Therapeutic potential of the hematopoietic growth factors.D Usha, UM Thatte, SA Dahanukar
Dept of Pharmacology, Seth GS Medical College, Parel, Bombay, Maharashtra.
Keywords: Animal, Bone Marrow Diseases, drug therapy,Bone Marrow Transplantation, Colony-Stimulating Factors, Recombinant, therapeutic use,Granulocyte Colony Stimulating Factor, Recombinant, therapeutic use,Granulocyte-Macrophage Colony-Stimulating Factor, therapeutic use,Hematopoietic Cell Growth Factors, therapeutic use,Human,
The development of semisolid culture systems in the recent past has driven home the fact that haematopoietic precursor cells proliferate and mature in vitro in the presence of stimulatory factors. These stimulatory factors were identified to be glycoproteins and their ability to stimulate precursor cells led to them being referred to as colony-stimulating factors (CSFs). Subsequently, such factors for almost every haematopoietic cell lineage were discovered to exist in vivo e.g. erythropoietin for erythrocytes, interleukin-2 for T-lymphocytes or macrophage - colony stimulating factor for macrophages, etc. Progress from the laboratory to the clinic was quick. This was contributed to, in a large measure, by the availability of recombinant DNA techniques, which allowed large amounts of the factors for clinical use. Today the clinical utility of some factors like erythropoietin is clearly established. The clinical status of two of the best characterised factors acting on the leucocyte series, namely granulocyte - macrophage colony - stimulating factor (GM-CSF) and granulocyte colony - stimulating factor (G-CSF) will be discussed in this article.
Native GM-CSF (mol wt - 23,000 Dalton) is a product of T-lymphocytes, macrophages, fibroblasts and endothelial cells. Native G-CSF (mol wt - 25,000 Dalton) on the other hand, is produced only by the latter three cell types.
The gene for GM-GSF is located on chromosome 5q 21-322 and that for G-CSF has been localised to 17q 11.2 - 212. This is important in view of the fact that in several cases of refractory anaemia, deletions along the chromosome regions 5q 21 - 32 have been detected.
GM-CSF and G-CSF have been found to play an important role in myelopoiesis, which is a complex series of events through which primitive self-renewing bone marrow stem cells differentiate. These CSFs also promote cell survival by suppressing apoptosis, which is the active process of self destruction characterised by specific DNA degradation and morphological changes in CSF deprived cells.
As shown in [Figure - 1], GM-CSF specifically stimulates the development of neutrophils and monocytes from the granulocytes and monocyte / macrophage colony-forming units respectively. In addition, it also stimulates (though to a lesser extent) the development of eosinophils, platelets and erythrocytes. G-CSF acts on the granulocyte - macrophagecolony-forming units and promotes their maturation to the granulocyte colony-forming units. G-CSF further acts on these granulocyte colony-forming units to promote the formation of functionally mature neutrophils. These CSFs also stimulate the release of mature cells from bone marrow into the peripheral circulation and reduce their maturation time,.
Lymphoid series is not shown in the figure.
In addition to having a proliferative effect on progenitor cells, these CSFs also act on mature leucocytes. Thus, the expression of chemotactic receptors is increased, which enhances chemotaxis. They also induce phagocytosis and microbial killing by neutrophils at sites of infection and inflammation.
GM-CSF and G-CSF do not act in isolation, as is already evident from the fact that they are synergistic with respect to their effects on the granulocyte - macrophage - colony forming units. These interactions are indicative of the fact that GIVI-CSF facilitates lineage commitment and subsequently supports or amplifies the clonogenic activity of lineage specific G-CSF.
The mechanism of action of both these CSFs is &subject of ongoing research. Receptor molecules have been identified for these CSFs on haematopoietic cells.
Though the number of these receptors are surprisingly few, degradation of the receptor - ligand complex is rather slow and this contributes to sustained action of these mediator molecules. In addition to these high affinity receptors, low affinity receptors too are known to exist for rGM-CSF
These CSFs have been postulated to transduce signals via the cyclic AMP and inosine triphosphate messenger pathways.
Based on encouraging results obtained in animals, clinical studies were initiated to evaluate the value of these CSFs in humans. In phase I clinical trials, administration of rG-CSF (recombinant G-CSF) and rGM-CSF (recombinant GM-CSF) by the intravenous or subcutaneous route produced a dose dependent increase in segmented and immature neutrophils. In addition rGM-CSF led to an increase in monocytes and eosinophils as well. This was seen after an initial brief period of leucopenia. This leucopenia was observed 10-30 minutes following administration and leucocyte recovery took place 1-2 hours later. The onset of and recovery from leucopenia was delayed when the CSFs were administered subcutaneously. Radionucleotide labelling studies indicate that this leucopenia is due to sequestration of leucocytes in the lungs due to increase in the production of an adhesive glycoprotein, and recovery is due to re-entry of these cells into the circulating pool.
The subsequent persistent increase in leucocyte counts is due initially to enhanced egress of mature cells from the bone marrow, shortened maturation time, enhanced de-margination of intra-vascular neutrophils and inhibited extra vascular migration. Further elevation, with continued exposure, reflects the CSF-induced increase in proliferative traction of haemopoietic cells in the bone marrow. Examination of bone marrow 5-14 days after treatment reveals an increase in cellularity and in the ratios of myeloid to erythroid progenitor cells. There is also an increase in the circulating levels of granulocyte and macrophage colony-forming units. Leucocyte levels normalise within 48 hours of cessation of treatment.
There are several clinical settings where these CSFs have been used in patients. However, the studies that have been carried out so far are initial ones, mainly to establish a dose response relationship with regard to its efficacy and toxicity. Larger, randomised placebo controlled studies are required to clarity the impact of these CSFs in clinical conditions. The main uses identified so far are listed in [Table:1].
The basis of using CSFs in this condition is that there could be a deficient production of CSFs following transplantation which can be supplemented by administering exogenous CSFs.
rGM-CSF has been more extensively used for this purpose. It has been tried in autologous and allogeneic, bone marrow transplantation. It was found to decrease the incidence of bacteremia and antibiotic use, with variable effects on platelet counts. It has also been used in cases of failure of bone marrow engraftment where increase in bone marrow cellularity was observed, though there was no effect on platelet and erythrocyte production. The U.S. FDA approved dosage schedule for this condition is represented in [Table:2].
rGM-GSF either alone or in combination with cyclophosphamide facilitates harvest of stem cells by apheresis for subsequent transplantation, because it can increase the number of progenitor cells in the circulation.
rGM-CSF, on the other hand has been mainly used in patients undergoing cytotoxic chemotherapy where bone marrow transplantation is carried out to overcome the dose limits of anticancer drugs. It has been found to decrease the period of neutropenia and frequency of bacteremia. The dosage schedule that has been tried in clinical trials of G-CSF is represented in [Table:2].
Aplastic Anaemia: A - plastic anaemia is characterised by bone marrow failure and pancytopenia. rGM-CSF has been used as an adjunct to existing immunomodulating therapy for the treatment of this condition. This resulted in a 2-8 fold increase in the peripheral granulocyte / neutrophil counts, fewer febrile days and a decrease in the number of blood transfusions needed. Bone marrow cellularity was increased, though the response of RBCs and platelets was equivocal. rGCSF has also been tried for this condition in various dosage regimens (as shown in [Table:3]) with encouraging results.
In the treatment of agranulocytosis, rG-CSF has found to be more effective than rGM-CSF in elevating the neutrophil counts.
Myelodysplasia: The myelodysplastic syndromes are a group of stem cell disorders characterised by maturation defects which result in refractory cytopenias.
Both rGM-CSF12 and rG-CSF13 have been tried in this condition with promising results [Table:3]. The number of blasts in myelodysplastic patients were found to decrease in the bone marrow, with subsequent increase in the number of mature elements following CSF administration. The possibility of these CSFs stimulating abnormal clones and promoting transformation to acute leukemia is an important consideration and is currently being addressed in phase III trials. Thus patient selection becomes an important criterion for CSF therapy. Those with initial high leukemic burden are not ideal candidates for CSF therapy.
Neutropenia : Both these CSFs have been tried in a variety of neutropenic states such as chronic idiopathic, cyclic and autoimmune neutropenias, Felty's syndrome and in glycogen storage disease,,,,,. rG-CSF was found to increase neutrophil counts more effectively than rGM-CSF, which incidentally was found to induce eosinophilia. There is some data to suggest that these two growth factors can be used in combination or sequentially to maximise production of mature neutrophils. [Table:3]
Neutropenia due to the disease per se and due to the deleterious effects of the drugs used such as azidothymidine, which cause myelosuppression, can be alleviated by the use of these two CSFs.
rGM-CSF was found to ameliorate leucopenia associated with HIV infection and azidothymidine induced neutropenia without affecting the disease course as determined by p24 antigen levels, CD4: CD8 ratios and recovery of HIV from mononuclear Cells.
In similar studies involving administration of rG-CSF in patients receiving azidothymidine, neutrophil levels was found to increase . In addition, subcutaneous administration of rG-CSF was found to increase erythropoietin levels, erythrocyte progenitors and also haemoglobin levels in patients with advanced HIV infection. The dosages tried in this condition is represented in [Table:3].
Cytotoxic drugs that are used to treat various cancers cause myelosuppression and thus cause an increase in the incidence of bacterial and fungal infections. In a number of studies, it was shown that rGM-CSF and rG-CSF decreased the severity of neutropenia in a dose dependent fashion with a higher neutrophil nadir,. Consequently, there was a decrease in antibiotic use. Although the incidence of bacterial/fungal infections did not decrease, the severity of these infections was definitely lesser as compared to the controls. Hence, these growth factors aid in maximising high dose chemotherapy.
Despite the concern that these CSFs can stimulate proliferation of residual malignant cells, they have widely been used in acute leukemic conditions and myelodysplasia for a variety of reasons. Both these CSFs, when administered along with intensive chemotherapy were found to decrease the period of recovery of neutrophils and incidence of infections ,. Also, they are found to recruit leukemic blasts to the S-phase and anti-neoplastic agents. Re-growth of leukemic blasts was observed in high risk patients and there was no evidence to prove the same in mild cases of acute myeloid leukemia and refractory multiple myeloma. But this aspect has yet to be studied more carefully and results should be interpreted with caution.
rGM-CSF has also been tried in the treatment of 8 patients who were accidentally exposed to Caesium 137 in a radiation accident in Brazil where it was found to accelerate bone marrow recovery.
The assessment of adverse effects of rGM-CSF and rG-CSF is variable, mainly because it has been used in desperately ill patients with complex patho-physiology.
rGM-CSF causes asthenia, rash, malaise and flu like syndrome, all of which are readily reversed once therapy has been withdrawn. Intravenous administration increases the incidence of phlebitis. Cutaneous infections have been reported. Respiratory distress too has reported and so care must be taken while administering it to patients with pre-existing respiratory disease. Also, capillary leak syndrome, leading to pleural/ pericardial effusion and hence hypoproteinaemia has also been reported. There is also reason to believe that children tolerate rGM-CSF better than adults.
rGM-CSF too has been well tolerated in clinical trials. Bone pain, which was readily relieved on cessation of treatment and analgesics, has been reported. Cutaneous eruptions, worsening of pre-existing psoriasis and nausea, fever, flu-like syndrome and capillary leak syndrome have also been reported. Serum levels of leucocyte alkaline phosphate, lactate dehydrogenase and uric acid may increase. For better understanding of these side effects, they have been listed in composite form in [Table:4].
What remains to be discussed now is the effective dosage and routes of administration of these cytokines in various disease states. Although continuous intravenous infusion has proved efficacious, subcutaneous administration has a definite advantage because the patient can self administer this therapy.
As regards dosage schedule, the relative efficacy of various dosage regimens remains to be evaluated in controlled comparative trials. Optimal dosage and duration of transplant and failure or delay of engraftment are not yet established.
Granulopoiesis stimulated by these CSFs may be very rapid and hence twice weekly complete blood count with differential count is recommended to avoid possible complications.
The cost of 10 day therapy with rGM-CSF is Rs. 70,000 for autologous bone marrow transplantation. rG-CSF is not available in India.
The use of these cytokines to accelerate granulocyte recovery is a novel therapeutic approach to the management of immunocompromised patients. The ability of these CSF's to intensity anti-neoplastic treatment is an exciting new therapeutic possibility. Although there is still controversy regarding their use in myelodysplasia and in patients with leukaemic clones, their use remains a potential field of exploitation.
In addition to the use of CSFs in these conditions, other potential targets for their use have been envisaged. Myeloid leukaemia stem cells have a high capacity for self-renewal without differentiation leading to progressive accumulation of leukaemic cells in the periphery and bone marrow. Hence a very pertinent clinical use of these CSF's is that they can induct differentiation of these leukemic cells and ultimately extinguish the clone. This has been confirmed in experimental conditions. However, when used in patients, they have been found to stimulate proliferation of residual malignant cells inpatients with acute myeloid leukaemia undergoing cancer chemotherapy and/or marrow transplantation.
Another potential use of these CSFs is that they can be used to overcome growth kinetic resistance of cell cycle S-phase specific cytotoxic agents since they can stimulate proliferation of leukaemic cells and thus render them susceptible to these agents.
We see therefore, that rGM-CSF and rG-CSF are useful additions in our therapeutic armamentarium.
[Figure - 1]