THE ONSET OF A REFRACTORY RESPONSE TO ANDROGENS IN THE ANEMIA OF BONE MARROW FAILURE FRANK H. GARDNER, M.D. AND (BY INVITATION) EMMANUEL C. BESA, M.D. GALVESTON
Clinical reports have accumulated during the past 15 years regarding the benefit of androgens in a variety of anemias. Physicians have had different opinions regarding the type of androgen used and claims have been made that one preparation has been better than another.' These opinions have at times reflected a preference for the use of oral androgens over parenteral preparations. In part this has been associated with a concern in the use of injectable testosterone esters in patients with thrombocytopenia. There has been no data available to predict if there would be a hematological response with any preparation. Historically the long acting injectable esters of testosterone have been thought to be more efficacious because of the slow release of the native hormone from the oil vehicle.2 Early in the evaluation of androgens it was evident that the rodent would respond to testosterone, possibly from an elevation of the erythropoietin levels.3 In these studies following the initial erythropoietin response, evidence of bone marrow stimulation by reticulocytosis or increased incorporation of radioactive iron into hemoglobin was demonstrated. Some efforts have been made to compare the various androgen preparations that have been used clinically.4'5 Several years ago our laboratory made a study to evaluate in the ex-hypoxic polycythemic mouse system, the various clinical androgens that have been acceptable for treatment of the anemia bone marrow failure.6 One may note, (Fig. 1), that there was a similar response for most of the clinical preparations tested. This was demonstrated both by the reticulocyte response and the incorporation of radioactive iron. It was of interest that one preparation, nandrolone decanoate, was much more stimulatory than other hormone preparations used. On the basis of this observation in our clinical From the Division of Hematology-Oncology, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77550. Emmanuel C. Besa, M.D., Division of Hematology and Oncology, Department of Medicine, The Medical College of Pennsylvania and Hospital, Philadelphia, Pennsylvania. Supported by Research Grant DHEW 7R01 CA 19997 01; The John A. Hartford Foundation, Incorporated; and a grant (RR-73) from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. 108
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FIG. 1. Heme incorporation in the ex hypoxic mouse following intraperitoneal injection of a variety of clinical androgens. All preparation given on an equimolar dosage. Nandrolone decanoate was most stimulatory and had highest serum erythropoietin levels (not graphed) in the group tested. Data derived from reference 6.
investigations this preparation has been used in a majority of patients in recent years.7 Despite these comparisons in the rodent it became obvious there was no predictable way to state that one androgen would be better than another for stimulating the bone marrow. Earlier studies by Bernard and his associates emphasized that patients who did not utilize a tracer dose of radioactive iron probably could not be responsive to any of the testosterone preparations.8 This evaluation was made on the basis of two observations: 1) Patients who incorporated less than 20% of a tracer dose of radioactive iron 10 days after injection into circulating hemoglobin were thought to have little or no residual marrow cellularity on which androgens might be expected to act. 2) Isotope surface scanning of the skeleton (sacrum) in patients who would respond showed evidence that the radioactive iron initially was localized in the marrow with a slow decline over about a 5 day interval as the isotope was incorporated in circulating hemoglobin. If neither of these observations suggested a residual nidus of erythroid stem cells, one would probably not see a response with any of the hormonal preparations. These observations have been repeated in our laboratory many times and we would agree that in the absence of such a residual marrow reservoir patients have had a poor prognosis.9 This observation is emphasized for in the absence of any predictable marrow activity, one probably cannot expect
FRANK H. GARDNER AND EMMANUEL C. BESA 110 to see a response to the various androgen preparations. In our experiences this type of physiological measurement has been more helpful in predicting a hematological response than one might expect from the cytology of a bone marrow core or aspiration biopsy.10 Multiple core biopsies have demonstrated that one may find marrow islands left in a hypoplastic or fibrotic marrow in different parts of the skeleton. However in most clinical studies numerous marrow samples cannot be obtained. Many treatment failures have been attributed to androgens when there was inadequate marrow reserves to expect a response. In studies reported herein all patients had evidence from the radioiron studies that they could respond to androgens. More recently skeletal scanning with 1llndium Chloride has been used to indicate marrow erythroid activity. This procedure has been used in place of radioiron studies, and the preliminary studies are encouraging.-" Utilizing information described above during the past years we have collected a group of patients who have been of special interest to us in that they did not respond to androgens as we might have anticipated. In each instance the patients received a three month course of androgen therapy. The oral or injectable testosterone preparation was given on a schedule of 5 mg/kg orally daily or parenterally weekly during the three month interval. In some instances the medication was changed to an oral preparation because of the patients dislike for injections. In most patients our laboratory has preferred to use parenteral androgens to avoid the risk of cholestatic hepatitis. 12 In the other patients the medication was changed since usually we have observed a hematological response in 3 months with these pharmacological doses. As noted in Table I, a group of patients with a variety of etiologies for bone marrow failure have been tabulated who failed to respond to one androgen but did have hematological improvement with a second preparation. One TABLE I
Patients with Bone Marrow Failure Responding to Different Androgens After No Response to the Initial Preparation Patient
Age
Sex
1 2 3 4 5 6 7 8 8A 9 10 11 12 13
62 41 10 52 68 56 22 64 64 63 66 30 70 26
F M
M M M M M
Aplasia-idio.
Aplasia-PNH Refractory anemia Refractory anemia Fanconi's anemia
M
M M M M
Diagnosis Aplasia-drug Aplasia-drug
F F
No Response
Response
Testosterone enanthate
Oxymetholone
Fluoxymesterone
Testosterone enanthate Nandrolone decanoate Testosterone cypionate (Plts) Nandrolone decanoate Nandrolone decanoate Oxymetholone
Oxymetholone Oxymetholone (Plts) Testosterone enanthate Oxymetholone Nandrolone decanoate
Myelofibrosis
Oxymetholone
Fluoxymesterone
Myelofibrosis Myelofibrosis Myelofibrosis
Fluoxymesterone
Nandrolone decanoate Testosterone enanthate Testosterone enanthate Etiocholanolone Etiocholanolone T.E. + Etiocholanolone
Myelofibrosis Myelofibrosis Myelofibrosis
Fluoxymesterone Nandrolone decanoate Nandrolone decanoate Nandrolone decanoate Testosterone enanthate (T.E.)
REFRACTORY RESPONSE TO ANDROGENS IN ANEMIA
illl
patient, No. 4, is listed who had had no improvement in his platelet count despite a nine month course of oxymetholone. There has been some slow decline in his blood count, but on changing to a parenteral injection there was a sudden and rapid improvement in his thrombocytopenia within two weeks and improvement in his hematocrit. In this collection of patients one may note that despite the original preparation there were responses to another androgen whether it be oral or parenteral. This improvement is quite dramatically outlined (Fig. 2) whereby a child with hypoplastic anemia required multiple platelet transfusions associated with severe epistaxis and anemia. When the androgen preparation was altered there was a rapid improvement in his hematocrit and a slower but significant elevation in his platelet count. It should be emphasized that despite the initial and usual erythroid response to TC
APLASTIC ANEMIA 10
2 OXYMETHOLONE
250MG/DAY E PREDNISOiE 80 MG /DAY NANDROLONE DECANOATE
4
6 MONTHS
8
10
/OK
FIG. 2. Treatment of Aplastic Anemia. Alteration in the androgen used was followed by a rapid increase in the hematocrit with a slower rise in the platelet count. Values continue to remain elevated at 14 months despite reduction in the dosage (100 mg/wk) of nandrolone decanoate. Data kindly furnished by Dr. Mary E. Haggard.
112
FRANK H. GARDNER AND EMMANUEL C. BESA
testosterone in bone marrow failure, the thrombocytopenia improves slowly over months. In these patients the androgen therapy is continued even though there is a complete erythroid response until the platelet count is at least in the low range of normal (above 100,000 mm3). Three patients have been observed with myelofibrosis who have become refractory to the androgen preparation after an initial response. In each instance the patient initially had improvement on testosterone therapy with marked increase in the red cell mass, and the achievement of relatively normal blood values. While the preparation was continued there was onset of anemia requiring resumption of blood transfusions. Increasing the dose of androgen was helpful in two patients but the third patient only improved with selection of another testosterone preparation. One patient, No. 8, has been tried on six androgen preparations. In three instances he became refractory with recurrence of anemia while receiving the testosterone, and he was unresponsive to two preparations. However by using the different testosterone esters he has been able to maintain a normal hematocrit. Two patients with myelofibrosis had a response to parenteral etiocholanolone after being unresponsive to nandrolone decanoate. Is there a mechanism that we can explain such a response? At the moment there are no measurements that will allow us to define the unresponsive patient. Clinically from these observations we should recognize that patients can respond to androgen if different preparations are tried. Indeed some of the problems associated in the interpretation of the past literature has been the decision to reject androgens if the patient has not responded to a single preparation, or to the physician's favorite androgen. It is hoped that these observations will encourage physicians to try numerous preparations in the patients who have bone marrow failure and isotopic laboratory evidence that they might be responsive to androgens. In the past 20 years a variety of drugs have been reported to induce or alter the enzyme function of liver. These responses enhance metabolic changes of liver microsomes so that the metabolism of other drugs may be rapidly changed by increased conjugation or hydroxylation. 13 In some instances these appear to be correlated with increase of cytochrome P450. It is of interest that several years ago Resan and his associates made an interesting observation that may have some bearing on these clinical problems. 14 Rats were treated with phenobarbital concurrently while they received testosterone propionate. With this therapy over a 6 week interval they did not have the expected increase in their red cell mass that was noted in control paired animals receiving only testosterone. Without the increase in red cell mass they also did not demonstrate an elevation of the serum erythropoietin levels that was observed in the controls that received only testosterone. From this data the altered
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microsomal enzyme changes enhanced the biotransformation of the testosterone so that it was not available for marrow stem cell receptors. A variety of drugs are metabolized more rapidly when given with phenobarbital. 15 Some drugs not only stimulate enhanced metabolism of other compounds, but also will increase the metabolism of the drug per se with continued administration. Phenobarbital, tolbutamide, and probenecid are examples of drugs that increase their own metabolic conjugation. In the rat liver testosterone will enhance hydroxylation to increase the secretion of 6,3 hydroxycortisol (,8-cortol). In contrast, estradiol will decrease the excretion rate. 16 Similarly both adult male and female rats receiving a variety of androgens had two to threefold increases in the liver microenzyme systems that metabolized hexobarbital and TPNH oxidation. When testosterone propionate, 19-nortestosterone, and methyl-testosterone were compared the animal studies suggested that the non-virilizing and more anabolic androgens as defined by 19-nortestosterone induced greater liver microsomal enzyme changes than did the classical virilizing preparations.17 Finally it must be emphasized that the stimulation of reductases and the activity of the liver microsomes to metabolize drugs are depressed in castrates of both sexes in the rat. When androgens are given to these animals there is a marked induction of these enzymes by testosterone.'8 These observations then may be likened to the absence of erythrocytosis when phenobarbital is administered along with testosterone. In certain patients androgens rapidly increase conjugation and excretion of the testosterone so that it is not available to bind to the receptor sites on the stem cell. The alteration in the manner of administration of the androgen or the type of ester used then may not be metabolized in a like fashion to the previous preparation. Hence the different androgen is available for circulation to the stem cell for erythroid stimulation. We do not have any data to confirm this concept in our current clinical studies, but believe that the clinical responses would fit this concept. Another proposal for these clinical observations might also be considered. As a general scheme of androgen metabolism, the unsaturated testosterone is metabolized by 5a and 5,8 reductase on ring A in two equal major excretion patterns; namely androsterone (5a derivatives), and etiocholanolone (5,8 derivatives). At the present time we have no data as to the androgen metabolite that binds to the receptor sites on the committed stem cell. Certainly the 5a dihydrotestosterone can stimulate erythropoiesis when administered in the rodent system, but there is no assurance that this preparation will specifically bind in that form to the erythroid precursor. During the past five years, evidence has accumulated in the rodent that the 5,8 pathway metabolites can be active in
14
FRANK H. GARDNER AND EMMANUEL C. BESA
inducing heme synthesis in the rodent as well as improving the anemia in patients with refractory anemias.19' 20 In the rodent's system as in man the administration of the 5,/ derivative (etiocholanolone) has not been associated with elevation of serum erythropoietin levels as is observed with the 5a compounds.2' Hence it has been suggested that the bone marrow stem cell responsive to hormonal stimulation may well initially bind to its receptor site 5,B derivatives, (Fig. 3). Small amounts of erythropoietin are necessary for maturation of these committed erythroid cells, but high levels of erythropoietin per se are not required for cellular division. Certainly the high erythropoietin levels in refractory anemias do not appear to be helpful. Many times the androgen will elevate erythropoietin without a hematologic response. How can we correlate this data with the refractory responses noted? All of our thoughts must be hypotheses at the present time. We would propose that the metabolism of some patie ts is such that there is an alteration in the amount of 5,3 reductase. Hence instead of the usual approximately 1 to 1 ratio between the two main derivatives of testosterone, there is a predominance of 5a reductase derivatives (androsterone). This is a stimulatory pathway for erythropoietin production but not necessarily a predominant pathway for a strong receptor binding metabolite to the marrow erythroid cell. Patients who have little or no 5,B metabolite derivatives formed or who have more rapid conjugation of such metabolites have inadequate hormones available for recognition by the marrow receptor sites. Hence these patients may have no response to that androgen preparation. These patients may then respond to etiocholanolone since the initial enzyme step is not needed to prepare Postulated Mechanism of Androgen Steroids on Erythropoiesis
5-H Steroidr Testosterone ESF
Uncommitted Stem Cell Compartment
* 0 ( *
Committed Stem Cell Compartment
Erythroid Precursors
FIG. 3. Testosterone or 5,B metabolites may act on stem cell receptors. Erythropoietin (ESF) is not needed to initiate stem cell proliferation in this concept. The active component of testosterone may be a 5,B metabolite that is formed prior to binding with the stem cell receptor. Redrawn from reference 22.
REFRACTORY RESPONSE TO ANDROGENS IN ANEMIA
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the 5,3 metabolite. In contrast the patients who become refractory after initial response may have an enzyme induction from the testosterone so to conjugate themselves out of availability for the marrow stem cell. The clinical observation on the response to etiocholanolone may suggest that this concept can be tested. SUMMARY Patients with bone marrow failure may have a hematopoietic response to an androgen preparation after they have no response from the use of a previous testosterone preparation. The failure to try a variety of hormone preparations may lead to improper conclusions as to a potential marrow proliferation for a clinical response. In some instances the use of a 5,8 steroid metabolite may initiate stem cell proliferation in patients previously refractory to testosterone or nontestosterone esters. These observations suggest that some patients refractory to testosterone may be unable to form adequate 5,8 metabolites by alterations in 5a reductase or enhanced metabolic transformation of the androgen. All patients should be evaluated with several androgen esters before concluding that their marrow cannot achieve a hematopoietic response. 1.
2. 3. 4.
5. 6.
REFERENCES ALLEN, D. M., FINE, M. H., NECHELES, T. F. AND DAMESHEK, W.: Oxymetholone therapy in aplastic anemia. Blood 32: 83, 1968. GARDNER, F. H., AND PRINGLE, J. C. JR.: Androgens and erythropoiesis I. Preliminary clinical observation. Arch. Intern. Med. 107: 846, 1961. FRIED, W., AND GURNEY, C. W.: The erythropoietic-stimulating effects of androgens. Ann. N.Y. Acad. Sci. 149: 356, 1968. DUARTE, L., SANCHEZ-MEDAL, L., LABARDINI, J. AND ARRIAGA, L.: The erythropoietic effect of anabolic steroids. Proc. Soc. Exptl. Biol. Med. 125: 1030, 1967. MOLINARI, P. F., AND ROSENKRANTZ, H. J.: Erythropoietic activity and androgenic implications of 29 testosterone derivatives in orchiectomized rats. J. Lab. Clin. Med. 78: 399, 1971. GORSHEIN, D., MURPHY, S., AND GARDNER, F. H.: Comparative study on the erythropoietic effects of androgens and their mode of action. J. Appl. Physiology 35: 376,
1973. 7. BESA, E. C., GORSHEIN, D., AND GARDNER, F. H.: Blood volume changes in normal and various anemic states in man following androgens. Arch. Int. Med. 133: 418, 1974. 8. NAJEAN, Y., DRESCH, C., SCHAISON, G., CHASSIGNEUX, J., JACQUILLAT, C. AND BERNARD, J.: Etude de l'effet de l'androgenotherapie au cours des insuffisances medullaires. Presse Med. 74: 2537, 1966. 9. GARDNER, F. H., AND NATHAN, D. G.: Androgens and erythropoiesis. III. Further evaluation of testosterone treatment of myelofibrosis. New Eng. J. Med. 274: 420, 1966. 10. GARDNER, F. H.: Aplastic anemia. Cont. Ed. for Fam. Phys. 4: 114, 1976. 11. BUNN, F. H., MCNEIL, B. J., ROSENTHAL, D. S., AND KRANTZ, S. B.: Bone marrow imaging in pure red cell aplasia. Arch. Int. Med. 136: 1169, 1976.
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12. GARDNER, F. H.: Use of androgens in the treatment of bone marrow suppression. In Dimitrov, N. V. and Nodine, J. H. (eds.) XXIX Hahnemann International Symposium on Drugs and Hematologic Reactions. Grune and Stratton, New York and London, p. 357, 1974. 13. KUNTZMAN, R., WELCH, R., AND CONNEY, A. H.: Factors influencing steroid hydroxylases in liver microsomes. Adv. Enzyme Regul. 14: 149, 1966. 14. RESAN, T. K., SHAHIDI, N. T., AND KORST, D. R.: The effect of phenobarbital on testosterone-induced erythropoiesis. J. Lab. Clin. Med. 79: 187, 1972. 15. CONNEY, A. H., AND BURNS, J. J.: Metabolic interactions among environmental chemicals and drugs. Science 178: 576, 1972. 16. KATO, R., TAKAHASHI, A., AND OMORI, Y.: Effect of androgen and estrogen on the hydroxylation of steroid hormones by rat liver microsomes. Endocrinol. Japon. 16: 653, 1969. 17. GILLIE, R. B., WUHMANN-WENNHOLD, A., JOHNSON, L. R. AND NELSON, D. H.: Effects of testosterone and estradiol in vivo on hepatic cortisol metabolism in vitro. Proc. Soc. Exp. Biol. and Med. 142: 54, 1973. 18. CONNEY, A. H., LEVIN, W., AND KUNTZMAN, R.: Effects of drugs and environmental chemicals on steroid metabolism. Clin. Pharmacol. Ther. 14: 738, 1973. 19. GORSHEIN, D., AND GARDNER, F. H.: Erythropoietic activity of steroid metabolites in mice. Proc. Natl. Acad. Sci. 65: 564, 1970. 20. BESA, E. C., DALE, D. C., WOLF, S. M. AND GARDNER, F. H.: Treatment of refractory anemia with etiocholanolone and prednisolone (etio-pred). Blood 46: 1001, 1975. 21. GORDON, A. S., ZANJANI, E. D., LEVERE, R. D. AND KAPPAS, A.: Stimulation of mammalian erythropoiesis by 5,8-H steroid metabolites. Proc. Natl. Acad. Sci. U.S.A. 65: 919, 1970. 22. NECHELES, T. F.: Studies on the control of hemoglobin synthesis: a model of erythroid differentiation based upon the in vitro effect of erythropoietin and 5,8-H steroid. Z. Gesante Blutforsch 10: 53 (suppl.), 1972.
DISCUSSION DR. RICHARD W. VILTER (Cincinnati): Frank, I happen to be a believer, but there are those who are not, as you well know. When one looks at the aplastic anemia patients as a group, there are those who go downhill and usually die unless they are handled by bone marrow transfusions. There are those that have an up-and-down fluctuation and then there's another very favorable group that goes on to spontaneous remission. Presumably the middle group is the one with which you and I are dealing. What is the evidence that this middle group, that you have shown so nicely on these slides, and mylofibrosis patients also -are not responding purely by chance to the androgen. There are many who claim that placebo effect of the same kind can be demonstrated in these two groups. DR. GARDNER: This is an important point. These patients have usually been defined as having abnormal marrow activity. The aplastic patient, following the observation of Jean Bernard about mid-sixties, (evidence of iron incorporation, poor evidence of iron intake and release from the skeleton) probably does not have enough marrow stem cells available on which to respond to any stimulus. With these patients we would immediately try to determine if a bone marrow transfusion could be done. Now, what I've tried to show with a few slides is evidence of response to androgen and etiocholanalone. When the drugs were stopped, he again had the onset of anemia. The anemia improved with reinstitution of the androgen. I think that if you don't evaluate the drug in this manner you cannot demonstrate that you have an effective agent. Now, we have tried a number of patients with placebo therapy, and we hope to have that paper published in the near future to comment on the observation of Dr. Kaplan, made in Minneapolis, in which he
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has observed a hematological response to sesame oil. We have a unique group of patients that could be tried on alternate schedules of testosterone and sesame oil. The cell mass was measured, and we did not see any response to the placebo sesame oil but did observe a large increase in the red cell mass with testosterone. Also we have been convinced that if the patient doesn't have adequate marrow by using physiological measurements (certainly the bone marrow biopsy is not adequate) that they shouldn't be studied with androgen. The aplastic patients ought to have bone marrow transplantation if they have compatible donors, available. I think that if you don't continue to replace or stop androgen therapy, to see if there a recurrence of anemia you may be fooled by a spontaneous remission. DR. CHARLES EDWIN BUTTERWORTH (Birmingham): Frank, I'd like to congratulate you on another lovely clinical study and ask you a couple of questions on a topic that disturbs me. As you know we talked on the beach yesterday. First, I think we're thinking nowadays of target tissue, tissue capable of responding to steroid hormones, as having specific binders which non-responding tissues lack. My question is: (perhaps you don't have an answer, but would like to speculate) why is bone marrow a target tissue in the first place, and if it is a target tissue that will bind steroid hormones, might this form the basis of some test in vitro that would help you predict responsiveness. Also, as you have shown for androgen, etiocholanalone is essentially non-androgenic, could you use such an in uitro test to develop some sort of steroid that might produce a response that eliminates this very troublesome problem of androgenic side-effects in the females. DR. GARDNER: One suggestion that the bone marrow is a receptor organ, is the statistical difference in male and female red cell mass. There are many studies on this fact from various laboratories, and we have the changes in red-cell mass within a variety of rodent studies when the animal is castrated. Now, the technique of measuring a receptor which we might use, as a diagnostic procedure, is of course right in the forefront. There is data that the hormone can act on marrow cell receptors. One of the problems, of course, is a sampling in man -where can you get enough marrow in the aplastic patient to find the cells? Well, this may well be not necessarily related to the use of the erythroblast. Indeed, we're hoping that some of the our culture studies with bone marrow fibroblasts, following the observations of different testosterone receptors in fibroblasts that the Hopkins group has brought out, may be useful as a predictor for responsiveness in our patients. Our data is too scanty to make a proposal that it will work now. I think that's the area where we should go, and I'm hopeful that some of the earlier data of Minguell and Manwaring will be useful models for the prediction. However we have to find a way to get adequate tissue in the marrow-failure patient to study for receptor sites. DR. NATHANIEL I. BERLIN (Chicago): Frank, you know, we've discussed this a number of times, and as I look at the slides again, I find that you have not discussed with us the role of the prednisone which some of these patients were given. You may remember those few patients that we treated for another purpose and from which we seemed to be able to develop a temporal relationship between the onset of prednisone therapy and a very nice hemalogical response. I realize this complicates your problem, but maybe you'd like to speculate on the role of prednisone in these patients. DR. GARDNER: The two patients discussed here -the young lady who would not take androgen further and received etiocholanolone had not responded to a similar dose of prednisone - and the man who had responded to etiocholanalone had an onset of anemia when he was given only prednisone in the past few months. We have others in the series, but I would emphasize that we have in the literature only a few rare patients that appear to have responded to prednisone. Never in my experience have I seen a refractory anemia respond to prednisone unless it was on the basis of altering the RE system to control a hemolytic component.
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FRANK H. GARDNER AND EMMANUEL C. BESA
DR. FRED KERN, JR. (Denver): I got the impression from your talk that there were receptor sites on stem cell that differentiates between the 5-alpha and 5-beta metabolites. Is that correct? DR. GARDNER: Well, no we can't say that has been shown at all. First of all, a lot of these materials aren't even available in isotope labeled forms to study. I should say that in the chick embryo blood island, it is interesting-and this doesn't answer your question completely-that these cells on incubation, will convert about 60% of the testosterone to 5 beta metabolites. But the method of proving that the steroid metabolite is inducing proliferation is very difficult in that culture system. We are only making this inference without adequate proof at present.
Clinical reports have accumulated during the past 15 years regarding the benefit of androgens in a variety of anemias. Physicians have had different opinions regarding the type of androgen used and claims have been made that one preparation has been better than another.' These opinions have at times reflected a preference for the use of oral androgens over parenteral preparations. In part this has been associated with a concern in the use of injectable testosterone esters in patients with thrombocytopenia. There has been no data available to predict if there would be a hematological response with any preparation. Historically the long acting injectable esters of testosterone have been thought to be more efficacious because of the slow release of the native hormone from the oil vehicle.2 Early in the evaluation of androgens it was evident that the rodent would respond to testosterone, possibly from an elevation of the erythropoietin levels.3 In these studies following the initial erythropoietin response, evidence of bone marrow stimulation by reticulocytosis or increased incorporation of radioactive iron into hemoglobin was demonstrated. Some efforts have been made to compare the various androgen preparations that have been used clinically.4'5 Several years ago our laboratory made a study to evaluate in the ex-hypoxic polycythemic mouse system, the various clinical androgens that have been acceptable for treatment of the anemia bone marrow failure.6 One may note, (Fig. 1), that there was a similar response for most of the clinical preparations tested. This was demonstrated both by the reticulocyte response and the incorporation of radioactive iron. It was of interest that one preparation, nandrolone decanoate, was much more stimulatory than other hormone preparations used. On the basis of this observation in our clinical From the Division of Hematology-Oncology, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77550. Emmanuel C. Besa, M.D., Division of Hematology and Oncology, Department of Medicine, The Medical College of Pennsylvania and Hospital, Philadelphia, Pennsylvania. Supported by Research Grant DHEW 7R01 CA 19997 01; The John A. Hartford Foundation, Incorporated; and a grant (RR-73) from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. 108
REFRACTORY RESPONSE TO ANDROGENS IN ANEMIA
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% 59FE INCORPORATION
p~~~~~~~~~ ; 9~~~ !1 ; CONTROL
4
'
I
[........
TESTOSTERONE ENANTHATE TESTOSTERONE TESTOSTERO
_____________________________-----.-----
CYPIONATE TESTOSTERONE
........
.
PROPIONATE OXYMETHOLONE Ei
ETHYLESTRENOL
Z
-
.: ;:-:
t-:;:;::
NANDROLONE .:.:-:-:-:::::: PHENYL PROPIONATE NANDROLONE
^
...............
..........................
DECANOATE
FIG. 1. Heme incorporation in the ex hypoxic mouse following intraperitoneal injection of a variety of clinical androgens. All preparation given on an equimolar dosage. Nandrolone decanoate was most stimulatory and had highest serum erythropoietin levels (not graphed) in the group tested. Data derived from reference 6.
investigations this preparation has been used in a majority of patients in recent years.7 Despite these comparisons in the rodent it became obvious there was no predictable way to state that one androgen would be better than another for stimulating the bone marrow. Earlier studies by Bernard and his associates emphasized that patients who did not utilize a tracer dose of radioactive iron probably could not be responsive to any of the testosterone preparations.8 This evaluation was made on the basis of two observations: 1) Patients who incorporated less than 20% of a tracer dose of radioactive iron 10 days after injection into circulating hemoglobin were thought to have little or no residual marrow cellularity on which androgens might be expected to act. 2) Isotope surface scanning of the skeleton (sacrum) in patients who would respond showed evidence that the radioactive iron initially was localized in the marrow with a slow decline over about a 5 day interval as the isotope was incorporated in circulating hemoglobin. If neither of these observations suggested a residual nidus of erythroid stem cells, one would probably not see a response with any of the hormonal preparations. These observations have been repeated in our laboratory many times and we would agree that in the absence of such a residual marrow reservoir patients have had a poor prognosis.9 This observation is emphasized for in the absence of any predictable marrow activity, one probably cannot expect
FRANK H. GARDNER AND EMMANUEL C. BESA 110 to see a response to the various androgen preparations. In our experiences this type of physiological measurement has been more helpful in predicting a hematological response than one might expect from the cytology of a bone marrow core or aspiration biopsy.10 Multiple core biopsies have demonstrated that one may find marrow islands left in a hypoplastic or fibrotic marrow in different parts of the skeleton. However in most clinical studies numerous marrow samples cannot be obtained. Many treatment failures have been attributed to androgens when there was inadequate marrow reserves to expect a response. In studies reported herein all patients had evidence from the radioiron studies that they could respond to androgens. More recently skeletal scanning with 1llndium Chloride has been used to indicate marrow erythroid activity. This procedure has been used in place of radioiron studies, and the preliminary studies are encouraging.-" Utilizing information described above during the past years we have collected a group of patients who have been of special interest to us in that they did not respond to androgens as we might have anticipated. In each instance the patients received a three month course of androgen therapy. The oral or injectable testosterone preparation was given on a schedule of 5 mg/kg orally daily or parenterally weekly during the three month interval. In some instances the medication was changed to an oral preparation because of the patients dislike for injections. In most patients our laboratory has preferred to use parenteral androgens to avoid the risk of cholestatic hepatitis. 12 In the other patients the medication was changed since usually we have observed a hematological response in 3 months with these pharmacological doses. As noted in Table I, a group of patients with a variety of etiologies for bone marrow failure have been tabulated who failed to respond to one androgen but did have hematological improvement with a second preparation. One TABLE I
Patients with Bone Marrow Failure Responding to Different Androgens After No Response to the Initial Preparation Patient
Age
Sex
1 2 3 4 5 6 7 8 8A 9 10 11 12 13
62 41 10 52 68 56 22 64 64 63 66 30 70 26
F M
M M M M M
Aplasia-idio.
Aplasia-PNH Refractory anemia Refractory anemia Fanconi's anemia
M
M M M M
Diagnosis Aplasia-drug Aplasia-drug
F F
No Response
Response
Testosterone enanthate
Oxymetholone
Fluoxymesterone
Testosterone enanthate Nandrolone decanoate Testosterone cypionate (Plts) Nandrolone decanoate Nandrolone decanoate Oxymetholone
Oxymetholone Oxymetholone (Plts) Testosterone enanthate Oxymetholone Nandrolone decanoate
Myelofibrosis
Oxymetholone
Fluoxymesterone
Myelofibrosis Myelofibrosis Myelofibrosis
Fluoxymesterone
Nandrolone decanoate Testosterone enanthate Testosterone enanthate Etiocholanolone Etiocholanolone T.E. + Etiocholanolone
Myelofibrosis Myelofibrosis Myelofibrosis
Fluoxymesterone Nandrolone decanoate Nandrolone decanoate Nandrolone decanoate Testosterone enanthate (T.E.)
REFRACTORY RESPONSE TO ANDROGENS IN ANEMIA
illl
patient, No. 4, is listed who had had no improvement in his platelet count despite a nine month course of oxymetholone. There has been some slow decline in his blood count, but on changing to a parenteral injection there was a sudden and rapid improvement in his thrombocytopenia within two weeks and improvement in his hematocrit. In this collection of patients one may note that despite the original preparation there were responses to another androgen whether it be oral or parenteral. This improvement is quite dramatically outlined (Fig. 2) whereby a child with hypoplastic anemia required multiple platelet transfusions associated with severe epistaxis and anemia. When the androgen preparation was altered there was a rapid improvement in his hematocrit and a slower but significant elevation in his platelet count. It should be emphasized that despite the initial and usual erythroid response to TC
APLASTIC ANEMIA 10
2 OXYMETHOLONE
250MG/DAY E PREDNISOiE 80 MG /DAY NANDROLONE DECANOATE
4
6 MONTHS
8
10
/OK
FIG. 2. Treatment of Aplastic Anemia. Alteration in the androgen used was followed by a rapid increase in the hematocrit with a slower rise in the platelet count. Values continue to remain elevated at 14 months despite reduction in the dosage (100 mg/wk) of nandrolone decanoate. Data kindly furnished by Dr. Mary E. Haggard.
112
FRANK H. GARDNER AND EMMANUEL C. BESA
testosterone in bone marrow failure, the thrombocytopenia improves slowly over months. In these patients the androgen therapy is continued even though there is a complete erythroid response until the platelet count is at least in the low range of normal (above 100,000 mm3). Three patients have been observed with myelofibrosis who have become refractory to the androgen preparation after an initial response. In each instance the patient initially had improvement on testosterone therapy with marked increase in the red cell mass, and the achievement of relatively normal blood values. While the preparation was continued there was onset of anemia requiring resumption of blood transfusions. Increasing the dose of androgen was helpful in two patients but the third patient only improved with selection of another testosterone preparation. One patient, No. 8, has been tried on six androgen preparations. In three instances he became refractory with recurrence of anemia while receiving the testosterone, and he was unresponsive to two preparations. However by using the different testosterone esters he has been able to maintain a normal hematocrit. Two patients with myelofibrosis had a response to parenteral etiocholanolone after being unresponsive to nandrolone decanoate. Is there a mechanism that we can explain such a response? At the moment there are no measurements that will allow us to define the unresponsive patient. Clinically from these observations we should recognize that patients can respond to androgen if different preparations are tried. Indeed some of the problems associated in the interpretation of the past literature has been the decision to reject androgens if the patient has not responded to a single preparation, or to the physician's favorite androgen. It is hoped that these observations will encourage physicians to try numerous preparations in the patients who have bone marrow failure and isotopic laboratory evidence that they might be responsive to androgens. In the past 20 years a variety of drugs have been reported to induce or alter the enzyme function of liver. These responses enhance metabolic changes of liver microsomes so that the metabolism of other drugs may be rapidly changed by increased conjugation or hydroxylation. 13 In some instances these appear to be correlated with increase of cytochrome P450. It is of interest that several years ago Resan and his associates made an interesting observation that may have some bearing on these clinical problems. 14 Rats were treated with phenobarbital concurrently while they received testosterone propionate. With this therapy over a 6 week interval they did not have the expected increase in their red cell mass that was noted in control paired animals receiving only testosterone. Without the increase in red cell mass they also did not demonstrate an elevation of the serum erythropoietin levels that was observed in the controls that received only testosterone. From this data the altered
REFRACTORY RESPONSE TO ANDROGENS IN ANEMIA
113
microsomal enzyme changes enhanced the biotransformation of the testosterone so that it was not available for marrow stem cell receptors. A variety of drugs are metabolized more rapidly when given with phenobarbital. 15 Some drugs not only stimulate enhanced metabolism of other compounds, but also will increase the metabolism of the drug per se with continued administration. Phenobarbital, tolbutamide, and probenecid are examples of drugs that increase their own metabolic conjugation. In the rat liver testosterone will enhance hydroxylation to increase the secretion of 6,3 hydroxycortisol (,8-cortol). In contrast, estradiol will decrease the excretion rate. 16 Similarly both adult male and female rats receiving a variety of androgens had two to threefold increases in the liver microenzyme systems that metabolized hexobarbital and TPNH oxidation. When testosterone propionate, 19-nortestosterone, and methyl-testosterone were compared the animal studies suggested that the non-virilizing and more anabolic androgens as defined by 19-nortestosterone induced greater liver microsomal enzyme changes than did the classical virilizing preparations.17 Finally it must be emphasized that the stimulation of reductases and the activity of the liver microsomes to metabolize drugs are depressed in castrates of both sexes in the rat. When androgens are given to these animals there is a marked induction of these enzymes by testosterone.'8 These observations then may be likened to the absence of erythrocytosis when phenobarbital is administered along with testosterone. In certain patients androgens rapidly increase conjugation and excretion of the testosterone so that it is not available to bind to the receptor sites on the stem cell. The alteration in the manner of administration of the androgen or the type of ester used then may not be metabolized in a like fashion to the previous preparation. Hence the different androgen is available for circulation to the stem cell for erythroid stimulation. We do not have any data to confirm this concept in our current clinical studies, but believe that the clinical responses would fit this concept. Another proposal for these clinical observations might also be considered. As a general scheme of androgen metabolism, the unsaturated testosterone is metabolized by 5a and 5,8 reductase on ring A in two equal major excretion patterns; namely androsterone (5a derivatives), and etiocholanolone (5,8 derivatives). At the present time we have no data as to the androgen metabolite that binds to the receptor sites on the committed stem cell. Certainly the 5a dihydrotestosterone can stimulate erythropoiesis when administered in the rodent system, but there is no assurance that this preparation will specifically bind in that form to the erythroid precursor. During the past five years, evidence has accumulated in the rodent that the 5,8 pathway metabolites can be active in
14
FRANK H. GARDNER AND EMMANUEL C. BESA
inducing heme synthesis in the rodent as well as improving the anemia in patients with refractory anemias.19' 20 In the rodent's system as in man the administration of the 5,/ derivative (etiocholanolone) has not been associated with elevation of serum erythropoietin levels as is observed with the 5a compounds.2' Hence it has been suggested that the bone marrow stem cell responsive to hormonal stimulation may well initially bind to its receptor site 5,B derivatives, (Fig. 3). Small amounts of erythropoietin are necessary for maturation of these committed erythroid cells, but high levels of erythropoietin per se are not required for cellular division. Certainly the high erythropoietin levels in refractory anemias do not appear to be helpful. Many times the androgen will elevate erythropoietin without a hematologic response. How can we correlate this data with the refractory responses noted? All of our thoughts must be hypotheses at the present time. We would propose that the metabolism of some patie ts is such that there is an alteration in the amount of 5,3 reductase. Hence instead of the usual approximately 1 to 1 ratio between the two main derivatives of testosterone, there is a predominance of 5a reductase derivatives (androsterone). This is a stimulatory pathway for erythropoietin production but not necessarily a predominant pathway for a strong receptor binding metabolite to the marrow erythroid cell. Patients who have little or no 5,B metabolite derivatives formed or who have more rapid conjugation of such metabolites have inadequate hormones available for recognition by the marrow receptor sites. Hence these patients may have no response to that androgen preparation. These patients may then respond to etiocholanolone since the initial enzyme step is not needed to prepare Postulated Mechanism of Androgen Steroids on Erythropoiesis
5-H Steroidr Testosterone ESF
Uncommitted Stem Cell Compartment
* 0 ( *
Committed Stem Cell Compartment
Erythroid Precursors
FIG. 3. Testosterone or 5,B metabolites may act on stem cell receptors. Erythropoietin (ESF) is not needed to initiate stem cell proliferation in this concept. The active component of testosterone may be a 5,B metabolite that is formed prior to binding with the stem cell receptor. Redrawn from reference 22.
REFRACTORY RESPONSE TO ANDROGENS IN ANEMIA
115
the 5,3 metabolite. In contrast the patients who become refractory after initial response may have an enzyme induction from the testosterone so to conjugate themselves out of availability for the marrow stem cell. The clinical observation on the response to etiocholanolone may suggest that this concept can be tested. SUMMARY Patients with bone marrow failure may have a hematopoietic response to an androgen preparation after they have no response from the use of a previous testosterone preparation. The failure to try a variety of hormone preparations may lead to improper conclusions as to a potential marrow proliferation for a clinical response. In some instances the use of a 5,8 steroid metabolite may initiate stem cell proliferation in patients previously refractory to testosterone or nontestosterone esters. These observations suggest that some patients refractory to testosterone may be unable to form adequate 5,8 metabolites by alterations in 5a reductase or enhanced metabolic transformation of the androgen. All patients should be evaluated with several androgen esters before concluding that their marrow cannot achieve a hematopoietic response. 1.
2. 3. 4.
5. 6.
REFERENCES ALLEN, D. M., FINE, M. H., NECHELES, T. F. AND DAMESHEK, W.: Oxymetholone therapy in aplastic anemia. Blood 32: 83, 1968. GARDNER, F. H., AND PRINGLE, J. C. JR.: Androgens and erythropoiesis I. Preliminary clinical observation. Arch. Intern. Med. 107: 846, 1961. FRIED, W., AND GURNEY, C. W.: The erythropoietic-stimulating effects of androgens. Ann. N.Y. Acad. Sci. 149: 356, 1968. DUARTE, L., SANCHEZ-MEDAL, L., LABARDINI, J. AND ARRIAGA, L.: The erythropoietic effect of anabolic steroids. Proc. Soc. Exptl. Biol. Med. 125: 1030, 1967. MOLINARI, P. F., AND ROSENKRANTZ, H. J.: Erythropoietic activity and androgenic implications of 29 testosterone derivatives in orchiectomized rats. J. Lab. Clin. Med. 78: 399, 1971. GORSHEIN, D., MURPHY, S., AND GARDNER, F. H.: Comparative study on the erythropoietic effects of androgens and their mode of action. J. Appl. Physiology 35: 376,
1973. 7. BESA, E. C., GORSHEIN, D., AND GARDNER, F. H.: Blood volume changes in normal and various anemic states in man following androgens. Arch. Int. Med. 133: 418, 1974. 8. NAJEAN, Y., DRESCH, C., SCHAISON, G., CHASSIGNEUX, J., JACQUILLAT, C. AND BERNARD, J.: Etude de l'effet de l'androgenotherapie au cours des insuffisances medullaires. Presse Med. 74: 2537, 1966. 9. GARDNER, F. H., AND NATHAN, D. G.: Androgens and erythropoiesis. III. Further evaluation of testosterone treatment of myelofibrosis. New Eng. J. Med. 274: 420, 1966. 10. GARDNER, F. H.: Aplastic anemia. Cont. Ed. for Fam. Phys. 4: 114, 1976. 11. BUNN, F. H., MCNEIL, B. J., ROSENTHAL, D. S., AND KRANTZ, S. B.: Bone marrow imaging in pure red cell aplasia. Arch. Int. Med. 136: 1169, 1976.
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12. GARDNER, F. H.: Use of androgens in the treatment of bone marrow suppression. In Dimitrov, N. V. and Nodine, J. H. (eds.) XXIX Hahnemann International Symposium on Drugs and Hematologic Reactions. Grune and Stratton, New York and London, p. 357, 1974. 13. KUNTZMAN, R., WELCH, R., AND CONNEY, A. H.: Factors influencing steroid hydroxylases in liver microsomes. Adv. Enzyme Regul. 14: 149, 1966. 14. RESAN, T. K., SHAHIDI, N. T., AND KORST, D. R.: The effect of phenobarbital on testosterone-induced erythropoiesis. J. Lab. Clin. Med. 79: 187, 1972. 15. CONNEY, A. H., AND BURNS, J. J.: Metabolic interactions among environmental chemicals and drugs. Science 178: 576, 1972. 16. KATO, R., TAKAHASHI, A., AND OMORI, Y.: Effect of androgen and estrogen on the hydroxylation of steroid hormones by rat liver microsomes. Endocrinol. Japon. 16: 653, 1969. 17. GILLIE, R. B., WUHMANN-WENNHOLD, A., JOHNSON, L. R. AND NELSON, D. H.: Effects of testosterone and estradiol in vivo on hepatic cortisol metabolism in vitro. Proc. Soc. Exp. Biol. and Med. 142: 54, 1973. 18. CONNEY, A. H., LEVIN, W., AND KUNTZMAN, R.: Effects of drugs and environmental chemicals on steroid metabolism. Clin. Pharmacol. Ther. 14: 738, 1973. 19. GORSHEIN, D., AND GARDNER, F. H.: Erythropoietic activity of steroid metabolites in mice. Proc. Natl. Acad. Sci. 65: 564, 1970. 20. BESA, E. C., DALE, D. C., WOLF, S. M. AND GARDNER, F. H.: Treatment of refractory anemia with etiocholanolone and prednisolone (etio-pred). Blood 46: 1001, 1975. 21. GORDON, A. S., ZANJANI, E. D., LEVERE, R. D. AND KAPPAS, A.: Stimulation of mammalian erythropoiesis by 5,8-H steroid metabolites. Proc. Natl. Acad. Sci. U.S.A. 65: 919, 1970. 22. NECHELES, T. F.: Studies on the control of hemoglobin synthesis: a model of erythroid differentiation based upon the in vitro effect of erythropoietin and 5,8-H steroid. Z. Gesante Blutforsch 10: 53 (suppl.), 1972.
DISCUSSION DR. RICHARD W. VILTER (Cincinnati): Frank, I happen to be a believer, but there are those who are not, as you well know. When one looks at the aplastic anemia patients as a group, there are those who go downhill and usually die unless they are handled by bone marrow transfusions. There are those that have an up-and-down fluctuation and then there's another very favorable group that goes on to spontaneous remission. Presumably the middle group is the one with which you and I are dealing. What is the evidence that this middle group, that you have shown so nicely on these slides, and mylofibrosis patients also -are not responding purely by chance to the androgen. There are many who claim that placebo effect of the same kind can be demonstrated in these two groups. DR. GARDNER: This is an important point. These patients have usually been defined as having abnormal marrow activity. The aplastic patient, following the observation of Jean Bernard about mid-sixties, (evidence of iron incorporation, poor evidence of iron intake and release from the skeleton) probably does not have enough marrow stem cells available on which to respond to any stimulus. With these patients we would immediately try to determine if a bone marrow transfusion could be done. Now, what I've tried to show with a few slides is evidence of response to androgen and etiocholanalone. When the drugs were stopped, he again had the onset of anemia. The anemia improved with reinstitution of the androgen. I think that if you don't evaluate the drug in this manner you cannot demonstrate that you have an effective agent. Now, we have tried a number of patients with placebo therapy, and we hope to have that paper published in the near future to comment on the observation of Dr. Kaplan, made in Minneapolis, in which he
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117
has observed a hematological response to sesame oil. We have a unique group of patients that could be tried on alternate schedules of testosterone and sesame oil. The cell mass was measured, and we did not see any response to the placebo sesame oil but did observe a large increase in the red cell mass with testosterone. Also we have been convinced that if the patient doesn't have adequate marrow by using physiological measurements (certainly the bone marrow biopsy is not adequate) that they shouldn't be studied with androgen. The aplastic patients ought to have bone marrow transplantation if they have compatible donors, available. I think that if you don't continue to replace or stop androgen therapy, to see if there a recurrence of anemia you may be fooled by a spontaneous remission. DR. CHARLES EDWIN BUTTERWORTH (Birmingham): Frank, I'd like to congratulate you on another lovely clinical study and ask you a couple of questions on a topic that disturbs me. As you know we talked on the beach yesterday. First, I think we're thinking nowadays of target tissue, tissue capable of responding to steroid hormones, as having specific binders which non-responding tissues lack. My question is: (perhaps you don't have an answer, but would like to speculate) why is bone marrow a target tissue in the first place, and if it is a target tissue that will bind steroid hormones, might this form the basis of some test in vitro that would help you predict responsiveness. Also, as you have shown for androgen, etiocholanalone is essentially non-androgenic, could you use such an in uitro test to develop some sort of steroid that might produce a response that eliminates this very troublesome problem of androgenic side-effects in the females. DR. GARDNER: One suggestion that the bone marrow is a receptor organ, is the statistical difference in male and female red cell mass. There are many studies on this fact from various laboratories, and we have the changes in red-cell mass within a variety of rodent studies when the animal is castrated. Now, the technique of measuring a receptor which we might use, as a diagnostic procedure, is of course right in the forefront. There is data that the hormone can act on marrow cell receptors. One of the problems, of course, is a sampling in man -where can you get enough marrow in the aplastic patient to find the cells? Well, this may well be not necessarily related to the use of the erythroblast. Indeed, we're hoping that some of the our culture studies with bone marrow fibroblasts, following the observations of different testosterone receptors in fibroblasts that the Hopkins group has brought out, may be useful as a predictor for responsiveness in our patients. Our data is too scanty to make a proposal that it will work now. I think that's the area where we should go, and I'm hopeful that some of the earlier data of Minguell and Manwaring will be useful models for the prediction. However we have to find a way to get adequate tissue in the marrow-failure patient to study for receptor sites. DR. NATHANIEL I. BERLIN (Chicago): Frank, you know, we've discussed this a number of times, and as I look at the slides again, I find that you have not discussed with us the role of the prednisone which some of these patients were given. You may remember those few patients that we treated for another purpose and from which we seemed to be able to develop a temporal relationship between the onset of prednisone therapy and a very nice hemalogical response. I realize this complicates your problem, but maybe you'd like to speculate on the role of prednisone in these patients. DR. GARDNER: The two patients discussed here -the young lady who would not take androgen further and received etiocholanolone had not responded to a similar dose of prednisone - and the man who had responded to etiocholanalone had an onset of anemia when he was given only prednisone in the past few months. We have others in the series, but I would emphasize that we have in the literature only a few rare patients that appear to have responded to prednisone. Never in my experience have I seen a refractory anemia respond to prednisone unless it was on the basis of altering the RE system to control a hemolytic component.
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DR. FRED KERN, JR. (Denver): I got the impression from your talk that there were receptor sites on stem cell that differentiates between the 5-alpha and 5-beta metabolites. Is that correct? DR. GARDNER: Well, no we can't say that has been shown at all. First of all, a lot of these materials aren't even available in isotope labeled forms to study. I should say that in the chick embryo blood island, it is interesting-and this doesn't answer your question completely-that these cells on incubation, will convert about 60% of the testosterone to 5 beta metabolites. But the method of proving that the steroid metabolite is inducing proliferation is very difficult in that culture system. We are only making this inference without adequate proof at present.
