The combination of zidovudine and didanosine has additive to synergistic inhibitory activity against HIV-1 in vitro [17, 18] and is being used clinically, because both agents are licensed and available. We did a clinical trial to characterize the safety and efficacy of a range of doses using combination zidovudine and didanosine therapy compared with zidovudine therapy alone. We found that this combination therapy is well tolerated and is associated with enhanced in-vivo activity as shown by higher and more prolonged increases of CD4⁺ cell counts, with more frequent decreases in plasma HIV-1 RNA titers, and with more stable hematologic measurements than zidovudine therapy alone.

Methods

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Between February 1990 and August 1991, 69 patients were enrolled in the study: 30 at University of Washington, 14 at University of Miami, 14 at New England Medical Center and Tufts University, and 11 at University of California, San Francisco. Patients had HIV-1 infection, CD4⁺ cell counts fewer than 400/mm³, fewer than 121 days of previous zidovudine therapy (Retrovir; Burroughs Wellcome Company, Research Triangle Park, North Carolina), and no previous didanosine therapy (Videx; Bristol Laboratories, Princeton, New Jersey). Patients also had granulocyte counts of 1200 cells/mm³ or more, hemoglobin levels greater than 90 g/L, platelet counts greater than 90 000/mm³, creatinine levels less than 1.5 times the upper limit of normal, and aspartate aminotransferase levels less than 5 times the upper limit of normal. Patients were excluded if they had visceral or progressive Kaposi sarcoma; more than four stools per day for 4 weeks; opportunistic infections requiring maintenance therapy; a history of pancreatitis, seizures, peripheral neuropathy, or zidovudine or didanosine intolerance; were pregnant or nursing; were taking experimental medications; or required chronic acyclovir therapy.

Study Design

The study was an open-label, partially randomized study of five different combination regimens of zidovudine and didanosine and one dosage regimen of zidovudine alone (Figure 1). Zidovudine was given as capsules three times per day. Didanosine was given twice daily as sachets containing a citrate/phosphate buffer with a neutralizing capacity of 30 to 40 mEq/dose. Because of initial concerns about the safety of this combination, enrollment was required in the lower dose groups (groups 1, 2, and 3) before enrollment in groups 4 and 5. In March 1991, after enrollment was completed in groups 1 to 5, the protocol was modified to enroll consecutively identified eligible patients in the zidovudine-alone regimen (group 6), to permit a comparison with standard therapy. Because standard zidovudine therapy in the United States at the time this trial was done was 500 to 600 mg daily, the groups that included zidovudine at 600 mg daily (groups 3, 5, and 6) were planned to have larger enrollment than the other groups. Treatment duration was 24 weeks. Patients were replaced if they discontinued treatment permanently before week six for reasons other than toxicity.

View larger version (25K): [in this window] [in a new window]   Figure 1. Outline of the study design. Enrollment into group 1 and groups 2 to 5 occurred concurrently. Enrollment started in groups 4 and 5 after enrollment was complete in groups 2 and 3. Didanosine was given in sachets; 500 mg of sachet form is equivalent to 400 mg in tablet form, and 334 mg of sachet form is equivalent to 200 mg in tablet form. ddI = didanosine; ZDV = zidovudine.

Patient Evaluation

The study protocol was approved by institutional review boards of participating institutions, and patients gave written informed consent. Patients had a standardized clinical and laboratory evaluation at enrollment and had weekly follow-up visits for 4 weeks and then every 2 weeks. Symptoms and signs were assessed at each visit and were graded as absent or within normal limits, or as mild, moderate, severe, or life-threatening severity. Patients at the University of Washington in groups 3 (n = 9) and 5 (n = 6) had blood samples collected at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, and 8 hours for pharmacokinetic assays, after a single 200-mg zidovudine dose, 2 days before starting study drugs, and after a single 167- or 250-mg didanosine dose on the day before starting combination therapy. In addition, 26 patients in groups 3 and 5 had plasma collected on a similar schedule during weeks 2 (n = 26) and 12 (n = 23). Doses of study drugs were modified if patients had moderate peripheral neuropathy or certain severe toxicities. Study drugs were permanently discontinued for severe or life-threatening toxicities.

Laboratory Measurements

CD4⁺ and CD8⁺ cells from peripheral blood were enumerated using monoclonal antibodies and flow cytometry [19]. Sera from each patient, frozen at –30°C, were assayed simultaneously for HIV-1 p24 antigen by an enzyme-linked immunosorbent assay (Abbott Laboratories, North Chicago, Illinois). A positive result for HIV-1 was defined as 12 pg/mL using an HIV-1 p24 antigen reference standard supplied by the AIDS Clinical Trials Group Virology Reference Laboratory. Sera from patients enrolled at the University of Washington were also assayed for immune-complex dissociated-HIV p24 antigen (Abbott Laboratories).

Plasma from patients enrolled at the University of Washington, stored at –70°C, was assayed for HIV-1 RNA by a semiquantitative polymerase chain reaction technique, as previously described [20]. All assays were done in duplicate on coded samples, and the semiquantitative assessment of the polymerase chain reaction-signal strength was graded using scanning densitometry without knowledge of any clinical data or treatment group.

Zidovudine concentrations in plasma were determined by radioimmunoassay [21]. Plasma didanosine concentrations were analyzed by high-performance liquid chromatography [22].

Data Analysis

Comparisons among groups were made using chi-square analysis or the Fisher exact test for discrete variables and using the Wilcoxon Mann-Whitney test and Kruskal-Wallis test among three or more groups for continuous variables. All P values were two-tailed.

To evaluate the effect of therapy for sequentially monitored variables, area-under-the-curve (AUC) analyses were done based on absolute change from pretreatment or time-averaged absolute change from pretreatment values. The baseline CD4⁺ cell count was an average of two pretreatment counts for each patient. The AUC for CD4⁺ cell counts was calculated by multiplying the average CD4⁺ cell count between two successive time points by the time elapsed between those time points, summing these areas for the study period, and dividing the result by the duration of the study period. This AUC was strongly related to the pretreatment CD4⁺ cell count. Analysis of covariance, adjusting for the initial CD4⁺ cell count, was used to examine the relation between the treatment group and the AUC. Residuals from the model were analyzed, and no pattern was discernible, suggesting the model was appropriate. Area-under-the-curve analysis, based on absolute CD4⁺ cell changes from pretreatment values, was used to compare these areas across groups using nonparametric statistics.

The proportion of patients with an increase in absolute CD4⁺ cells of 50 and 100 cells/mm³, on two consecutive measurements above the pretreatment value, was evaluated using contingency tables. The duration of response was defined as the time to two consecutive counts at or below baseline and was analyzed using Kaplan-Meier survival techniques.

Pharmacokinetic parameter estimates were calculated by standard methods and included the peak concentration (C_max), the time to peak concentration (T_max), the elimination half-life (t_1/2), the AUC, and the apparent oral clearance. The evaluation of a pharmacokinetic drug interaction between zidovudine and didanosine was done by paired t-test analysis of parameter estimates obtained for those patients in groups 3 and 5 who received single doses of zidovudine alone (day 2) and didanosine alone (day 1) followed by coadministration of both compounds with pharmacokinetic evaluations in weeks 2 and 12. Parameter estimates were considered statistically different for P values less than 0.05.

Results

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Study Patients

Sixty-seven (97%) patients were men; 61 (88%) were white, 2 (3%) were black, and 6 (9%) were of other races. The mean age (±SD) was 34 ± 6 years. Fifty-four (78%) were homosexual or bisexual men, 8 (12%) were homosexual or bisexual men who also used injection drugs, 2 (3%) were injection drug users, 3 (4%) had heterosexually acquired HIV-1, 1 (1%) had hemophilia, and 1 (1%) had unknown risk behavior for HIV. Thirty-eight (55%) patients were asymptomatic, 22 (32%) had constitutional symptoms, and 9 (13%) had the acquired immunodeficiency syndrome (AIDS). The median pretreatment CD4⁺ cell count was 259 cells/mm³, and the median Karnofsky performance score was 100. No statistical differences were seen in demographic, clinical, or laboratory characteristics among the six dosing groups, except for the expected difference in frequency of detectable HIV-1 p24 antigen (Table 1). The median HIV-1 p24 antigen level for patients in group 1 was 140 pg/mL (range, 17 to 282 pg/mL). Thirty-one patients had taken previous zidovudine therapy; the mean duration of therapy (±SD) was 65 days (± 36 days).

Fifty-eight (84%) patients completed 24 weeks of study treatment and 11 (16%) discontinued study treatment, 5 before week 6. Pretreatment characteristics of patients who discontinued treatment were similar to those who completed therapy. The reasons for study medication discontinuation were toxicity (3 patients), administrative reasons (4 patients), lost to follow-up (2 patients), moved out of area (1 patient), and alcohol use (1 patient). Of 58 patients completing the study, 28 had previously received zidovudine and 30 had had no previous nucleoside treatment.

Prophylaxis for Pneumocystis carinii pneumonia was required for patients with CD4⁺ cell counts fewer than 200/mm³ and was used by 56% of patients receiving combination therapy and 58% of patients receiving zidovudine alone. Other standard treatments for HIV-associated conditions (such as antifungal agents for oral candidiasis) were commonly used, and rates of use were similar in all treatment groups.

Pharmacokinetic Interactions

Pharmacokinetic parameter estimates for zidovudine and didanosine administered alone and in combination are shown in Table 2. For both agents, the maximum concentration, the time to peak concentration, the elimination half-life, the AUC, and the apparent oral clearance were similar for the drugs administered alone and in combination. Neither zidovudine nor didanosine appeared to affect the pharmacokinetic disposition of the other compound. Peak zidovudine concentrations after 200-mg doses averaged 1.16 µg/mL. Mean peak didanosine concentrations after 167- and 250-mg doses were 0.41 and 0.72 µg/mL, respectively.

CD4⁺ Cell Counts

The median peak CD4⁺ cell increase among those receiving combination regimens was 166 cells/mm³ (range, 21 to 507 cells/mm³) compared with 77 cells/mm³ (range, 0 to 230 cells/mm³) among those receiving zidovudine alone (P = 0.001). Similar trends were seen among different combination regimens and were seen if CD4⁺ percentages were analyzed.

Analyses of CD4⁺ cell counts (using AUC techniques) indicated that patients receiving combination therapy had better CD4⁺ cell responses than did those receiving zidovudine alone (Table 3). Median calculated AUC values (during weeks 0 to 12, 13 to 24, and 0 to 24) were 22, –2,and –1,respectively, for zidovudine alone (group 6) compared with 67, 57, and 67, respectively, for the combination regimens (groups 1 to 5). When adjusted for the initial CD4⁺ cell count and previous zidovudine therapy, the median calculated AUC was higher for combination therapy (groups 1 to 5) than for zidovudine alone (group 6) (P < 0.001). Similarly, AUC values in groups 3 and 5 (zidovudine, 600 mg/d, with 334 or 500 mg/d of didanosine) were greater than group 6 (zidovudine alone) (P = 0.001). Although the median change in CD4⁺ cell-AUC tended to be higher during weeks 13 to 24 for groups 3 and 5 than the other combination groups, no statistical differences in AUC were noted among groups 1 to 5 during the study.

Thirty-three (72%) patients receiving combination regimens (groups 1 to 5) who completed the protocol had a CD4⁺ cell response (increase of >50 cells/mm³ on two consecutive measurements) compared with 4 (33%) patients receiving zidovudine alone (group 6) (P = 0.02). The proportion of patients receiving combination regimens having this magnitude of CD4⁺ cell response was 71%, 75%, 75%, 71%, and 67%, respectively, in groups 1 to 5. Similar trends were seen if the analyses included only those who received at least 12 weeks of therapy or if the response criteria were an increase of 100 cells/mm³ after consecutive determinations. More patients receiving combination regimens with 600 mg/d of zidovudine (groups 3 and 5) had an increase in CD4⁺ cells of more than 50 cells/mm³ compared with patients receiving 600 mg/d of zidovudine alone (71% versus 33%, respectively) (P = 0.032). No statistical relation was noted between pretreatment CD4⁺ cell counts and proportion of responders.

To assess the durability of CD4⁺ cell increases, we evaluated the time the CD4⁺ cell counts were maintained at or above entry level during therapy (Figure 2). CD4⁺ cell counts tended to remain above pretreatment values longer with combination regimens (groups 1 to 5) than with zidovudine alone (group 6) [P = 0.06, Figure 2, top]. No statistical differences among the five combination regimens were noted Figure 2, bottom).

View larger version (19K): [in this window] [in a new window]   Figure 2. Survival analyses that show the time to the development of two consecutive CD4+ cell counts at or below baseline. Top. Comparison of all five combination zidovudine and didanosine groups (1 to 5) with zidovudine alone (group 6) (P = 0.06). The numbers of patients treated with a combination regimen or with zidovudine alone at various time points are shown at the bottom of the figure. Bottom. Survival analyses for all six individual treatment groups. Groups 1 to 5 are combination regimens with zidovudine and didanosine (see Table 1 and Figure 1 for doses), and group 6 is zidovudine therapy alone (600 mg). ddI = didanosine; ZDV = zidovudine.

Patients who had not previously received zidovudine had a more marked increase in CD4⁺ cell count than did those who had previously received zidovudine. The median peak CD4⁺ cell increase was 211 cells/mm³ in patients who previously had not received zidovudine compared with 112 cells/mm³ in patients who previously had received zidovudine (P = 0.003); a similar pattern was seen with all combination regimens. Although no difference was seen in the proportion of zidovudine-naive and zidovudine-experienced patients having CD4⁺ cell responses using the 50-cell criteria, patients who previously had not received zidovudine were more likely to have a 100-cell response than were patients who previously had received zidovudine (20 of 30 patients versus 8 of 28, P = 0.004). Similarly, the CD4⁺ cell AUC was 83 for zidovudine-naive and 24 for zidovudine-experienced patients (P = 0.015).

Virologic Markers of HIV-1 Infection

Sequential plasma samples at 0, 12, and 24 weeks from 25 University of Washington patients were assayed blinded to treatment group for plasma HIV-1 RNA by polymerase chain reaction by a semiquantitative assay. Seventeen patients had a one log or more decrease in virion-associated HIV-1 RNA copy number during therapy, 7 had no change, and 1 had an increase. Nine patients had a decrease in virion RNA from pretreatment levels at both 3 and 6 months, 5 had a decrease between 3 and 6 months, and 3 had a decrease at 3 months that was not sustained at 6 months. Of 17 patients who had a decrease in plasma RNA titers, 15 were treated with a combination regimen. Overall, 15 (83%) of 18 patients receiving combination regimens had a decrease in plasma HIV-1 RNA titers compared with 2 (29%) of 7 patients receiving zidovudine alone (P = 0.017). Ten of 12 patients receiving zidovudine (600 mg/d) with didanosine had a decrease in plasma HIV-1 RNA titers compared with 2 of 7 patients receiving 600 mg daily of zidovudine alone (P = 0.04). Among patients with detectable virion-associated RNA in plasma before treatment, RNA was not detectable at 3 and 6 months in 7 and 9 patients, respectively, in 16 recipients receiving combination therapy compared with 1 of 6 patients receiving zidovudine-alone. Median pretreatment CD4⁺ cell counts were similar, 263 and 273 cells/mm³, respectively, in patients who had decreased HIV-1 RNA signals and in those who did not (P > 0.05). Patients with a decrease in plasma HIV-1 RNA titers had a greater increase in CD4⁺ cell counts during therapy than did those with no decrease. The median change in CD4⁺ cell AUC was 86 for 17 patients with decreases in HIV-1 RNA titers during weeks 1 to 24 compared with an AUC of 8.5 for 7 patients with no change in HIV-1 RNA titers (P = 0.04).

Among the 7 patients in group 1 (with detectable levels of HIV-1 p24 antigen at study entry), 3 had decreases to below detectable levels, 1 had a 50% decrease, and 3 had no change in antigen level during this low-dose therapy. Among 25 patients who had sera assayed for immune-complex dissociated-HIV p24 antigen, 7 had detectable antigen at baseline compared with only 2 using the conventional assay. Four of these 5 patients treated with combination regimens had antigen that decreased to levels that were not detectable, whereas 1 of the 2 patients receiving zidovudine alone had a 50% decrease in antigen levels.

Beta-2-µglobulin values decreased from pretreatment levels during the study by a mean ±SD of 0.6 ± 0.2 overall (P < 0.0001). No statistical differences in trends were noted among the six treatment groups or among the five combination regimens compared with zidovudine alone.

Clinical Status and Weight

Only three patients developed new AIDS-defining illnesses; no deaths occurred and median Karnofsky performance scores remained similar in all groups during the study. Weight increased by a mean of 1.3 kg (P < 0.001), with 14 (25%) of 57 patients completing 24 weeks having an increase of more than 2.3 kg and only 4 patients having a weight loss of this magnitude. Trends in weight did not differ by treatment group. However, patients with no previous zidovudine therapy gained more weight (mean, 2.0 kg) compared with those who had previously received zidovudine (mean, 0.5 kg) (P = 0.02).

Toxicity

Three patients had study medication permanently terminated because of toxicity: 1 patient for lethargy and irritability; 1 for persistent nausea, headache, and fatigue; and 1 for anemia. Six other patients had dose modifications for study drug-associated toxic effects. Mild peripheral neuropathy developed in five patients. Neither pancreatitis nor reproducible increases in amylase levels developed in any patient. Rates of study drug-associated toxicity were similar in all treatment groups (P > 0.2).

Hemoglobin levels decreased slightly (median change, –3 g/L; P = 0.034) (Table 4). However, hemoglobin levels decreased less with combination regimens containing 600 mg/d of zidovudine (median, –1.5 g/L for groups 3 and 5) than with 600 mg of zidovudine alone (group 6) (P = 0.03). Overall, lymphocyte counts increased by a median of 165 (P = 0.004), and platelet counts increased by 22 000 (P < 0.001). No differences among treatment groups were seen for neutrophil, lymphocyte, or platelet counts or for erythrocyte mean corpuscular volume trends.

Discussion

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Among patients who were starting or had recently begun antiretroviral therapy, a combination of zidovudine and didanosine produced higher and more sustained increases in CD4⁺ cell counts, more frequent reduction in HIV-1 RNA titers in plasma, and better stabilization of hemoglobin levels than standard doses (600 mg/d) of zidovudine alone. The effect of combination therapy on CD4⁺ cell counts was apparent when analyzed several ways. Peak CD4⁺ cell and percentage increases, the proportion of patients with CD4⁺ cell counts remaining above pretreatment values and the increases in CD4⁺ cell counts as reflected in AUC calculations, were each better among patients receiving combination therapy compared with those receiving 600 mg of zidovudine alone. In addition, CD4⁺ cell responses, as measured by sustained increases (50 or 100 cell/mm³) and by duration of time that CD4⁺ cell counts were maintained above entry level values, were better in patients receiving combination therapy compared with those receiving zidovudine alone.

Although our results are encouraging, CD4⁺ cell counts are an incomplete surrogate marker for HIV disease [23, 24], and the clinical effects of this combination therapy remain to be determined. No statistical differences were seen in CD4⁺ cell trends among the different combination dosing regimens. However, with the inherent variability in CD4⁺ cell measurements, even in quality-controlled laboratories, 50 patients per treatment group would be necessary to show a 50-cell difference between any two of the combination groups with 95% power.

Although both zidovudine and didanosine have been shown to increase CD4⁺ cell counts, decrease HIV-1 viral replication, and delay progression of disease, our study was the first to evaluate these two agents administered in combination and to compare combination therapy with zidovudine monotherapy. Because the trial was initiated before didanosine was shown to have a clinical benefit, didanosine monotherapy was not evaluated; in retrospect, this feature would have been useful. Evidence that combination therapy produced better inhibition of HIV-1 replication than zidovudine monotherapy included the CD4⁺ cell responses, the higher frequency of decreases in HIV-1 plasma RNA titers, and the association between decreases in plasma-virion RNA and more sustained CD4⁺ cell responses. In addition, smaller decreases in hemoglobin levels were seen among patients treated with combination therapy than with an identical dose of zidovudine monotherapy, despite in-vitro observations suggesting additive inhibition of bone marrow progenitors [17]. These data suggest that the anemia associated with HIV-1 may, in part, occur because of the effect of HIV-1 on erythrocyte production or destruction and that effective antiviral therapy may ameliorate this effect.

Whether combination therapy delays development of in-vitro resistance to either drug was not evaluated. Several studies [8-10] have suggested that resistance to zidovudine develops over time and it has been hoped that combination therapy might delay this process. However, the benefits of combination therapy in our study are probably related to better inhibition of HIV-1 replication than to effects on antiviral resistance. Patients in our study had relatively high CD4⁺ cell counts, were just starting antiretroviral therapy, and the effects on CD4⁺ cell counts and plasma HIV-1 RNA titers were seen shortly after therapy was initiated. Thus, a delay in zidovudine resistance is an unlikely explanation for our results. The HIV-1 plasma RNA signal in our assay was stable after daily serial determinations for several days among patients with detectable plasma virion levels who were receiving chronic zidovudine therapy [25], suggesting that the decrease seen with the combination regimen was not due to assay variability. In addition, the decreases in HIV p24 antigen levels in most patients with the lowest dose combination support the added antiviral effect of this combination.

Although the patients were not randomized among all of our treatment groups, all groups were similar in demographic and clinical characteristics. The laboratory determinations measuring antiviral activity and toxicity were objective measurements that were not affected by the open-label study design. Regulatory concerns about the safety of concurrent zidovudine and didanosine use precluded randomization among all combination therapy groups. Although patients in the zidovudine monotherapy group were enrolled after those in groups 1 to 5, consecutive newly identified eligible patients were enrolled, preventing any potential bias in therapy assignment. In addition, CD4⁺ cell counts were done in laboratories certified by an ongoing quality-control program.

Combination zidovudine and didanosine therapy can be administered safely. Neither zidovudine nor didanosine affected the other agent's plasma pharmacokinetic characteristics, although we did not measure intracellular levels of either drug. The observation that combination therapy had no measurable additive toxic effects is clinically important.

Our data show the importance of differentiating between patients who have received previous nucleoside therapy and those who have not in evaluations of new antiretroviral agents. We observed differences between persons with no previous zidovudine therapy compared with those who had limited previous zidovudine therapy (6 to 120 days; mean, 65 days). Patients who had previously been treated with zidovudine had 50% of the CD4⁺ cell increase and 25% of the weight gain of those with no previous therapy.

Combination zidovudine and zalcitabine therapy has been shown to have greater and more persistent CD4⁺ cell responses than using 150 mg daily of zidovudine alone in patients with advanced HIV-1 infection without previous nucleoside therapy [26]. Both our study and the zidovudine and zalcitabine combination studies were done in patients just initiating antiretroviral therapy. Determining whether the benefits that we found for viral replication will be sustained enough to produce clinical effects on HIV-disease progression or will apply to persons with prolonged, previous antiretroviral therapy will require further study. Larger trials of both these nucleoside combinations compared with zidovudine alone and didanosine alone are underway and should eventually provide such clinical information.

Our data indicate that for selected patients, the concomitant administration of zidovudine and didanosine appears safe. We were not able to show statistical differences among any of our dosage regimens. However, to have a regimen that has antiviral efficacy even if doses are missed, we suggest that reasonable starting doses of this combination for an average-sized adult should be the same as for each agent used as monotherapy (zidovudine, 600 mg daily and didanosine, 500 mg daily in sachet form [400 mg daily in tablet form]). If patients are unable to tolerate these doses, lower doses may be acceptable.

Abbreviation

AUC: area under the curve