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Deoxycytidine therapy in two patients with adenosine deaminase deficiency and severe immunodeficiency disease
CI.INICAL
IMML’NOLOCiY
AND
IMMt’NOP41‘HOLOGY
37, 30-36 (1985)
Deoxycytidine Therapy in Two Patients with Adenosine Deaminase Deficiency and Severe lmmunodeficiency Disease’ MORTON J. COWAN, DIANE W. WARA, AND ARTHUR J. AMMANN Pediatric
Immunology.
Unit.ersity
of Californitr.
San Frcmcisro.
Cul(fhrnia
94143
Two children with adenosine deaminase (ADA) deficiency and combined immunodeficiency disease were given parenteral deoxycytidine in order to reverse the severe T-cell immunodeticiency associated with this disease. One patient received a total of three courses of parenteral deoxycytidine. On two occasions deoxycytidine (50 mgikgi day) was infused intravenously continuously for 2 weeks. During one of the infusions she received the deoxycytidine deaminase inhibitor tetrahydrouridine (THU). Steadystate levels of plasma deoxycytidine increased 4-fold with THU. RBC dCTP/dATP increased more than IO-fold after 48 hr of deoxycytidine infusion. Immunologic studies following the intravenous infusion of deoxycytidine showed transient improvement in T-cell immunity. The third course of deoxycytidine (50 mgikgiday) was administered subcutaneously during a IO-hr night-time infusion. After 6 and I2 weeks of nightly subcutaneous infusions. there was minimal improvement in the in ~,itro immunologic studies and no clinical improvement. The second patient received a single 2-week course of continuous intravenous deoxycytidine (50 mgikgiday) following which there was no significant change in T-cell immunity. This study defines some of the pharmacologic parameters of human deoxycytidine metabolism and suggests that some patients with ADA deficiency may respond to deoxycytidine therapy with improvement in T-cellmediated immunity. although the changes are small and the effect on clinical status appears to be limited. 1 1985 Acndemlc Prrx\. Inc.
INTRODUCTION
Adenosine deaminase (ADA) deficiency was the first abnormality in purine metabolism to be associated with primary immunodeficiency disease. Although all of the pathogenic mechanisms responsible for the immunodeficiency in ADA deficiency are not known, one which has received a great deal of attention involves the inhibition of ribonucleotide reductase by elevated intracellular levels of dATP generated by the high kinase activities within lymphocytes ( 1, 2). Ribonucleotide reductase is an enzyme which is essential for the synthesis of DNA precursors such as dCTP, dGTP. and TTP. While all three deoxynucleotides are affected by ribonucleotide reductase inhibition, the one which appears to be most dependent upon this enzyme is dCTP (2). In cell culture models of ADA deficiency addition of deoxycytidine (which can be phosphorylated to dCTP independently of ribonucleotide reductase) partially reverses the inhibitory effects of deoxyadenosine (2-4). The treatment of choice for patients with ADA deficiency and severe combined immune deficiency disease (SCID) is bone marrow transplantation. However, ’ This work was supported by: Pediatric Clinical Research Center (MO lRR00079) Immunology Research Foundation, and NIH All8168 30 0090-1229185 $1.50 Copyright All nphf\
‘t 1985 by Academic Pre\b. Inc. of reproduction in an\ fkrm reerred
DEOXYCYTIDINE
THERAPY
FOR
ADA
DEFICIENCY
31
when a suitable histocompatible bone marrow donor is unavailable approaches to therapy are limited. One approach has been to provide active enzyme by transfusion of irradiated normal erythrocytes (5). This therapy has been only partially and temporarily successful (6, 7). Major concerns with chronic erythrocyte infusions are the possible complications associated with transfusion reactions, hemosiderosis, and transmission of infectious agents into immunodeficient children. Another possible therapeutic approach is the infusion of deoxycytidine. Although reversal of deoxyadenosine toxicity in ADA inhibited lymphocytes has been shown, the use of deoxycytidine in vivo is limited. In this paper we report the use of deoxycytidine infusions in two children with adenosine deaminase deficiency, one of whom had SCID and the other had late onset CID with 2% of normal ADA activity in her peripheral blood lymphocytes. The deoxycytidine was infused both intravenously and subcutaneously. We were able to determine some of the pharmacologic parameters of human deoxycytidine metabolism as well as assess its efficacy following subcutaneous administration over a 3-month period. Case No. I. The patient was relatively healthy until 5 years of age when she manifested initial signs of what was thought to be allergic lung disease. She was seen at the University of California in San Francisco at 8 years of age because of severe lymphopenia and was diagnosed as having ADA deficiency with combined immunodeficiency disease. Immunologic studies revealed an absolute Tcell lymphopenia and severely low in vitro lymphocyte responses to phytohemagglutinin (PHA) and to alloantigen in the mixed lymphocyte reaction (MLR). She had normal quantitative immunoglobulins with decreased antibody responses to immunization with pneumococcal polysaccharide and keyhole limpet hemocyanin. She did not have a histocompatible donor for bone marrow transplantation. Following informed consent from both the patient and her parents she was treated with a total of three infusions of parenteral 2’-deoxycytidine. On two occasions she received 2 weeks of continuous intravenous deoxycytidine at a dose of 50 mglkgiday and on the third occasion she received subcutaneous deoxycytidine at the same dose (150 mgiml normal saline) at a rate of 1 mlihr over a IO-hr period at night. During the initial intravenous infusion period she received the deoxycytidine deaminase inhibitor tetrahydrouridine (THU) at a dose of 150 mg/kg/day intravenously. There were no major side effects or systemic abnormalities noted during any of the infusions. A minor side effect was the development of nontender subcutaneous swellings at each site of subcutaneous infusion with some bruising. Case No. 2. The patient was a term male infant who at 2 weeks of age developed severe oral thrush, candida dermatitis, and a chronic cough unresponsive to conventional antibiotic therapy. At 6 months of age he was diagnosed as severe combined immunodeficiency disease with ADA deficiency. He did not have a histocompatible donor available and at 8 months of age he received a soybean lectin processed haploidentical bone marrow transplant from his mother. There was no evidence for engraftment at 4 months post-transplantation and his mother refused further transplant therapy. Following informed consent he received a 2week course of continuous intravenous deoxycytidine (50 mg/kg/day). There was no toxicity associated with his therapy.
v_-
C‘OWAN.
WAKA.
AND
AMMANh
METHODS
Immunologic stIrdies. In lqitro immune tests included T-cell quantitation by the erythrocyte rosetting technique, lymphocyte response to phytohemagglutinin. and lymphocyte response to alloantigen in the mixed lymphocyte reaction (8). Efliyme ~~ISSN~.Adenosine deaminase was measured using a spectrophotometric assay of uric acid production (9). Determinatio,r yf’dr~o.u~c.gtidirle, dATP. and dCTP by high pressrrre liquid chromarographg (HPLC). Samples were measured using a Beckman model 322 HPLC system. Deoxynucleoside and deoxynucleotide peaks were analyzed by a simultaneous dual wavelength detector at 254 and 280 nm. Erythrocyte samples for deoxynucleotide analysis were prepared by perchloric acid precipitation with trioctylamine-freon extraction. They were analyzed using a IO pm SAX Beckman column with isocratic elution using 0.4 M NH,H,PO, (pH 3.4) 1.5% acetonitrile buffer. Plasma deoxycytidine was analyzed on a reverse-phase Beckman ultrasphere-ODS column with 2 mM potassium acetate (pH 7.5). 0.5% methanol buffer for elution. RESULTS
Enzyme Actillities In both patients, erythrocyte ADA activity was undetectable. Sufficient PBMC were available in patient No. I to measure ADA activity which was 17.2 nmlhri mg protein or about 1.9% of normal control activity (897 5 295 nm/hr/mg protein). Parents of each patient had activity consistent with obligate heterozygotes (data not shown). Deoxycytidine
and dCTP Levels in Plasma
and RBC, Respectivel>
Erythrocyte levels of ATP, dATP and dCTP in each patient are shown in Table I. While ATP levels were not remarkably different between patients and controls, there was a significant elevation in dATP levels. The dATP concentrations in patient No. 1 were significantly lower than in patient No. 2 which is consistent with her more benign clinical course. The ratio of dATP/ATP in patients No. I and No. 2 were 0.09 and 0.48, respectively (normal <0.0003). Deoxy CTP levels in erythrocytes were unmeasurable prior to deoxycytidine infusions. However, after 1 week of intravenous infusions (without THU) RBC dCTP rose to 2. I nmi ml RBC and 0.35 rim/ml RBC in patients No. 1 and No. 2, respectively. ErythTABLE ERYTHROCYTE
(DEOXY)NUCLEOTIDE ATP (nmiml PRBCJ
Case No. 1 pre I week IV 6 weeks subcutaneous I2 weeks subcutaneous Case No. 2 pre 2 days IV Normal (9)
271 301 214 167 316 I25 348 -t 90
LEVELS dATP (am/ml PRBC) 26 32 17 22 152 5x co. I
I DURING
DEOXYCYTILXNE
INFUSIONS
dATP/ATP
dCTP (rim/ml PRBC)
0.09 0.1 I 0.08 0.13 0.48 0.46 0
co. I 2.1 I.? 0.6 10. I 0.35 co. I
dCTP/dATP 0 0.07 0.07 0.03 0 0.01 0
DEOXYCYTIDINE
THERAPY
FOR ADA DEFICIENCY
33
rocyte dCTP was also elevated after 6 and 12 weeks of subcutaneous infusions in patient No. 1. As part of the study of deoxycytidine metabolism in these patients we measured plasma deoxycytidine levels and erythrocyte dCTP/dATP at varying times during and after completing the infusions. In addition, we evaluated the effect of THU. an inhibitor of deoxycytidine deaminase on deoxycytidine and dCTP levels. During the initial infusion of deoxycytidine in patient No. 1, there was a gradual increase in plasma deoxycytidine which reached a plateau between 10 and 20 hr (Fig. 1). The level achieved was between 0.02 and 0.03 mM. When the deoxycytidine infusion was discontinued (arrow) there was a rapid decline in plasma deoxycytidine with an apparent half life of
Immunologic
Studies
We measured absolute T-cell numbers and lymphocyte responses to PHA and alloantigen before, during, and after deoxycytidine infusions. The results of these studies are shown in Table 2. Prior to infusion the absolute T-cell counts were significantly below the lower limit of normal for our laboratory. Lymphocyte responses to PHA and alloantigen were 4% and ~30% of the lower limits of normal, respectively. With deoxycytidine infusions (intravenous and subcutaneous) there were transient increases in the lymphocyte responses to mitogen (intravenous infusions only) and alloantigen in patient No. I. The only in vitro study which returned entirely to normal was the MLR, which consistently im100 r
i
4 \0
I 120
I
130
t INFUSION
TIME
(hrs)
FIG. I. Deoxycytidine infusion in patient No. 1-deoxycytidine. Plasma deoxycytidine concentrations (per milliliter of packed RBC) at various times during the infusion of deoxycytidine (solid line) or deoxycytidine and tetrahydrouridine (dashed line). Arrow indicates when infusion stopped.
34
C‘OWAK.
WAR/I.
A%I)
.,\,MMANN
2.5
I
-.
dcvt
O---O
dcyt
/* ,0---p k
+TH”
INFUSION
TIME (hours)
FIG. 2. Deoxycytidine infusion in patient No. I-dCTP. Erythrocyte dCTP levels expressed as percentage of dATP at various times during the infusion of deoxycytidine (solid line) or deoxycytidine and tetrahydrouridine (dashed line). Arrow indicates when infusion stopped.
proved with each deoxycytidine infusion. We observed no significant response to deoxycytidine infusion in patient No. 2.
in I~YI
DISCUSSION
The mechanism(s) for the immunodeficiency in ADA deficiency is (are) not known. Of the many possibilities, at least two are likely: (1) inhibition of ribonucleotide reductase by elevated levels of dATP, and (2) inhibition of essential methylation reactions by S-adenosyl homocysteine (10). It is likely that more than one mechanism of toxicity exists in ADA deficiency to explain the severe immunodeficiency disease. Results of studies in in vitro models of ADA defiTABLE Ih!
Vmo
T-CELL
IMMUNITY
DURING
post
Normal
(31
DEOXYCYTIDINE
THEKAP)
-.
PHA”
MLR”
221 -t 18 4.58 t 30 2% 211 181 177 2 59
346 L 243 1201 f 962 2441 656 1.55 312 f 255
1340 2 53 4692 2 3632 6498 5673 4222 1504 k 1170
44 rfr 37 53 54 _t 14 P-720
229 k 349 0 1930 -+ 1287 >I6000
1468 2 1633 2306 i 1218 915 + 457 >_5000
ATC”
Case No. 1 pre (2) 2 weeks IV No. I (2) 2 weeks IV No. 2 6 weeks subcutaneous 12 weeks subcutaneous post (5) Case No. 2 Pre (6) 2 weeks IV
2
0 Abbreviations: ATC, absolute T-cell count (per mm3); PHA, lymphocyte response to phytohemagglutinin (cpm): MLR. mixed lymphocyte reaction, one-way tcpm): ( j. number of studies.
DEOXYCYTIDINE
THERAPY
FOR
ADA
DEFICIENCY
35
ciency using both PBMC and cell lines suggest that deoxycytidine could bypass the effects of ribonucleotide reductase inhibition on lymphocyte function (2-4). However, studies in several patients with PNP deficiency indicate that deoxycytidine does not significantly affect their immune deficiency (1 l- 13). In these limited studies infusion methods and doses have varied and plasma or erythrocyte levels of deoxycytidine or dCTP were not reported. In our study patient No. I responded minimally to deoxycytidine in all three courses of therapy while patient No. 2 failed to show any improvement. In fact. over the 3 years that we have been following patient No. 1 and during the multiple times that we have measured her in vitro immunity, it has been only during the infusions of deoxycytidine that she has had measurable in vitro responses to PHA or normal responses to alloantigen. In each case her in 13ituo laboratory studies returned to preinfusion levels once the deoxycytidine was stopped. Unfortunately, these responses were transient with no demonstrable clinical improvement. There are at least three explanations for these findings: (I) deoxycytidine did not reach the potential target cells; (2) the target cells were no longer present, or (3) the major mechanism for the toxicity in ADA deficiency is not limiting amounts of dCTP by inhibition of ribonucleotide reductase. In both patients we measured increases in dCTP in erythrocytes indicating that deoxycytidine was available to the cells. It is possible, however, that the severity of the ADA deficiency could play a role in determining availability of target cells; i.e.. patients with more severe disease have fewer viable, potentially salvagable lymphocytes and are less likely to respond to deoxycytidine. The ADA activity which we measured in the first patient’s PBMC (approximately 2% of normal) was higher than has been reported in most patients with ADA deficiency. her erythrocyte dATP somewhat lower, and her immunodeficiency disease and clinical course more benign. It is possible that this patient had more lymphocytes capable of responding to deoxycytidine than patient No. 2 who has had a more severe clinical course, although the responses to deoxycytidine by patient No. 1 were limited. Finally, based on these results, one must seriously question the importance of ribonucleotide reductase inhibition in the pathogenesis of ADA deficiency and consider the likelihood that other mechanisms exist for the immune deficiency. Our studies demonstrate that high levels of deoxycytidine can be achieved in plasma, and dCTP in red cells. Furthermore, significantly higher levels can be achieved with the use of the deoxycytidine deaminase inhibitor tetrahydrouridine. However, the plasma half-life of deoxycytidine is extremely short and although prolonged by THU, the effect is probably not clinically relevant. Because it is impractical to expect a long-term course of therapy to be given intravenously, we performed a trial of subcutaneous deoxycytidine infusions. We were able to demonstrate that measurable amounts of deoxycytidine could be delivered to the patient’s RBC even without THU present. The only side effect which we found during any of the infusions was the result of repeated subcutaneous infusions over the abdomen resulting in some bruising and discomfort at the sight of the injections. While in viva deoxycytidine resulted in transient improvement in in vitro immunity in one of two patients with ADA deficiency there was no clinical improve-
36
cow.2i\r.
W,ZR/\.
4NI>
,\MILI:Zhh
ment. Deoxycytidine alone. therefore. is insufficient to restore significant immunity in ADA deficient patients and should be eliminated as a therapeutic modality. Our results suggest that either the “salvagable” lymphocytes arc no longer present or other more important mechanisms for the immune deficiency cxi4t. Further studies to determine these mechanisms and alternative means of’therapy are necessary. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. I I. 12. 13. 14. 15.
Carson. D. A.. Kaye. J.. and Seegmiller. J. E.. PUJC. .Ytrr/. Act&. SC,;.USA 74. 5677. IY77. Ullman, B.. Gudas, L. J.. Cohen. A., and Martin. D. W.. Jr.. Cell 14, 365. 1978. Bluestein. H. G., and Seegmiller. J. E. Fed. PUK. 37. 1465. 1978. Thompson. L. E. Fed. PIW. 37, 1465. 1978. Polmar. S. H., et ~1.. N. Enal. J. Md. 295, 1337. 1976. Rubinstein. A.. Hirschhorn. R.. Sicklick. M.. and Murphy. R. A.. N. Enal. J. Med. 300, 387. 1979. Schmalstieg, E C.. Mills. G. C.. Nelson, J. A.. rt u/.. J. Prdi~rr. 93, 597. 1978. Cowan, M. J., Fujiwara. P.. and Ammann. A. J.. C/in. /fur,~r~~o/. Irlzfntrf~opcrthol. 17, 595. 1980. Cowan, M. J.. Fraga, M.. Andrew. J.. Lameris-Martin. N.. and Ammann. A. J.. Cell /m~ur&. 67, 121, 1982. Kredich. N. M.. and Martin. D. W.. Jr.. Crll 12, 931. 1977. Rijkers. G. T.. Zegers. B. J. M., Spaapen. L.. et ul.. Ad),. E.rp. Med. Bid. 165B. 171. 1983. Watson, A. R., Simmonds. H.. Webster. D.. TV trl.. A&. Erp. Med. Bid. 165A, 53, 1983. Stoop, J. W.. Zegers. B. J. M.. Spaapen, L.. et r/l.. Ah. hp. Bid. Mrd. 165A. 61. 1983. Jenkins. T., et ul.. J. Pediurr. 89, 732. 1976. Reem, G. H.. Borkowsky. W.. and Hirschhorn. R.. Pedirrtr. Rcs. 13, 649. 1979.
Received October 23, 1984: accepted with revision April 18. I985
IMML’NOLOCiY
AND
IMMt’NOP41‘HOLOGY
37, 30-36 (1985)
Deoxycytidine Therapy in Two Patients with Adenosine Deaminase Deficiency and Severe lmmunodeficiency Disease’ MORTON J. COWAN, DIANE W. WARA, AND ARTHUR J. AMMANN Pediatric
Immunology.
Unit.ersity
of Californitr.
San Frcmcisro.
Cul(fhrnia
94143
Two children with adenosine deaminase (ADA) deficiency and combined immunodeficiency disease were given parenteral deoxycytidine in order to reverse the severe T-cell immunodeticiency associated with this disease. One patient received a total of three courses of parenteral deoxycytidine. On two occasions deoxycytidine (50 mgikgi day) was infused intravenously continuously for 2 weeks. During one of the infusions she received the deoxycytidine deaminase inhibitor tetrahydrouridine (THU). Steadystate levels of plasma deoxycytidine increased 4-fold with THU. RBC dCTP/dATP increased more than IO-fold after 48 hr of deoxycytidine infusion. Immunologic studies following the intravenous infusion of deoxycytidine showed transient improvement in T-cell immunity. The third course of deoxycytidine (50 mgikgiday) was administered subcutaneously during a IO-hr night-time infusion. After 6 and I2 weeks of nightly subcutaneous infusions. there was minimal improvement in the in ~,itro immunologic studies and no clinical improvement. The second patient received a single 2-week course of continuous intravenous deoxycytidine (50 mgikgiday) following which there was no significant change in T-cell immunity. This study defines some of the pharmacologic parameters of human deoxycytidine metabolism and suggests that some patients with ADA deficiency may respond to deoxycytidine therapy with improvement in T-cellmediated immunity. although the changes are small and the effect on clinical status appears to be limited. 1 1985 Acndemlc Prrx\. Inc.
INTRODUCTION
Adenosine deaminase (ADA) deficiency was the first abnormality in purine metabolism to be associated with primary immunodeficiency disease. Although all of the pathogenic mechanisms responsible for the immunodeficiency in ADA deficiency are not known, one which has received a great deal of attention involves the inhibition of ribonucleotide reductase by elevated intracellular levels of dATP generated by the high kinase activities within lymphocytes ( 1, 2). Ribonucleotide reductase is an enzyme which is essential for the synthesis of DNA precursors such as dCTP, dGTP. and TTP. While all three deoxynucleotides are affected by ribonucleotide reductase inhibition, the one which appears to be most dependent upon this enzyme is dCTP (2). In cell culture models of ADA deficiency addition of deoxycytidine (which can be phosphorylated to dCTP independently of ribonucleotide reductase) partially reverses the inhibitory effects of deoxyadenosine (2-4). The treatment of choice for patients with ADA deficiency and severe combined immune deficiency disease (SCID) is bone marrow transplantation. However, ’ This work was supported by: Pediatric Clinical Research Center (MO lRR00079) Immunology Research Foundation, and NIH All8168 30 0090-1229185 $1.50 Copyright All nphf\
‘t 1985 by Academic Pre\b. Inc. of reproduction in an\ fkrm reerred
DEOXYCYTIDINE
THERAPY
FOR
ADA
DEFICIENCY
31
when a suitable histocompatible bone marrow donor is unavailable approaches to therapy are limited. One approach has been to provide active enzyme by transfusion of irradiated normal erythrocytes (5). This therapy has been only partially and temporarily successful (6, 7). Major concerns with chronic erythrocyte infusions are the possible complications associated with transfusion reactions, hemosiderosis, and transmission of infectious agents into immunodeficient children. Another possible therapeutic approach is the infusion of deoxycytidine. Although reversal of deoxyadenosine toxicity in ADA inhibited lymphocytes has been shown, the use of deoxycytidine in vivo is limited. In this paper we report the use of deoxycytidine infusions in two children with adenosine deaminase deficiency, one of whom had SCID and the other had late onset CID with 2% of normal ADA activity in her peripheral blood lymphocytes. The deoxycytidine was infused both intravenously and subcutaneously. We were able to determine some of the pharmacologic parameters of human deoxycytidine metabolism as well as assess its efficacy following subcutaneous administration over a 3-month period. Case No. I. The patient was relatively healthy until 5 years of age when she manifested initial signs of what was thought to be allergic lung disease. She was seen at the University of California in San Francisco at 8 years of age because of severe lymphopenia and was diagnosed as having ADA deficiency with combined immunodeficiency disease. Immunologic studies revealed an absolute Tcell lymphopenia and severely low in vitro lymphocyte responses to phytohemagglutinin (PHA) and to alloantigen in the mixed lymphocyte reaction (MLR). She had normal quantitative immunoglobulins with decreased antibody responses to immunization with pneumococcal polysaccharide and keyhole limpet hemocyanin. She did not have a histocompatible donor for bone marrow transplantation. Following informed consent from both the patient and her parents she was treated with a total of three infusions of parenteral 2’-deoxycytidine. On two occasions she received 2 weeks of continuous intravenous deoxycytidine at a dose of 50 mglkgiday and on the third occasion she received subcutaneous deoxycytidine at the same dose (150 mgiml normal saline) at a rate of 1 mlihr over a IO-hr period at night. During the initial intravenous infusion period she received the deoxycytidine deaminase inhibitor tetrahydrouridine (THU) at a dose of 150 mg/kg/day intravenously. There were no major side effects or systemic abnormalities noted during any of the infusions. A minor side effect was the development of nontender subcutaneous swellings at each site of subcutaneous infusion with some bruising. Case No. 2. The patient was a term male infant who at 2 weeks of age developed severe oral thrush, candida dermatitis, and a chronic cough unresponsive to conventional antibiotic therapy. At 6 months of age he was diagnosed as severe combined immunodeficiency disease with ADA deficiency. He did not have a histocompatible donor available and at 8 months of age he received a soybean lectin processed haploidentical bone marrow transplant from his mother. There was no evidence for engraftment at 4 months post-transplantation and his mother refused further transplant therapy. Following informed consent he received a 2week course of continuous intravenous deoxycytidine (50 mg/kg/day). There was no toxicity associated with his therapy.
v_-
C‘OWAN.
WAKA.
AND
AMMANh
METHODS
Immunologic stIrdies. In lqitro immune tests included T-cell quantitation by the erythrocyte rosetting technique, lymphocyte response to phytohemagglutinin. and lymphocyte response to alloantigen in the mixed lymphocyte reaction (8). Efliyme ~~ISSN~.Adenosine deaminase was measured using a spectrophotometric assay of uric acid production (9). Determinatio,r yf’dr~o.u~c.gtidirle, dATP. and dCTP by high pressrrre liquid chromarographg (HPLC). Samples were measured using a Beckman model 322 HPLC system. Deoxynucleoside and deoxynucleotide peaks were analyzed by a simultaneous dual wavelength detector at 254 and 280 nm. Erythrocyte samples for deoxynucleotide analysis were prepared by perchloric acid precipitation with trioctylamine-freon extraction. They were analyzed using a IO pm SAX Beckman column with isocratic elution using 0.4 M NH,H,PO, (pH 3.4) 1.5% acetonitrile buffer. Plasma deoxycytidine was analyzed on a reverse-phase Beckman ultrasphere-ODS column with 2 mM potassium acetate (pH 7.5). 0.5% methanol buffer for elution. RESULTS
Enzyme Actillities In both patients, erythrocyte ADA activity was undetectable. Sufficient PBMC were available in patient No. I to measure ADA activity which was 17.2 nmlhri mg protein or about 1.9% of normal control activity (897 5 295 nm/hr/mg protein). Parents of each patient had activity consistent with obligate heterozygotes (data not shown). Deoxycytidine
and dCTP Levels in Plasma
and RBC, Respectivel>
Erythrocyte levels of ATP, dATP and dCTP in each patient are shown in Table I. While ATP levels were not remarkably different between patients and controls, there was a significant elevation in dATP levels. The dATP concentrations in patient No. 1 were significantly lower than in patient No. 2 which is consistent with her more benign clinical course. The ratio of dATP/ATP in patients No. I and No. 2 were 0.09 and 0.48, respectively (normal <0.0003). Deoxy CTP levels in erythrocytes were unmeasurable prior to deoxycytidine infusions. However, after 1 week of intravenous infusions (without THU) RBC dCTP rose to 2. I nmi ml RBC and 0.35 rim/ml RBC in patients No. 1 and No. 2, respectively. ErythTABLE ERYTHROCYTE
(DEOXY)NUCLEOTIDE ATP (nmiml PRBCJ
Case No. 1 pre I week IV 6 weeks subcutaneous I2 weeks subcutaneous Case No. 2 pre 2 days IV Normal (9)
271 301 214 167 316 I25 348 -t 90
LEVELS dATP (am/ml PRBC) 26 32 17 22 152 5x co. I
I DURING
DEOXYCYTILXNE
INFUSIONS
dATP/ATP
dCTP (rim/ml PRBC)
0.09 0.1 I 0.08 0.13 0.48 0.46 0
co. I 2.1 I.? 0.6 10. I 0.35 co. I
dCTP/dATP 0 0.07 0.07 0.03 0 0.01 0
DEOXYCYTIDINE
THERAPY
FOR ADA DEFICIENCY
33
rocyte dCTP was also elevated after 6 and 12 weeks of subcutaneous infusions in patient No. 1. As part of the study of deoxycytidine metabolism in these patients we measured plasma deoxycytidine levels and erythrocyte dCTP/dATP at varying times during and after completing the infusions. In addition, we evaluated the effect of THU. an inhibitor of deoxycytidine deaminase on deoxycytidine and dCTP levels. During the initial infusion of deoxycytidine in patient No. 1, there was a gradual increase in plasma deoxycytidine which reached a plateau between 10 and 20 hr (Fig. 1). The level achieved was between 0.02 and 0.03 mM. When the deoxycytidine infusion was discontinued (arrow) there was a rapid decline in plasma deoxycytidine with an apparent half life of
Immunologic
Studies
We measured absolute T-cell numbers and lymphocyte responses to PHA and alloantigen before, during, and after deoxycytidine infusions. The results of these studies are shown in Table 2. Prior to infusion the absolute T-cell counts were significantly below the lower limit of normal for our laboratory. Lymphocyte responses to PHA and alloantigen were 4% and ~30% of the lower limits of normal, respectively. With deoxycytidine infusions (intravenous and subcutaneous) there were transient increases in the lymphocyte responses to mitogen (intravenous infusions only) and alloantigen in patient No. I. The only in vitro study which returned entirely to normal was the MLR, which consistently im100 r
i
4 \0
I 120
I
130
t INFUSION
TIME
(hrs)
FIG. I. Deoxycytidine infusion in patient No. 1-deoxycytidine. Plasma deoxycytidine concentrations (per milliliter of packed RBC) at various times during the infusion of deoxycytidine (solid line) or deoxycytidine and tetrahydrouridine (dashed line). Arrow indicates when infusion stopped.
34
C‘OWAK.
WAR/I.
A%I)
.,\,MMANN
2.5
I
-.
dcvt
O---O
dcyt
/* ,0---p k
+TH”
INFUSION
TIME (hours)
FIG. 2. Deoxycytidine infusion in patient No. I-dCTP. Erythrocyte dCTP levels expressed as percentage of dATP at various times during the infusion of deoxycytidine (solid line) or deoxycytidine and tetrahydrouridine (dashed line). Arrow indicates when infusion stopped.
proved with each deoxycytidine infusion. We observed no significant response to deoxycytidine infusion in patient No. 2.
in I~YI
DISCUSSION
The mechanism(s) for the immunodeficiency in ADA deficiency is (are) not known. Of the many possibilities, at least two are likely: (1) inhibition of ribonucleotide reductase by elevated levels of dATP, and (2) inhibition of essential methylation reactions by S-adenosyl homocysteine (10). It is likely that more than one mechanism of toxicity exists in ADA deficiency to explain the severe immunodeficiency disease. Results of studies in in vitro models of ADA defiTABLE Ih!
Vmo
T-CELL
IMMUNITY
DURING
post
Normal
(31
DEOXYCYTIDINE
THEKAP)
-.
PHA”
MLR”
221 -t 18 4.58 t 30 2% 211 181 177 2 59
346 L 243 1201 f 962 2441 656 1.55 312 f 255
1340 2 53 4692 2 3632 6498 5673 4222 1504 k 1170
44 rfr 37 53 54 _t 14 P-720
229 k 349 0 1930 -+ 1287 >I6000
1468 2 1633 2306 i 1218 915 + 457 >_5000
ATC”
Case No. 1 pre (2) 2 weeks IV No. I (2) 2 weeks IV No. 2 6 weeks subcutaneous 12 weeks subcutaneous post (5) Case No. 2 Pre (6) 2 weeks IV
2
0 Abbreviations: ATC, absolute T-cell count (per mm3); PHA, lymphocyte response to phytohemagglutinin (cpm): MLR. mixed lymphocyte reaction, one-way tcpm): ( j. number of studies.
DEOXYCYTIDINE
THERAPY
FOR
ADA
DEFICIENCY
35
ciency using both PBMC and cell lines suggest that deoxycytidine could bypass the effects of ribonucleotide reductase inhibition on lymphocyte function (2-4). However, studies in several patients with PNP deficiency indicate that deoxycytidine does not significantly affect their immune deficiency (1 l- 13). In these limited studies infusion methods and doses have varied and plasma or erythrocyte levels of deoxycytidine or dCTP were not reported. In our study patient No. I responded minimally to deoxycytidine in all three courses of therapy while patient No. 2 failed to show any improvement. In fact. over the 3 years that we have been following patient No. 1 and during the multiple times that we have measured her in vitro immunity, it has been only during the infusions of deoxycytidine that she has had measurable in vitro responses to PHA or normal responses to alloantigen. In each case her in 13ituo laboratory studies returned to preinfusion levels once the deoxycytidine was stopped. Unfortunately, these responses were transient with no demonstrable clinical improvement. There are at least three explanations for these findings: (I) deoxycytidine did not reach the potential target cells; (2) the target cells were no longer present, or (3) the major mechanism for the toxicity in ADA deficiency is not limiting amounts of dCTP by inhibition of ribonucleotide reductase. In both patients we measured increases in dCTP in erythrocytes indicating that deoxycytidine was available to the cells. It is possible, however, that the severity of the ADA deficiency could play a role in determining availability of target cells; i.e.. patients with more severe disease have fewer viable, potentially salvagable lymphocytes and are less likely to respond to deoxycytidine. The ADA activity which we measured in the first patient’s PBMC (approximately 2% of normal) was higher than has been reported in most patients with ADA deficiency. her erythrocyte dATP somewhat lower, and her immunodeficiency disease and clinical course more benign. It is possible that this patient had more lymphocytes capable of responding to deoxycytidine than patient No. 2 who has had a more severe clinical course, although the responses to deoxycytidine by patient No. 1 were limited. Finally, based on these results, one must seriously question the importance of ribonucleotide reductase inhibition in the pathogenesis of ADA deficiency and consider the likelihood that other mechanisms exist for the immune deficiency. Our studies demonstrate that high levels of deoxycytidine can be achieved in plasma, and dCTP in red cells. Furthermore, significantly higher levels can be achieved with the use of the deoxycytidine deaminase inhibitor tetrahydrouridine. However, the plasma half-life of deoxycytidine is extremely short and although prolonged by THU, the effect is probably not clinically relevant. Because it is impractical to expect a long-term course of therapy to be given intravenously, we performed a trial of subcutaneous deoxycytidine infusions. We were able to demonstrate that measurable amounts of deoxycytidine could be delivered to the patient’s RBC even without THU present. The only side effect which we found during any of the infusions was the result of repeated subcutaneous infusions over the abdomen resulting in some bruising and discomfort at the sight of the injections. While in viva deoxycytidine resulted in transient improvement in in vitro immunity in one of two patients with ADA deficiency there was no clinical improve-
36
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ment. Deoxycytidine alone. therefore. is insufficient to restore significant immunity in ADA deficient patients and should be eliminated as a therapeutic modality. Our results suggest that either the “salvagable” lymphocytes arc no longer present or other more important mechanisms for the immune deficiency cxi4t. Further studies to determine these mechanisms and alternative means of’therapy are necessary. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. I I. 12. 13. 14. 15.
Carson. D. A.. Kaye. J.. and Seegmiller. J. E.. PUJC. .Ytrr/. Act&. SC,;.USA 74. 5677. IY77. Ullman, B.. Gudas, L. J.. Cohen. A., and Martin. D. W.. Jr.. Cell 14, 365. 1978. Bluestein. H. G., and Seegmiller. J. E. Fed. PUK. 37. 1465. 1978. Thompson. L. E. Fed. PIW. 37, 1465. 1978. Polmar. S. H., et ~1.. N. Enal. J. Md. 295, 1337. 1976. Rubinstein. A.. Hirschhorn. R.. Sicklick. M.. and Murphy. R. A.. N. Enal. J. Med. 300, 387. 1979. Schmalstieg, E C.. Mills. G. C.. Nelson, J. A.. rt u/.. J. Prdi~rr. 93, 597. 1978. Cowan, M. J., Fujiwara. P.. and Ammann. A. J.. C/in. /fur,~r~~o/. Irlzfntrf~opcrthol. 17, 595. 1980. Cowan, M. J.. Fraga, M.. Andrew. J.. Lameris-Martin. N.. and Ammann. A. J.. Cell /m~ur&. 67, 121, 1982. Kredich. N. M.. and Martin. D. W.. Jr.. Crll 12, 931. 1977. Rijkers. G. T.. Zegers. B. J. M., Spaapen. L.. et ul.. Ad),. E.rp. Med. Bid. 165B. 171. 1983. Watson, A. R., Simmonds. H.. Webster. D.. TV trl.. A&. Erp. Med. Bid. 165A, 53, 1983. Stoop, J. W.. Zegers. B. J. M.. Spaapen, L.. et r/l.. Ah. hp. Bid. Mrd. 165A. 61. 1983. Jenkins. T., et ul.. J. Pediurr. 89, 732. 1976. Reem, G. H.. Borkowsky. W.. and Hirschhorn. R.. Pedirrtr. Rcs. 13, 649. 1979.
Received October 23, 1984: accepted with revision April 18. I985