cell tumour

Dose

  1. Chronic Lymphocytic leukaemia:
    1. Single Agent: 100mg/m2 day 1 and 2 every 4 weeks
    2. With rituximab: 90mg/m2 day 1 and 2 when used with rituximab
  2. Non-Hodgkin Lymphoma:
    1. Single Agent: 120mg/m2 day 1 day 2 of a 21 day cycle.
    2. With Rituximab:  90mg/m2 o day 1 and day 2 of a 28 day cycle

Dosing in Special Populations

  1. Pregnancy:  Bendamustine is mutagenic.
  2. Paediatric Patients: Safety of bendamustine in paediatric population is not established
  3. Renal Failure: Bendamustine should not be used in patients with creatine clearance of less than 40ml/min
  4. Hepatic failure: Bendamustine should not be used in patients with moderate or severe hepatic impairment (bilirubin >3 X upper limit of normal or bilirubin 1.5-3 X upper limit of normal with AST or ALT 2.5-10X normal)

 

Hydroxyurea – Drug Information


Hydroxyurea was synthesised in Germany in 1860 and was found inhibit granulocyte production. It was only a hundred years after this that its potential as an anticancer drug was realized.

Hydroxyurea Mechanism of Action

Mechanism of Action of Hydroxyurea

Hydroxyurea enters the cell by passive diffusion. It inhibits of ribonucleotide reductase (RR). RR converts ribonucleotide diphosphates to deoxyribonucleotide diphosphates. Deoxyribonucleotide diphosphates are converted to deoxyribonucleotide triphosphates  and incorporated into DNA. Depletion of deoxyribonucleotide triphosphates results in impaired DNA synthesis. RR has two subunits M-1 and M2.  The M-2 subunit is the catalytic subunit and contains iron. Hydroxyurea inhibits RR by chelating iron. Hydroxyurea is an S phase specific drug. The cells exposed to hydroxyurea progress normally through the cell cycle, have a normal G1-S transition but accumulate in the S phase because of an inability to synthesise DNA. They then undergo apoptosis by p53 dependent and independent mechanisms.  Hydroxyurea may be transformed to nitric oxide. Nitric oxide is also an inhibitor of RR and may be responsible for drugs ability to induce foetal haemoglobin. This is important for treatment of sickle cell anaemia. Resistance to HU develops by elevated cellular activity of RR.

Pharmacokinetics of Hydroxyurea

Oral bioavailability of hydroxyurea is 80-100%. Parenteral formulation has no advantage over oral formulation. The drug is well distributed. It enters breast milk, cerebrospinal fluid and third space collections. The ratio of plasma to CSF levels is 4-9:1 and plasma to ascites levels is 2-7.5:1. The elimination half-life is 3.5-4.5 hours. Renal elimination is the main pathway of elimination. Sixty to eighty per cent of the dose eliminated by kidney unchanged. Patients with creatinine clearance of 10-50ml/hr should receive 50% and those with creatinine clearance of less than 10ml/hr should receive 20% of the planned dose. Hydroxyurea is metabolized but the metabolic pathways are not known.

Indications

  1. Myeloproliferative diseases
    1. Chronic myeloid leukaemia
    2. Essential thrombocytosis
    3. Polycythaemia Vera
  2. Acute leukaemia to control counts
  3. Sickle cell anaemia

Dose

Myelosuppression is the dose limiting effect of hydroxyurea. The dose of hydroxyurea needs to be titrated to the leucocyte and platelet counts. The acceptable lower limits of these counts will depend on the indication but generally speaking a leukocyte count less than 2.5X109/L or a platelet count less than 100X109/L is an indication for discontinuing therapy. With the abovementioned provisions in mind the dose of hydroxyurea for different indications are as follows:

  1. Myeloproliferative diseases: The usual dose is 20-30mg/kg/day.
  2. Acute leukaemia: 50-100mg/kg per day
  3. Sickle cell anaemia: 15-20mg/kg/day

Drug interactions

  1. HU inhibits formation of deoxynucleotides and enhanced the effect of agents damaging the DNA, as no nucleotides are available for repair. The effects of purine and pyrimidine analogues. When hydroxyurea is combined with any of these agents it should be done as a part of a protocol whose toxicity has been evaluated. This will prevent unacceptable toxicity.
  2. It has been shown to be synergistic with agents damaging the DNA like cisplatin, alkylating agents and topoisomerase II inhibitors.
  3. It has been used as a radiosensitizing agent in the treatment of head and neck and cervical cancer. It depletes the deoxynucleotide pool needed for DNA repair after radiation-induced damage.
  4. Enhanced anti HIV activity of azidothymidine, dideocytidine and dideoxyinosine

Toxicity

  1. Myelosuppression: The dose limiting toxicity of hydroxyurea is myelosuppression. Hydroxyurea causes rapid fall in leucocyte counts. When used in non-haematological malignancies the fall in leucocyte counts is evident by days 2-5. When used in patients with leukaemia the fall is evident faster, sometimes within a day. This property of hydroxyurea is useful in myeloid leukaemia with very high leucocyte count. Hydroxyurea is the treatment of choice for patients with chronic myeloid leukaemia presenting with very high counts. Though used in acute myeloid leukaemia with hyperleucocytosis, benefit from its use has not been proven in clinical trials.
  2. Gastrointestinal: Oral ulceration and gastrointestinal tract effects may be seen in some patients. They are particularly common in patients who receive chemoradiation with hydroxyurea.
  3. Skin: Dermatological changes may be seen with prolonged use. These include
    1. Skin Pigmentation and rash: Hyperpigmentation, erythema of the face and hands, diffuse maculopapular rash and dry skin. Severe reactions may resemble lichen planus.
    2. Nail Changes: The nails may show atrophy and formation of multiple pigmented bands.
    3. Leg Ulcers: Leg ulcerations may be seen in patients with prolonged therapy with hydroxyurea.
    4. Alopecia: Alopecia may occasionally be seen with the use of hydroxyurea
    5. Radiation Recall: Erythema or pigmentation of previously radiated skin may be seen in some patients.
  4. Mutagenicity and Teratogenicity: Hydroxyurea is a proven teratogen and contraindicated in women are pregnant or are planning a pregnancy. Women in the reproductive age group must be advised about contraception. The carcinogenic potential of hydroxyurea is uncertain. In view of the mechanism of action it is prudent not to use hydroxyurea for non-malignant disease.

Discovery of Vitamin K and It’s Targets and Antagonists


Henrik Dam and Edward Doisy were awarded the 1943 Nobel Prize for Physiology or Medicine. Dam discovered vitamin K and Doisy isolated two forms of vitamin K. Vitamin K was discovered by Henrik Dam while carrying out experiments to study cholesterol metabolism in chicken.  Evidence available in the early twentieth century suggested that chicken unlike rats, mice and dog could not synthesize cholesterol. Henrik Dam fed chickens with artificial cholesterol free diet to see if they could synthesize cholesterol. Chicks developed haemorrhage after 2-3 weeks of cholesterol free diet. Their blood did not clot well. Dam supplemented the diet to determine the component of cholesterol free diet responsible for the haemorrhage.

  1. supplementing the diet with pure cholesterol did not stop the haemorrhages indicating that the lack of cholesterol was not the cause of bleeding
  2. supplementing with  linseed oil or triolin did not prevent the symptoms indicating that low fats were not the cause of the symptoms
  3. Chicken does not need vitamin C indicating that the bleeding was not because of scurvy

Holst and Halbrook at the University of California corrected the bleeding caused by cholesterol free diet by feeding chicken cabbage. They questioned the claims that chicken do not need vitamin C and proposed that it was the vitamin C in the cabbage that corrected the bleeding. Dam was able to disprove this hypothesis by demonstrating the failure of injectable vitamin C to correct bleeding. In 1934 Dam proposed that the bleeding was because of an unrecognized dietary factor, in 1935 he called it vitamin K (K from “koagulation” the Scandinavian and German for clotting) an in 1939 along with Karrar and co-workers made the first pure preparation of vitamin K (Henrik Dam’s Noble Prize Lecture).

In 1920 Northern US and Canada has an epidemic of a bleeding disease in cattle. Cows were bleeding spontaneously or following minor trauma. Twelve of the twenty-five bulls subjected to castration died of bleeding. The following year the disease was found to be due to ingestion of fodder containing moldy sweet clover. Unspoiled sweet clover was not found to cause the disease. The disease was demonstrated to be due to the deficiency of prothrombin in 1929. The substance causing the bleeding disease, 3,3′-methylenebis-(4-hydroxycoumarin) or dicumarol,  was not isolated until 1940. Dicumarol was the first oral anticoagulant to be used.

The first four letters of Warfarin are an acronym for Wisconsin Alumni Research Foundation that funded the research into the molecule. Warfarin was not developed as an oral anticoagulant but as a rodenticide. It was an unsuccessful suicide attempt by an US army recruit that was responsible for introduction of warfarin as an oral anticoagulant.  Warfarin was found to be more potent than dicumarol and has completely replaced the latter as the preferred oral anticoagulant.  4-Hydroxycoumarin derivatives that have short half-lives are used as oral anticoagulants as the effects are easy to titrate. These include warfarin, acenocoumarol  and phenprocoumon. Superwarfarins,  which have a substantially longer (weeks to months) half-life than warfarin,  are used as rodenticides. The suerwarfarins include difenacoum, flocoumafen, bromadiolone, tioclomarol and brodifacoum.

It became apparent early in the development of oral anticoagulants that their actions were antagonized by vitamin K. The function of vitamin K was however not discovered until 1974. In this year three laboratories reported that prothrombin from cows treated with warfarin had 10 glutamic acid residues at the amino end that were replaced by an unusual amino acid, gamma carboxy glutamic acid. Vitamin K was found to be a cofactor in the gamma carboxylation of glutamate that was essential for the action of prothrombin. Prothrombin is not the only protein that undergoes a post-translational modification. Others include coagulations factors (factors VII, IX, X), inhibitors of coagulation (protein C, protein S), proteins involved in bone mineralization (osteocalcin, matrix Gla protein (MGP), and protein S) and cell growth regulation (Gas6). Many non-coagulation vitamin K dependent proteins have been identified by the presence of gamma-carboxyl glutamic acid.