In erythropoiesis, erythropoietin is the most important regulator of the proliferation of committed progenitors (BFU-E and CFU-E).

Severe anemia invariably present in its absence

Erythropoiesis is controlled by a highly responsive feedback system in which a sensor in the kidney can detect changes in oxygen delivery to increase the secretion of erythropoietin which then stimulate a rapid expansion of erythroid progenitors.

Erythropoetin is produced primarily by peritubular interstitial cells of the kidney under control of a single gene on human chromosome 7. The gene product is a protein containing 193 amino acids of which the first 27 are cleaved during secretion. The final hormonal peptide is heavily glycosylated and has a molecular weight of approximately 30,000 daltons.

Once released, erythropoietin travels to the bone marrow where it binds to a receptor on the surface of committed erythroid progenitors and is internalized. With anemia or hypoxia, renal synthesis rapidly increases by 100 fold of more, serum erythropoietin levels rise and marrow progenitor cell survival, proliferation and maturation are dramatically stimulated.

Recombinant human erythropoietin (epoetin alfa), produced using a mammalian cell line (Chinese hamster ovary cells), is virtually identical to endogenous hormone. Small differences in the carbohydrate portion of the molecule do not appear to affect the kinetics, potency or immunoreactivity.

Currently available preparations of epoetin alfa include EPOGEN and PROCRIT for subcutaneous or intravenous administration. The subcutaneous route of administration os preferred. When injected intravenously, epoetin alfa is cleared from the plasma with half-life of 10 hours. The effect on marrow progenitors is sufficiently sustained that it need not be given more often than three times a week to achieve adequate response. No significant allergic reactions have been associated with the intravenous or subcutaneous administration of epoetin alfa and antibodies have not been detected even after prolonged administration.

Recombinant erythropoietin therapy can be highly effective in a number of anemias, especially those associated with a poor erythropoietic response. There is a clear dose response relationship between epoetin alfa dose and the rise in hematocrit in anephric patients with eradication of their anemia at higher doses. Epoetin alfa has been shown to be effective in the treatment of anemias associated with surgery, AIDS, cancer chemotherapy, prematurity, prematurity, and certain chronic inflammatory illnesses.

Several principles govern the administration of epoetin alfa (EPO):

The anemia of chronic renal failure is generally regarded as multi-factorial, but the striking response to EPO indicates that the other factors must be of lesser importance.

EPO has other hematologic benefits

• Increasing the platelet count
• Correcting the abnormality in platelet function, thereby lowering the bleeding time and lessening the risk of uremic bleeding This probably explains in part the two-thirds reduction in the number of transfusion-dependent patients associated with the appropriate use of EPO.

EPO appears to have additional beneficial actions (via uncertain mechanisms):

• Relief of a variety of uremic signs and symptoms, such as pruritus, sexual dysfunction in men, and impaired carbohydrate and cortisol metabolism.
• Improved quality of life parameters, particularly vitality and sleep.
• A modest reduction in total cholesterol and triglyceride levels (7 and 11 percent, respectively).
• A reduction in macular edema in diabetic patients.
• Increased cognitive function and cerebral blood flow

Successful treatment of anemia can also have profound effects on the cardiovascular and hemodynamic abnormalities that accompany end-stage renal disease.

• The majority of dialysis patients have left ventricular hypertrophy; EPO therapy has been shown to reduce the left ventricular mass index by 10 to 30 percent.
• Patients with anemia have a high cardiac output in association with a fall in systemic vascular resistance. These changes return toward normal with EPO-induced correction of the anemia.
• Exercise-mediated cardiac ischemia is also ameliorated with correction of the anemia.

These beneficial effects may be negated by worsening blood pressure control.

Target hematocrit: The currently recommended target hematocrit ranges between 30% and 36%.

The most common side effect of epoetin alfa therapy is aggravation of hypertension, seen in 20% to 30% of patients and most often associated with a too rapid rise in hematocrit. .

Hypertension, occasionally severe enough to be associated with encephalopathy and seizures, is the most important complication of EPO therapy.

Among patients with end-stage renal disease, 20 to 50 percent of patients who receive EPO intravenously develop an elevation in diastolic pressure of 10 mmHg or more.

The blood pressure is less likely to rise after subcutaneous therapy, possibly because this route of administration does not elevate plasma endothelin levels.

The risk of hypertension can be ameliorated by raising the hematocrit slowly and by aiming for an hematocrit of 30 to 35 percent, a level that is sufficient to relieve symptoms without producing a significant elevation in blood pressure.

Patients who still become hypertensive can be treated with fluid removal (via dialysis or, if the patient has only chronic renal failure, diuretics) and the administration of antihypertensive agents.

Beta-adrenergic blockers and vasodilators should be considered as agents of first choice. Calcium channel blockers and angiotensin converting enzyme inhibitors also may be effective.

The dose of EPO should be reduced or discontinued for several weeks in severe cases or when other therapeutic measures are ineffective.

Aside from hypertension and its related problems, the most common side effects of EPO treatment are headache which occurs in 15 percent of cases and an influenza-like syndrome affecting 5 percent.

The influenza-like syndrome is of unknown etiology, but is responsive to anti-inflammatory drugs and does not seem to occur with subcutaneous EPO administration.

EPO also improves the hemostatic defect caused by uremia and raises the platelet count and may be associated with an increase in the risk of thrombotic events. An increased tendency to vascular access thrombosis in hemodialysis patients has also been reported. Changes that may contribute are the increase in hematocrit, improved platelet function, and enhanced release of plasminogen activator inhibitor-I by vascular endothelial cells. Correction of anemia with EPO requires an increase in heparin dose of approximately 25 percent in most patients.

Resistance to EPO: Some patients are relatively resistant to EPO and require doses greater than 100 U/kg. The most common cause is iron deficiency induced in part by the rapid increase in erythropoiesis.

Additional causes include the following:

• Bone marrow fibrosis due to secondary hyperparathyroidism; this should be suspected when EPO resistance occurs in iron replete patients.
• Occult malignancy and unsuspected hematologic disorders (such as folate deficiency). When evaluating a patient for possible folate deficiency, the red cell not serum folate concentration is the most reliable assay.
• Chronic inflammation (with inhibition possibly due to enhanced cytokine production). In one study of 128 patients, for example, erythropoietin resistance was most closely associated with high levels of C-reactive protein, a serum marker of inflammation.
• The accumulation of aluminum in bone.
• Malnutrition, which is a common finding in dialysis patients.
• Hemoglobinopathies, as patients with sickle cell disease or trait have an inadequate response to the administration of EPO; a predominance of hemoglobin F containing reticulocytes is observed in these patients.
• The administration of angiotensin converting enzyme inhibitors and/or angiotensin II receptor antagonists. Some reports, but not all, suggest that these agents may result in relative EPO resistance.

Myeloid Growth Factors

The myeloid growth factors are glycoproteins that stimulate the proliferation and differentiation of one or more myeloid cell lines. They also enhance the function of mature granulocytes and monocytes.

Recombinant forms of several of the growth factors have now been produced including GM-CSF, G-CSF, M-CSF or CSF-1, IL-3, SCF and thrombopoietin.

The myeloid growth factors are produced naturally by a number of different cells including fibroblasts, endothelial cells, macrophages and T cells. They are active at extremely low concentrations.

GM-CSF is capable of stimulating the proliferation, differentiation, and function of a number of myeloid cell lineages. It acts synergistically with other growth factors, including erythropoietin, at the level of the BFU.

CM-CSF stimulates the CFU-GEMM (granulocyte/erythrocyte/macrophage/megakaryocyte), CFU-GM, CFU-G, CFU-M, CFU-E, and the CFU-Meg (megakaryocyte) to increase cell production.

GM-CSF enhances the migration, phagocytosis, superoxide production, and antibody-dependent cell media toxicity of neutrophils, monocytes and eosinophils.

The activity of G-CSF is more focused with its principal action being to stimulate the proliferation, differentiation and function of the granulocyte lineage.

It acts primarily on CFU-G, although it can also play a synergistic role with IL-3 and GM-CSF in stimulating other cell lines.

G-CSF enhances phagocytic and cytotoxic activities of neutrophils but unlike GM-CSF, G-CSF has little effect on monocytes, macrophages and eosinophils.

G-CSF reduces inflammation by inhibiting IL-1, tumor necrosis factor, and interferon gamma.

Granulocyte/Macrophage Colony Stimulating Factor (GM-CSF)

Recombinant GM-CSF (sargramostim) is a 127 amino acid glycoprotein produced in yeast. Except for the substitution of a leucine at position 23 and variable levels of glycosylation, it is identical to endogenous GM-CSF.

The primary therapeutic effect of sargramostim, like natural GM-CSF, is stimulation of myelopoiesis.

Initial clinical application was in autologous bone marrow transplantation. By shortening the duration of neutropenia, transplant morbidity was significantly reduced without a change in long-term survival or risk of inducing an early relapse of the malignant process.

The role in allogenic transplantation is less clear. The effect of growth factor on neutrophil recovery is less pronounced in patients receiving prophylactic treatment for graft-versus-host disease. Studies have failed to show a significant effect on transplant mortality, long-term survival, appearance of GVHD, or disease relapse.

GM-CSF has been used to mobilize CD34-positive progenitor cells for peripheral blood stem cell collection for transplantation following myeloablative chemotherapy and it has been used to shorten the period of neutropenia and reduce morbidity in patients receiving intensive chemotherapy.

It will stimulate myelopoiesis in some patient with cyclic neutropenia, myelodysplasia, aplastic anemia or AIDS-associated neutropenia..

Sargramostim is administered by subcutaneous injection or slow intravenous infusion at a dose of 125 to 500 micrograms per square meter. Plasma levels rise rapidly after subcutaneous injection and then decline with a half-life of 2 to 3 hours. When given intravenously, the infusion should me maintained over 3 to 6 hours.

At lower doses the response is primarily neutrophilic, while at larger dose , monocytosis and eosinophilia are observed.

With initiation of therapy, there is a transient absolute decrease in the absolute leukocyte count secondary to margination and sequestration in the lungs followed by a dose-dependent biphasic increase in leukocyte count over the next 7 to 10 days.

Side effects of sargramostim, especially at high doses, include bone pain, malaise, flulike symptoms, fever, malaise, arthralgias, capillary leak syndrome (characterized by peripheral edema, and pleural or pericardial effusions), diarrhea, dyspnea, and rash.

Patients can be extremely sensitive to GM-CSF demonstration acute reactions to the first dose characterized by flushing, hypotension, nausea, vomiting, and dyspnea with a fall in arterial oxygen saturation due to sequestration of granulocytes in the pulmonary circulation. With prolonged administration of GM-CSF, a few patients may develop a capillary leak syndrome with peripheral edema and both pleural and pericardial effusions.

Granulocyte Colony Stimulating Factor (G-CSF)

Recombinant human G-CSF (filgrastim, NEUPOGEN) is a 175-amino acid glycoprotein produced in Escherichia coli. Unlike natural G-CSF, filgrastim is not glycosylated and carries an extra N-terminal methionine.

The principal action of filgrastim is the stimulation of CFU-G to increase neutrophil production. It also enhances the phagocytic and cytotoxic functions of neutrophils.

Filgrastim is effective in the treatment of severe neutropenia following autologous bone marrow transplantation and high-dose chemotherapy. Filgrastim, like GM-CSF, shortens the period of severe neutropenia and reduces mortality secondary to bacterial and fungal infections.

When used as part of an intensive chemotherapy regimen, it can decrease the frequency of both hospitalization for febrile neutropenia and interruptions in the chemotherapy protocol.

G-CSF has proven to be effective in the treatment of severe congenital neutropenias. While not eliminating the neutropenic cycle, G-CSF will increase the level of neutrophils and shorten the length of the cycle sufficiently to prevent recurrent bacterial infections in patients with cyclic neutropenia.

Filgrastim therapy can improve neutrophil counts in some patients with myelodysplasia or marrow damage (moderately severe aplastic anemia or tumor infiltration of the arrow).

The neutropenia of AIDS patients receiving zidovudine can be partially or completely reversed with filgrastim.

Filgrastim is routinely used in patients undergoing peripheral blood stem cell collection and stem cell transplant. It encourages the release of CD#$-positive progenitor cells from the marrow, reducing the number of collections necessary for the transplant.

Filgrastim is administered by subcutaneous injection or intravenous infusion over at least 30 minutes at a dose of 1 to 20 microgram/kg per day. The distribution and clearance rate from plasma (half-life) is 3.5 hours for both routes of administration.

Adverse effects from filgrastim include mild to moderate bone pain in those patients receiving high doses over a protracted period, local skin reactions following subcutaneous injections, and rarely, a cutaneous necrotizing vasculitis.

Patients with a history of hypersensitivity to proteins produced by E. coli should not receive filgrastim.

Mild to moderate splenomegaly has been observed in patients on long-term filgrastim therapy.

Monocyte/Macrophage Colony Stimulating Factor

Monocyte/Macrophage Colony Stimulating Factor (M-CSF) may play a role in stimulating monocyte and macrophage production, though with significant side effects including splenomegaly and thrombocytopenia.

Megakaryocyte Growth Factors

Interleukin-11

Interleukin-11 id a 65 - 85 kDalton protein produced by fibroblasts and stromal cells in the bone marrow.

Oprelvekin, the recombinant form of interleukin-11 approved for clinical use, is produced by expression in E. Coli.

Interleukin-11 is the first growth factor to gain FDA approval for the treatment of thrombocytopenia. It is approved for the secondary prevention of thrombocytopenia in patients receiving cytotoxic chemotherapy for treatment of nonmyeloid cancers.

Interleukin-11 is given by subcutaneous inhection at a dose of 50 microgram/kg per day. Its half-life is 7 -8 hours when given by subcutaneous injection. It is started 6 - 24 hours after completion of chemotherapy and continued for 14 - 21 days or until the platelet count passes the nadir and rises to greater than 50,000 cells per microliter.

The most common side effects of Interleukin-11 are fatigue, headache, dizziness, hypokalemia, and cardiovascular effects (probably caused by fluid retention secondary to increased renal reabsorption of sodium) which include dilution anemia, dyspnea as a result of pleural fluid, and transient atrial. All the side effects are reversible arrhythmias.

Thrombopoietin

Recombinant human thrombopoietin is a cytokine that selectively stimulates megakaryocytopoiesis.

Thrombopoietin was discovered in 1994 and currently its use is investigational.

In limited studies it has been effective in rapidly increasing platelet counts and in reducing the duration of severe thrombocytopenia and the need for platelet transfusion.

The potential clinical applications of the recombinant thrombopoietins are suggested by analyzing the current usage of platelet transfusions.

• Approximately 40 percent are used in hematology and oncology: 8 percent during chemotherapy for solid tumors, 16 percent during treatment of leukemia, 7 percent during bone marrow or peripheral blood stem cell transplantation, and 9 percent during the treatment of other chronic thrombocytopenic disorders.

• Another 40 percent are used in surgery: 20 percent for cardiovascular surgery, 17 percent for general surgery, and 3 percent for trauma.

• The remaining 20 percent are used to support the treatment of a wide range of conditions including chronic liver disease.

Two recombinant thrombopoietins have been subjected to intensive clinical investigation:

• A glycosylated molecule produced in CHO cells consisting of the full-length, native human amino acid sequence (recombinant thrombopoietin, rHuTPO) which has a circulatory half-life of 20 to 40 hours.

• A nonglycosylated, truncated molecule produced in Escherichia coli composed of the first 163 amino acids of the native molecule and chemically coupled to polyethylene glycol (pegylated, recombinant megakaryocyte growth and development factor, PEG-rHuMGDF).

This PEG-rHuMGDF) half of the native molecule is 50 percent similar to erythropoietin, contains all of the receptor-binding domain, but has a very short circulatory half-life and no biologic activity in vivo due to the absence of the remaining, carbohydrate-rich portion of the native molecule.

Potential risks associated with thrombopoietin include antibody formation, bone marrow fibrosis, thrombosis and thrombocytosis induction and stimulation of tumor growth.

Iron and Iron Salts

The effectiveness of iron therapy is best evaluated by tracking the reticulocyte response and the rise in the hemoglobin or the hematocrit. The reticulocyte count is observed in 4 to 7 or more days after beginning therapy. A measurable increase in the hemoglobin level takes even longer. At least 3 to 4 weeks after therapy has been started should pass before a decision as to effectiveness should be made.

The average dose for the treatment of iron-deficiency anemia is about 200 mg of iron per day (2 to 3 mg/kg), given in three equal doses of 65 mg. Children weighing 15 to 30 kg can take half the average adult dose, while small children and infants can tolerate relatively large doses - 5 mg/kg.

Orally administered ferrous sulfate, the least expensive form, is the preferred choice for iron deficiency. Ferrous salts are absorbed about three times as well as ferric salts.

All the ferrous salts (sulfate, fumarate, succinate, gluconate, etc.) Are absorbed to approximately the same extent.

When oral therapy fails, parenteral iron (Iron dextran injection) administration may be an effective alternative. Predictable indications are iron malabsorption (sprue, short bowel, etc.) severe oral iron intolerance, as a routine supplement to total parenteral nutrition, and in patients with renal disease who are receiving erythropoietin.

Parenteral iron has also been given to iron-deficient patients and pregnant women to create iron stores, something that would take months to achieve by the oral route.

Iron dextran injection is the parenteral preparation currently in general use in the USA. It is a colloidal solution of ferric oxyhydroxide complexed with polymerized dextran (MW approx. 180,000)resulting in a dark brown viscous liquid containing 50 mg/mL of elemental iron that is given by IV or deep IM routes.

A test dose of 0.5 mL (25 mg) must be given before initiation of therapy by the IM route.

The IV route is preferred and give more reliable response. In doses less than 500 mg, there is exponential clearance with a plasma half-life of 6 hours.

Side Effects:

Intolerance to oral preparations of iron is primarily a function of the amount of soluble iron in the upper gastrointestinal tract and of psychological factors. Side effects include heartburn, nausea, upper gastric discomfort, constipation, and diarrhea.

Chronic administration with iron overload can result in hemochromatosis.

Iron Poisoning: Large amounts of ferrous salts of iron are toxic. In adults fatalities are rare. Most deaths occur in childhood, particular between the ages of 12 and 24 months. As little as 1 to 2 grams of iron may cause death, but 2 to 10 grams is usually ingested in fatal cases.

Signs and symptoms of severe poisoning may occur within 30 minutes or may be delayed for several hours after ingestion. Signs and symptoms consist of abdominal pain, diarrhea, or vomiting of brown or bloody stomach contents containing pills.

Particular concern should exist if the following exists: pallor or cyanosis, lassistude, drowsiness, hyperventilation due to acidosis, and cardiovascular collapse. Shock, dehydration and acid-base abnormalities may exist.

Corrosive injury to the stomach may result in pyloric stenosis or gastric scarring.

If death does not occur within 6 hours, there may be a transient period of apparent recovery, followed by death in 12 to 24 hours. Hemorrhagic gastroenteritis and hepatic damage are prominent finding at autopsy.

When plasma concentration of iron is greater than total iron-binding capacity, deferoxamine should be administered. 10 to 15 mg/kg per hour deferoxamine intravenously by constant infusion is preferred for severe toxicity (serum iron levels greater than 500 micrograms per deciliter). Rates up to 45 mg/kg/hour have been used in a few cases.

For moderately toxic levels (350 to 500 micrograms/deciliter) intramuscular doses can be administered at a dose of 50 mg/kg with a maximum dose of 1 gram.

Hypotension has been observed with rapid IV boluses and with IM injections.

Vitamin B₁₂

Vitamin B₁₂ is available in pure form for injection or oral administration or in combination with other vitamins and mineral for oral or parenteral administration.

Oral preparations may be useful in supplementing deficient diets but they are of little value in the treatment of patients with deficiency of intrinsic factor or ileal disease.

Although small amounts of vitamin B₁₂ may be absorbed by simple diffusion, the oral route of administration cannot be relied upon for effective therapy in the patient with a marked deficiency of vitamin B₁₂ and abnormal hematopoiesis or neurological deficits.

The preparation of choice for the treatment of a vitamin B₁₂-deficiency state is cyanocobalamin, and it should be administered by intramuscular or deep subcutaneous injection.

Cyanocobalamin injection, a clear aqueous solution with a characteristic red color, is safe when given by the intramuscular or deep subcutaneous route, but it should never be given intravenously.

Following injection of cyanocobalamin, there have been rare reports of transitory exanthema and anaphylaxis. If a patient reports a previous sensitivity to injections of vitamin B₁₂, an intradermal skin test should be carried out before full dose is administered.

Cyanocobalamin is administered in doses of 1 to 1000 micrograms. Tissue uptake, storage and utilization depend on the availability of transcobalamin II.

Doses above 100 microgram are rapidly cleared from plasma into the urine (one of the water soluble vitamins) and administration of larger amounts of vitamin B₁₂ will not result in greater retention of the vitamin.

Administration of 1000 microgram is of value in the performance of the Schilling test, which is used to quantitate the absorption of vitamin B₁₂ and delineate the mechanism of disease. By performing the Schilling test with and without added intrinsic factor, it is possible to discriminate between intrinsic factor deficiency, by itself, and primary ileal disease.

Intrinsic factor is necessary for vitamin B₁₂ to be absorbed from the GI tract.

The sensitivity of the hematopoietic system to vitamin B₁₂ deficiency relates to its high rate of turnover of cells. As a result of the deficiency of vitamin B₁₂ DNA replication becomes highly abnormal.

Once a hematopoietic stem cell is committed to enter a programmed series of cell divisions, the defect in chromosomal replications results in an inability of maturing cells to complete nuclear divisions while cytoplasmic maturation continues at a relatively normal rate.

This results in a state of ineffective hematopoiesis in which there is production of morphologically abnormal cells and death of cells during maturation.

When the deficiency is marked, all blood cell lines, not just red blood cell are affected and a pancytopenia develops.

Vitamin B₁₂ deficiency in irreversible damage to the nervous system. Progressive swelling of myelinated neurons, demyelination, and neuronal cell death are seen in the spinal column and cerebral cortex.

A wide range of neurological signs and symptoms may develop including paresthesias of the hands and feet, diminution of vibration and position senses with resultant unsteadiness, decreased deep tendon reflexes, and, in the later stages, confusion, moodiness, loss of memory, and even a loss of central vision. The patient may also exhibit delusions, hallucinations, or overt psychosis.

Neurological damage may be dissociated from changes in the hematopoietic.

The degree and rate of improvement of neurological signs and symptoms depend on the severity and the duration of the abnormalities. Those that have been present for only a few months are likely to disappear relatively rapidly. When a defect has been present for months or years, full return to normal function may never occur.

Folic Acid

Folic has at one time was referred to as Vitamin M

Folate deficiency is a common complication of diseases of the small intestine, which interfere with the absorption of folate from food and the recirculation of folate through the enterohepatic cycle.

In acute or chronic alcoholism, daily intake of folate in food may be severely restricted and the enterohepatic cycle of the vitamin may be impaired by toxic effects of alcohol on hepatic parenchymal cells. This is perhaps the most common cause of folate-deficient megaloblastic erythropoiesis, as well as the one most amenable to therapy as reinstitution of a normal diet is sufficient to overcome the effects of alcohol.

Folate deficiency has been implicated in the incidence of neural tube defects, including spina bifida, encephaloceles, and anencephaly , even in the absence of folate-deficient anemia and alcoholism.

Less than adequate intake of folate can result in elevated levels of plasma homocysteine. Hyper-homocysteinemia is considered an independent risk factor coronary artery and peripheral vascular disease and for venous thrombocytosis.

Folate has a role as a methyl donor in converting homocysteine to methionine.

The megaloblastic anemia that results from folate deficiency cannot be distinguished from that caused by vitamin B₁₂ deficiency, however, folate deficiency is rarely, if ever, associated with neurological abnormalities.

The appearance of megaloblastic anemia after deprivation of folate is much more rapid than that caused by interruption of vitamin B₁₂ and reflects the fact that stores of folate are limited in vivo.

Folate deficiency is best diagnosed by measuring folate in plasma and in red cells.

Folate is marketed as oral tablets containing 0.4, 0.8 and 1 mg pteroylglutamic acid, as an aqueous solution for injection containing 5 mg/mL, and in combination with other vitamins and minerals.

Side effects: There have been rare reports of reactions to parenteral injections of both folic acid and leucovorin.

Oral folic acid is usually not toxic and even with doses as high as 15 mg/day, there have been no substantial reports of side effects.

Folate acid in large amounts may counteract the antiepileptic effect of phenobarbital, phenytoin, and primidone and increase the frequency of seizures in susceptible children.