A pilot study of diethyldithiocarbamate in patients with acquired immune deficiency syndrome (AIDS) and the AIDS-related complex

Life Sciences, Vol. 45, pp. 2509-2520 Printed in the U.S.A.

Pergamon Press

AIDS RESEARCH CO~UNICATIONS

A PILOT STUDYOF DIETHYLDITHIOCARBAMATE IN PATIENTS WITH ACQUIRED IMMUNE DEFICIENCY SYNDROME (AIDS) AND THE AIDS-RELATED COMPLEX

Gary W. Brewton," Evan M. Hersh,+ Adan Rios,* Peter W.A. Mansell', Blaine Hollinger, # and James M. Reuben" "The Department of Clinical Immunologyand Biological Therapy, Division of Medicine, M.D. Anderson Cancer Center, Houston, Texas 77030. +Section of Hematology and Oncology, Arizona Cancer Center, Tucson, Arizona 85724. #Department of Microbiology, Baylor College of Medicine, Houston, Texas 77030 (Received in final form October 20, 1989)

Summary We investigated the use of diethyldithiocarbamate (DTC, or Imuthiol r, Merieux Institute) as a therapeutic agent in patients with Acquired Immune Deficiency Syndrome (AIDS) and AIDS-Related Complex (ARC). Patients were prospectively s t r a t i f i e d and randomized to receive DTC 200mg/mL intravenously weekly for 16 weeks or no therapy, followed by crossover to the opposite arm for an equal period. Fortyfour patients were entered and forty were evaluable. There was a s t a t i s t i c a l l y significant decrease in symptoms in the DTC treated patients compared to the controls (p=.O02). There was a significant improvement in lymphadenopathy in the treated patients compared to the controls (p=.O05). One patient showed disappearance of splenomegaly, one clearing of antifungal agent-reslstant perianal moniliasis, and one clearing of hairy leukoplakia. No significant differences in progression were noted. No changes were seen in any of the immunological parameters measured. There was no significant t o x i c i t y . Because of the changes in symptoms and in lymphadenopathy, we suggest that further study of DTC. both alone and in combination with other agents, may be indicated. I Since the f i r s t cases were recognized in 1981, the Acquired Immune Deficiency Syndrome (AIDS) has been diagnosed in more than 50,000 individuals in the United States alone ( I ) . Affected individuals have multiple immunologic abnormalities including depletion of CD4 + ("helper") T-cells, decreased T- and B- cell responses to antigens, impaired lymphocyte blastogenesis to mitogens and to specific antigenic stimulation (2), deficient production of or response to interleukin-2 (3), and interferongamma (4), impaired delayed-type hypersensitivity, deficient natural k i l l e r , I The study was approved by an institutional review board (human subjects committee), and that written, informed consent was obtained from the patients. Please address requests for reprints to Evan M. Hersh, Section of Hematology Oncology. Arizona Cancer Center, 1501N. Campbell Ave. Tucson, AZ 85724. 0024-3205/89 $3.00 + .00 Copyright (c) 1989 Pergamon Press plc

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and cytotoxic T-cell a c t i v i t y (5), polyclonal B-cell activation (6), as well as high levels of circulating immune complexes (7), beta-2-microglobulin (8), and acld-labile interferon (9). As a consequence, patients are vulnerable to infection with a variety of v i r a l , fungal, protozoan, and bacterial pathogens (10), and to the development of malignancies including Kaposi's sarcoma and non-Hodgkin's lymphoma (11). Beyondtreatment of opportunistic infections and secondary neoplasms, more definitive approaches to therapy of AIDS and related conditions may be broadly divided into two categories. The f i r s t is antiviral treatment such as azldothymidine (12) directed at the underlying Human Immunodeficiency Virus (HIV) infection. This has shown beneficial clinical effects (13). The second category of approach is immunorestorative or immunomodulatory therapy directed at correcting one or more aspects of the abnormal or deficient immune response in these patients. The use of immune interventions in patients with AIDS and related conditions has not to date produced evidence of consistent benefit. Diethyldithlocarbamate (DTC or Imuthiol r) is a low molecular weight dithiol compound f i r s t used c l i n i c a l l y in 1957 as a chelating agent (14) for the treatment of nickel poisoning. As such, i t was both effective and safe. Its a c t i v i t y as an immunorestorative was f i r s t described by Renoux in the 1970's (15). In animal models DTC restores T cell dependent immune responses in aged (16), athymlc (17), azathioprine-treated (18), irradiated (19), and l e f t hemi-decorticated mice (20). DTC is not mitogenlc in vitro. I t does not affect T-cell-independent B-cell functions. In humans i t has been claimed to have a c t i v i t y In rheumatoid a r t h r i t i s (21), tuberculosis (22), chronic bronchitis (22), and post-surglcal healing (23). I t has a favorable therapeutic index but produces disulfuram-llke reactions when alcohol is consumed in temporal proximity to a dose of DTC. The plasma half l i f e after a single intravenous dose of DTC Is 20 minutes, with 60% excreted within three hours, half by the lung (as carbon disulfide gas) and half by the kidney (as the glucuronide salt, sulfate, and other metabolic byproducts). Orally, DTC is variably absorbed and plasma drug levels vary after single oral doses (24). The duration of effect of a single intravenous dose in animal models is approximately seven days (as judged by augmentation of blastogenic response to PHA). The long duration of effect as compared to i t s short plasma half l i f e is currently unexplained. DTC is l i p o p h i l i c and readily penetrates the blood/braln barrier as determined by 35S-labeled drug isotope imaging (24). The mechanism of action to augment T-cell number and function is not known, although Renoux has proposed that DTC induces the l i v e r to produce a 12,000 dalton protein he called "hepatosin" with thymic hormone-like properties (25). Similar activity was seen in cell culture of hepatocytes exposed to DTC in vitro. Because of i t s immunologic activity, Lang et al. conducted an openlabel p i l o t study of weekly oral DTC (Imuthiol, Merleux Institute) in ARC and AIDS patients (26). They observed increases in CD4+ cell number, CD4+/CD8 ratio and skin test reactivity together with clinical improvement. Subsequently, Pompidou, et al. reported limited anti-viral a c t i v i t y against HIV in vitro (27). Recently, and subsequent to this study, Lang, et al. reported a double blind placebo controlled t r i a l of weekly oral DTC in HIV infected patients(28). Significant benefits were observed. With these results, we elected to further study the effect of DTC in symptomatic AIDS and ARC patients by the intravenous route. This paper reports the results of that study.

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Treatment of ARC and AIDS With DTC

251]

Methods

Forty-four patients wlth AIDS and ARC were enrolle~ In a randomized unblinded 32-week crossover study comparing DTC 200mg/m~ intravenously once a week versus no treatment. Patients were entered on study from Aprll, 1985 through February, 1986. AZT was not available to the patients at the time of the study. The sample size was based on the a b i l i t y to detect a difference of at least 25% in treated vs. control subjects. Eligible patients included those with AIDS (meeting the requirements of the Centers for Disease Control case definition) or ARC defined as: (I) HIV seropositivity, (2) two or more of the following symptoms or signs persisting at least 3 months without other explanation: fever, night sweats, weight loss of 10% ideal body weight, diarrhea (two or more unformed stools per day at least three days a week), fatigue, lymphadenopathy involving at least two discontiguous extra-inguinal sites, or thrush occurring in the absence of antibiotic therapy. All patients had at least two of the following Immunologlc abnormalities: 1) CD4+ cell number less than 500 per microliter, 2) CD4+/CD8+ ratio less than 0.5, 3) one or fewer positive skln test reactions measured on the standard seven antigen skin test battery (Multltest-CMIr, Merieux Institute), 4) Lymphocyte blastogenesis response (LBR) t9 phytohemagglutin (PHA) less than 20,000 counts per minute (cpmI per I0D cells, 5) LBR to pokeweed mitogen (PWM) less than 10,000 cpm p)r 10 cells, or 6) LBR to concanavilln-A (con-A) less than 20,000 cpm per 10° c e l l s . Most patients met all slx c r i t e r i a for immune deficiency. Exclusion c r i t e r i a included the following: 1) concurrent active opportunistic infection, 2) progressive Kaposi's sarcoma, defined as the occurrence of new cutaneous lesions or an increase of 50% in size of existing lesions over the four week period prior to enrollment, 3) hemoglobln less than 10gm/dl, 4) leukocyte count less than 2,000 cells per microllter, 5) platelet count less than 100,000 per microliter, 6) blood urea nitrogen greater than 30mg/dl, 7) serum creatinine greater than 2mg/dl, 8) and serum aspartate aminotransferase greater than 50Iu/L. After enrollment, patients were s t r a t i f i e d according to the following three c r i t e r i a : I) disease status (ARC vs. AIDS), 2) CD4+ cell number (~200 versus >200 per microliter), and 3) number of symptoms (~2 versus >2) as listed above under the definition of ARC. Within each stratum, patients were randomized to receive either i n i t i a l treatment with DTC 200mg/m intravenously weekly for 16 weeks versus no treatment. After this i n i t i a l 16 week period, patients were then crossed over to the opposite arm for an equal period. Clinical assessment was made every four weeks with particular attention to symptoms as indicated under the definition of ARC given above and to the measurement of lymphadenopathy, recorded as the sum of the products of the nodal cross-sectional diameters. Completeblood counts, lymphocyte cell surface markers, and lymphocyte blastogenesis responses were measured by flow cytometry using a panel of standard monoclonal antibodies including OKT-11, OKT-3, OKT-4, OKT-8, OKT-IO, B-1 and anti-DR as provided by Ortho Pharmaceuticals. Lymphocytebla)togenesis responses to the mltogens PHA, PWMand Con-A were measured by ( H)-thymldlne incorporation, as previously described. Serum chemistries were obtained every four weeks by multichannel automated analysis. Quantitative Immunoglobulin levels and delayed type hypersensitivity to a standard seven antigen battery (Multitest-cMIr), Merieux Institute) were performed every eight weeks.

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Progression was defined as 1) new or recurrent opportunistic infection, 2) new or progressive Kaposi's sarcoma, or 3) new or recurrent non-Hodgkin's lymphoma. Eight weeks was prospectively defined as an adequate t r i a l on the protocol. Patients who progressed were removed from the study and did not receive further treatment regardless of which arm (treatment versus no treatment) of the protocol they were on at the time of progression. Patients who progressed sooner than eight weeks after entry onto the protocol were excluded from statistical analysis regardless of which arm of the protocol they were on. For statistical analysis, the "symptom score" was defined as the number of symptoms or signs present (out of seven possible) as defined previously. Lymphadenopathy was included in the symptom score, but also i t was analyzed separately. Results were analyzed using the following statistical tools: chisquare, Fisher's exact test, Mann-Whitney test for nonparametric data, Kaplan-Meier l i f e table analysis, and the Gehan-Breslow test for equality of survival. Because ten patients progressed during the f i r s t half of the study leaving only 30 evaluable patients in the second half, statistical analysis was performed only on the f i r s t sixteen weeks of the study, except for l i f e table analysis and associated tests. In addition, eight consecutive patients during the f i r s t 16 weeks of the study (four on treatment and four on no treatment) had serial HIV cultures performed at four time points: on two occasions prior to beginning therapy, at 10 weeks, and at 14 weeks. Ability to culture HIV in vitro was determined by measurement of reverse transcriptase activity twice a week in supernatants of normal donor lymphocytes in RPMI into which the patient's cells had been Inoculated, supplemented with interleukin-2, PHA, and periodic feeding with additional normal donor lymphocytes. Cultures were considered positive i f the reverse transcriptase level consistently exceeded 5000cpm. Results Forty-four patients were entered and forty were evaluable after at least eight weeks on the protocol. The four patients who progressed early and were excluded from further analysis included two with Kaposi's sarcoma who developed multiple new lesions within the f i r s t four weeks (one patient on treatment and the other on observation), and one patient each with Pneumocystis carinii pneumonia and disseminated Mycobacterium aviumintracellularein--i-n-irection at seven weeks and six weeks respective--~both on i n i t i a l treatment). Randomization was successful and symmetric along all three criteria of stratification (Table 1) except there was a slight preponderance of AIDS patients among those receiving i n i t i a l treatment versus i n i t i a l observation. Progression rates among only ARC patients were: Two of 16 (12%) on DTC and 5 of 16 (31%) on observation (XZ=1.65, p=0.19). The diagnosis leading to a finding of progression were similar in both treated and untreated patients and included six patients with Pneumocystis carinii pneumonia (PCP), two with de-novo Kaposi s sarcoma (KS), and one ~ t h disseminated herpes zoster and non-Hodgkin's lymphoma plus disseminated Mycobacterium aviumintracellulare infection.

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Treatment of ARC and AIDS With DTC

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TABLE I Stratlflcatlon Of Evaluable Patients (N=40)

Category

Drug

Control

Total

ARC AIDS T4 ~200 T4 >200 Symptoms<2 Symptoms ~2

16 6 11 11 9 13

16 2 8 10 5 13

32 8 19 21 14 26

Total Patients

22

18

40

Ten patients progressed during the i n l t i a l 16 weeks: four of 22 (18%) on DTC and 6 of 18 (33%) on observation (XZ=I.21, p=O.23)(Table 2).

TABLE I I Evaluation Of Dlethyldlthlocarbamate In ARC And AIDS

Progression Number (percent Progressed) Group

Drug

Control

X2

P

All patients

4/22 (18)

6/18 (33)

1.21

0.23

ARC

2/16 (12)

5/16 (31)

1.65

0.19

AIDS

2/6

1/2

0.00

(33)

(50)

10 Progressions; 6: PCP, I: disseminated zoster, 1: NHL and M. avium.

.774

2: KS, and

Kaplan-Meier l i f e table analysis also showed no significant difference in progression-free survival among patients receiving i n i t i a l DTC, both among all patients (Figure 1) and among ARC patients only (Figure 2). The Gehan-Breslow test for equality of progression free survival was not s t a t i s t i c a l l y significant among all patients (p=0.354) or among ARC patients only (p=0.211).

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Progression Free Survival Data Analysis by Method of Kaplan and Meier l ' O " r ' ~ ~

,

0.9-

I

[ L],,

0.8u) 0.7o (3. "5

nq

0.6-

l

-

i |

i

0.5LL

g

0.4-

oQ.

0.3-

2 (3.

0.2-

ARC + AIDS Evaluable patients only (2 8 weeks on study) Log rank test p - .3651 Gehan Breslow test p : .312

0.1-

• 1 Imuthiol with 17 censored, 5 uncensored • 2 Control with 12 censored, 6 uncensored

0.0 0

i

i

5

10

i

i

i

i

15 20 25 30 Weeks

i

i

i

35

40

45

FIG. I .

The proportion of subjects in the progression free survival groups are plotted against weeks on study. The squares indicate the patients i n i t i a l l y in the treatment group and the circles those i n i t i a l l y in the the control group.

Survival Data Analysisby Method of Kaplan and Meier Survival Variable, Group Variable 10

g 0o89-

,,, , i 1 i ]

i

i ii

1

I

0.7

~_ 0.6 "5

i i

i

o.s

04 o o a..

0.3 0.2 0.1 0.0

ARC Evaluable patients only (> 8 weeks on study) Log rank test p ~ .177 Gehan Breslow test p ~ .109 • 1 Imuthiol with 14 censored, 2 uncensored • 2 Control with 11 censored, 5 uncensored

0 ; t'0 1; 2o

3'0

,o

Weeks

FIG. 2.

The proportion of subjects surviving are plotted against time in weeks. The squares indicate the i n i t i a l l y treated patients and the circles the patients i n i t i a l l y in the control group.

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Treatment of ARC and AIDS With DTC

2515

Patients receiving i n i t i a l treatment showed improvement in symptomatology (defined as any decrease in symptom score) at a high level of statistical significance. Among 22 DTC patients 16 (73%) had a reduction in symptom score versus four of 18 (22%) controls (xZ=lO.1, p=O.O02). The median decline in symptom score was 1.00 among treated patients versus zero among controls (p=0.0051 by Mann-Whitney). The data are shown in Tables 3 and 4.

TABLE I I l

Evaluation Of Dlethyldlthloca~amate In ARC And AIDS Symptomatology

Drug Number Improved* Percent

Control

16/22

4/18

73

22

X2

10.1

P (Fisher)

• 002

*Disappearance of I or more symptoms.

TABLE IV

Evaluation Of Olethyldlthlocarbamate In ARC AND AIDS Symptomatology

Median Symptom Score*

P (Mann-Whitney)

Parameter

Drug

Control

at entry

2.5

3.0

.289

at 16 weeks

1.4

4.0

.005

-1.0

0.0

change

*One point for each of six symptoms

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There was significant regression of previously abnormal lymphadenopathy among treated patients(Table 5). A response was defined as at lease 50% reduction in the sum of the products of lymph node cross sectional diameters. Among 34 patients with lymphadenopathy at entry, 10 of 19 (53%) DTC patients versus one of 15 (7%) controls had a response (xL=8.09, p=O.O05). In almost all cases, this regression of lymphadenopathy was accompanied by symptomatic improvement. Responders were not more likely to experience disease progression.

TABLE V

Evaluation Of Dlethyldlthlocarbamate In ARC And AIDS Lymphadenopathy

Number improved* Percent

Drug

Control

10/19

1/15

53

7

X2

8.09

P (Fisher)

.005

*50% or greater decrease in size of all measured nodes. Includes 1 patient with KS-involved lymph nodes.

Additional evidence of clinical improvement was seen in 3 patients on Imuthlol: disappearance of marked (Scm) splenomegaly, hairy leukoplakia, and perianal moniliasis refractory to maximal oral antifungal therapy. No such changes were seen in control patients. One patient with Kaposi's sarcoma at diagnosis confined to lymph nodes and confirmed by needle biopsy, experienced a 50% reduction in lymph node size. Repeat lymph node biopsy of this regression showed one microscopic nodule of active sarcoma and two nodular areas of scarring with the appearance of previous, healed sarcomatous involvement. This patient had not received any other therapy for his disease. This observation must be interpreted cautiously because Kaposi's lesions can wax and wane spontaneously. Lymphocyte surface markers and blastogenic responses to mitogens were similar at entry and showed no significant changes at 16 weeks between DTC and control patients (Table 6 and 7).

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Treatment of ARC and AIDS With DTC

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TABLE Vl Evaluation Of Dlethyldlthlocarbamate In ARC And AIDS Lymphocyte Markers

Median Number of Cells/ul

Parameter

Mann-Whitney

Drug

Control

Lymphocytes At entry Change

1245 -109

1185 -120

.7 .g

OKT4+ cells At entry Change

232 -25

192 -30

.6 .9

OKT8+ cells At entry Change

606 -12

482 +16

.5 .5

DR+ cells At entry Change

433 -45

357 -go

.8 .6

0.37 -.06

.9 .3

H/S Ratio At entry Change

0.39 -.03

P

TABLE VII Evaluation Of Dtethyldlthlocarbamate In ARC And AIDS

Lymphocyte Blastogenesls Parameter

Median CPM x 103

Mann-Whltney P

Drug

Control

At entry Change

57.4 +6.1

55.8 -8.3

.9 .3

CON-A At entry Change

24.0 +5.0

21.2 +6.0

.7 .5

6.9 +.4

4.0 -.4

.7 .5

PHA

PWM At entry Change

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No significant changes in the quantitative levels of IgG, IgA, or IgM were seen (data not shown). Neither was there any significant change in skin test reactivity under the current protocol (Table 8). TABLE VIII Evaluatlon Of Dlethyldlthlocarbamate In ARC And AIDS Delayed Hypersensitivity* Number of Patients (%) Category

Drug

Anergic to reactive

3/11 (27) 0/10 (0)

Reactive to anergic

5/11 (46) 3/8

Total reactive Before therapy After therapy

Control

(37)

11/22 (50) 8/18 (44) 9/22 (41) 5/18 (27)

*Multitest CMI battery of 7 antigens

No unexpected adverse side effects were seen in the treated patients. Mild transient nausea and retrosternal burning chest pain were seen in patients receiving DTC. Additionally, most patients noticed a characteristic alteration of taste and smell lasting 6-48 hours after drug administration. The taste and smell were variously described as metallic or sulfurous in nature. Only one patient experienced a mild disulfuram reaction when he consumed 120ml of beer eight hours after a dose of the drug. There was no evidence of a change in hematological or biochemical parameters during the study. Among the selected patients who had HIV cultures performed serially, no antiviral effect of DTC was observed. One treated patient had two positive cultures pre-enrollment and a negative culture at week 10, but the culture at week 14 was positive again. All other cultures in the remaining seven patients were positive both before and after treatment. Discussion

Thls study provides evidence of clinical activity for DTC in symptomatic ARC and AIDS patients. In this study there was s t a t i s t i c a l l y significant improvement in the clinical status of patients. The lack of objective improvement in rates of progression and results of immunologic testing prevent any final conclusions about the usefulness of DTC in ARC and AIDS patients. However, there are several possible explanations for this. First, the most effective dose, schedule, duration and route of administration of DTC are not known. A Phase I study might have been done, but since animal studies did not show a strong response effect and Lang's dose seemed to be active, we chose a related dose (as outlined below). The

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Treatment of ARC and AIDS With DTC

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earlier work by Lang u@ed an oral form of DTC at a dose of lOmg/kg (approximately 370mg/m~) weekly for six months. We chose the intravenous route to avoid hypothetical problems with malabsorption since diarrhea is a common problem in ARC and AIDS patients. We also elected to give roughly half the dosage per week that Lang used to insure safety and minimize adverse effects, i f any, although Lang's work shows that his dose was no more toxic than placebo. This dose was also chosen since blood levels of DTC after single IV doses are more than twice those after single oral dosing and doses of 5mg/kg were found to be effective in some of the systems described above. We chose to shorten the duration of treatment to four months (on each arm) to improve patient compliance. Second, the number of patients enrolled on each arm of this protocol may have been too small to detect real differences between the groups with available statistical methods. Additional studies are underway both in Europe and in the United States which will attempt to resolve this issue in longer-term randomized placebo controlled t r i a l s . Finally, at the time of this study,, we chose to enroll only a subset of individuals with advanced HIV infection based on clinical and immunologic grounds. We excluded patients with no or limited symptoms, since l i t t l e was known about DTC. I t is possible that patients with less advanced disease might, in particular, show a greater improvement in immunologic testing. Objections to our results can be raised based on the fact that the study was not blinded. Because of DTC's characteristic taste and odor i t is unlikely that even with a placebo infusion the patients would remain truly blinded throughout the study. Moreover, the validity of serial lymph node measurements should not be affected by the unblinded status of the investigator, and here we saw clear evidence of benefit by DTC treatment. The lack of precise understanding of the mechanism of DTC activity in augmenting T cell number and function is a serious limitation but should not prevent further clinical investigation while mechanisms are being studied. This is because DTC has minimal toxicity so long as ethanol is avoided. With the availability of at least one effective antiviral agent, i t now seems feasible to conduct studies of immunorestorative and other therapy in combination with antivirals. DTC may be a candidate for such studies. Acknowledgement This work was supported by a grant from the Merieux Institute, Miami, Florida. References 1. 2. 3. 4. 5. 6. 7. 8.

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E.M. EYSTER, J.J. GOEDERT, M.C. POON and O.T. PREBLE, N. Engl. J. Med.

309 583-586 (1983). 10. 11. 12.

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(1983).