POLYCLONAL ANTI-TUMOR NECROSIS FACTOR- Fab USED AS AN ANCILLARY TREATMENT FOR SEVERE MALARIA

Transcription

1 Am. J. Trop. Med. Hyg., 61(1), 1999, pp Copyright 1999 by The American Society of Tropical Medicine and Hygiene POLYCLONAL ANTI-TUMOR NECROSIS FACTOR- Fab USED AS AN ANCILLARY TREATMENT FOR SEVERE MALARIA S. LOOAREESUWAN, L. SJOSTROM, S. KRUDSOOD, P. WILAIRATANA, R. S. PORTER, F. HILLS, AND D. A. WARRELL Department of Clinical Tropical Medicine and Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Therapeutic Antibodies, Inc., Medical College of St. Bartholomew s Hospital, London, United Kingdom; Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom Abstract. Single doses (250, 500, 1,000, or 2,000 units/kg) of an ovine polyclonal-specific Fab fragment directed against tumor necrosis factor- (TNF- ) were given to 17 adult patients with severe falciparum malaria immediately before treatment with artesunate in a pilot study to assess safety and optimal dosage with a view to future studies. Clinical and laboratory variables were compared with 11 controls. In the groups given Fab, there was a tendency for a faster resolution of clinical manifestations and reduction of fever but also a tendency towards longer parasite clearance times. Adverse events were more common in the control group and no early anaphylactic or late serum sickness reactions occurred in the Fab treated patients. On admission all patients had markedly elevated levels of TNF- (85 1,532 ng/l) and interleukin-6 (IL-6) (30 27,500 ng/l). Also, 86% had elevated interferon- (IFN- ) levels, 75% had increased IL-2 levels, 36% had increased IL-8 levels, and 21% had increased IL-1 levels. Antibody treatment reduced IFN- concentrations in a dose-related manner, but had no obvious effects on levels of other cytokines in this small study, although unbound TNF- was undetectable after Fab treatment. Circulating concentrations of soluble E-selectin, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were not affected by Fab treatment. The Fab exhibited a two-compartment, dose-proportional kinetics with an average elimination halflife of 12.0 hr, with about 20% being excreted renally. These results encourage a randomized, placebo-controlled trial in patients with cerebral malaria and provide some guidance about dosage. Malaria remains a major global health problem and in Thailand, 300,000 cases are reported annually with 2,000 3,000 deaths mainly from cerebral malaria. 1 Efforts to reduce morbidity and mortality have concentrated on early identification and treatment of infected patients. Many ancillary treatments have been tried in combination with conventional antimalarial drugs but, to date, there is little evidence to support the use of any ancillary treatment in severe cases of Plasmodium falciparum infection. 2 In African children with cerebral malaria, circulating levels of tumor necrosis factor- (TNF- ) and some other cytokines were found to be greatly elevated and to correlate with clinical severity, outcome (mortality) and the incidence of neurological sequelae. 3,4 This association suggests that TNF- and other cytokines may play a key role in the pathophysiology of cerebral and other severe forms of falciparum malaria, especially since children with the TNF2 variant allele of the TNF- promoter region of the genome had a 4.0 times greater risk of developing cerebral malaria, and a 7.7 times greater risk of dying of it. 5 If high levels of circulating cytokines have a role in the pathology of malaria, neutralizing them might improve the outcome. In a pilot study of 41 children with cerebral malaria in The Gambia, three different doses of a murine monoclonal antibody directed against TNF- were administered. 6 There was a dose-dependent reduction in fever, and a dosedependent increase in plasma TNF- levels, suggesting that the monoclonal antibody might be retaining TNF- in the circulation. A later trial in 302 Gambian children with cerebral malaria compared the use of 5mg/kg of a similar monoclonal anti-tnf- antibody with a control group. 7 This study confirmed the anti-pyretic effect but case fatality was not reduced and there was a significantly increased incidence of neurologic sequelae in the antibody-treated children. There are several reasons why a polyclonal anti-tnf- 26 Fab might be expected to be more effective than a monoclonal antibody; its smaller size allows greater and more rapid penetration into the interstitial (tissue) fluid and its neutralization of many epitopes and higher binding affinities should inhibit the biological activity of TNF- more effectively. This approach has been used in the Jarisch-Herxheimer reaction of louse-borne relapsing fever in which an explosive release of TNF- and other cytokines is precipitated by antibiotic treatment of the Borrelia recurrentis infection. 8 This treatment suppressed the reaction and significantly reduced circulating levels of TNF-, IL-6, and IL-8. The objective of the present pilot study was to test the safety and pharmacokinetics of the polyclonal anti-tnf- Fab as an adjunct to optimal antimalarial therapy with artesunate in patients with severe (but not necessarily cerebral) malaria. It was an open-label study in which the clinical response and parasite and fever clearance times in patients given a single intravenous dose (250, 500, 1,000, or 2,000 units/kg) of the antibody before the first dose of artesunate were compared with a control group, receiving artesunate alone. Inhibiting TNF- may also affect circulating levels of adhesion molecules such as endothelial leukocyte adhesion molecule-1 (E-selectin), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM- 1) whose expression may be increased by TNF- in vascular endothelium. The pharmacokinetics of the Fab was studied as a basis for designing an optimal therapeutic regimen. PATIENTS AND METHODS Procedure. The study was conducted in the Intensive Care Unit for severe malaria at the Hospital for Tropical Diseases in Bangkok, Thailand. Approval was given by the Ethics Committee of Mahidol University, Bangkok, Thailand. Patients 14 years of age or older were included if they or a responsible family member or friend gave informed con-

2 ANTI-TNF- Fab TREATMENT FOR SEVERE MALARIA 27 sent. Patients fulfilled at least one of the following criteria of severe malaria: 9 unrousable coma (score on Glasgow Coma Scale 8 of 15), severe anemia (hematocrit 15%), renal failure (serum creatinine 265 mol/l), hyperparasitemia ( 5% infected erythrocytes), hypoglycemia (blood glucose 2.2 mmol/l), shock (systolic BP 70 mm of Hg), repeated generalized convulsions, spontaneous bleeding or pulmonary edema. Patients who appeared to be severely ill clinically and likely to die within the next few hours and/ or with obvious additional acute or chronic disease processes (e.g., lobar pneumonia or diabetes mellitus), had received quinine within the previous 24 hr, had taken experimental medication within the previous four weeks, or were pregnant were excluded. Plasmodium falciparum infection was confirmed by detection of asexual parasites in peripheral blood smears. A full history and results of medical history and laboratory studies were recorded on standard proformas. Patients received standard intensive care support including fluids, antibiotics, and vasoactive agents as required. To prevent reinfection, patients were encouraged to remain in the hospital for 28 days. Antibody and antimalarial treatment. The anti-tnf antibody (CytoTAb ; Therapeutic Antibodies, Inc., Nashville, TN) is a sterile, non-pyrogenic, affinity-purified and lyophilized preparation of ovine Fab obtained from the blood of sheep immunized with recombinant human TNF-. The product has been tested in animal models and in patients with sepsis or louse-borne relapsing fever. 8 It is standardized by measuring its ability to neutralize the lytic effects of TNF- on a L929 murine fibroblast cell line (1 unit protects against 13,000 IU of TNF- ); each vial contained 5,000 units. There were four patients in each treatment group consecutively given 250, 500, or 1,000 units/kg. Five were given 2,000 units/kg. Eleven patients were enrolled in the control group and given only saline placebo (100 ml). After baseline measurements and blood and urine samples had been obtained, the antibody was administered over a 30-min period as an intravenous 0.9% saline infusion (100 ml). At the end of the Fab infusion (time zero), an additional blood sample was taken and standard antimalarial treatment used in this unit was started (120 mg artesunate intravenously and then 60 mg every 12 hr, switching to 50 mg oral doses every 12 hr to a total of 600 mg). Oral mefloquine (750 mg) was given 12 hr after the last dose of artesunate followed by 500 mg 6 hr later (total dose 1,250 mg). Clinical and laboratory evaluation. Clinical signs and symptoms (weakness, chill and rigors, headache, myalgia, dizziness, abdominal pain, anorexia, nausea, vomiting, diarrhea, palpitations, insomnia, pruritus, coughing, tinnitus, dehydration, and liver and spleen enlargement) were recorded daily during the first week and then weekly until patients were discharged. Clinical efficacy was assessed by recording the duration of coma in cerebral malaria and the development of other severe manifestations or complications. Body temperature, pulse, blood pressure, and respiratory rate were measured every 4 hr during the first week and then daily. Fever clearance time (FCT) was defined as the time taken for a patient s temperature to decrease to 37.0 C and remain there for at least 24 hr. Routine laboratory examinations were performed on day 0, 1, 7, 14, 21, and 28. Thick and thin blood films were obtained from fingerpricks and stained with Giemsa. Parasite density was determined by counting the number per 1,000 red blood cells in a thin film or by the number per 200 white blood cells in a thick film. Blood films were deemed negative if no parasites were seen in 200 oilimmersion fields on a thick film. Blood smears were examined every 6 hr from initiation of treatment until they were negative on two consecutive occasions; thereafter, smears were done daily. Parasite clearance time was expressed in hours from the start of treatment until the parasite counts decreased below the level of detection. Blood (10 ml) was collected into endotoxin-free, lithiumheparin coated glass tubes before and after administering Fab and then at 1, 2, 4, 6, 8, 24, and 48 hr after the antimalarial treatment. After centrifugation, the plasma was aliquoted and stored at 20 C until it was sent on dry ice to England for analysis using commercial immunoassay kits according to the manufacturer s instructions. Medgenix (Fleurs, Belgium) kits were used to measure levels of IL-1, IL-2, IL-6, IL-8, IFN-, TNF-, and ICAM-1 and kits from R&D Systems (Minneapolis, MN) to measure VCAM-1and E-selectin. Total ovine Fab concentrations (i.e., both Fab that is present in the free form and Fab that is bound to TNF- ) was also determined using a validated radioimmunoassay. Urine was collected between 0 and 8 hr and 8 and 24 hr and after recording the volume, a 10-ml aliquot was stored frozen for measuring Fab. Pharmacokinetic and statistical analysis. Pharmacokinetic parameters were determined for individual patient data using the Fig.P computer program (Biosoft, Cambridge, United Kingdom). Graphs of the log plasma Fab concentrations against time best fitted the equation Cp Ae t Be t where Cp is the Fab concentration in plasma at time t, A and B are the intercepts of the distribution and elimination phases, and and are the distribution and elimination rate constants. 10 The area under the curve (AUC) was estimated by the trapezoidal method and extrapolated to infinity using C t /. Systemic clearance (CL) was calculated from dose/ AUC 0 and renal clearance (CL R ) by dividing the amount excreted in 24 hr by the AUC 0 24hr. The volume of the central compartment (V c ) was obtained by dividing the dose by (A B) and the volume of distribution at steady state (V ss ) by dividing the product of dose and area under the moment curve (AUMC) by (AUC) 2. To calculate the dose in mg/kg, protein determination showed that 1 mg of Fab equaled 50 units in the L929 cytotoxicity assay. Differences in clinical and laboratory data between the groups were analyzed by the Mann-Whitney U test. Proportional data was tested either by the chi-square test or by Fischer s (two-tailed) exact test; a P value 0.05 was considered significant. RESULTS Pretreatment evaluation. Clinical and laboratory data were similar in the control and treated groups on admission (Table 1). On enrollment, 68% of the patients had an elevated temperature ( 37.5 C) and pulse rate ( 100 beats/ min) and 71% had a low blood pressure (systolic 100 mm Hg). Most had thrombocytopenia (86% /L) and some had a low white blood cell count (36% /L).

3 28 LOOAREESUWAN AND OTHERS TABLE 1 Clinical and laboratory characteristics on admission* Criteria Age (years) Weight (kg) Women:men Oral temperature ( C) Systolic blood pressure (mm of Hg) Pulse rate (per min) Respiratory rate (per min) Geometric mean parasite count (per l) Hemoglobin (mmol/l) [ ] Creatinine ( mol/l) [53 124] Total bilirubin ( mol/l) [ ] Jaundice Hepatomegaly Increased transaminase ( 80 U/L) Hyperparasitemia ( 5% infected RBC) Hypotension (systolic BP 70 mg of Hg) Cerebral malaria (GCS 8/15) Splenomegaly Low hematocrit ( 15%) Acidosis (ph 7.25) High creatinine ( 265 mol/l) Low glucose ( 2.2 mmol/l) Control group (n 11) 19 (14 37) 50 (42 89) 2: ( ) 100 (70 120) 104 (80 130) 24 (20 60) 84,000 (700 1,290,000) 7.4 ( ) 133 (65 433) 117 (16 402) 8 (73%) 6 (55%) 7 (64%) 4 (36%) 4 (36%) 4 (36%) 0 1 (9%) 2 (18%) 2 (18%) 1 (9%) * Data are median values (range) or frequency (proportion). Normal ranges for laboratory data are shown in brackets. RBC red blood cells; BP blood pressure; GCS Glasgow Coma Scale. Either serum aspartate or alanine aminotransferase. Treated groups (n 17) 23 (14 66) 47 (30 70) 8: ( ) 80 (62 150) 110 (82 130) 28 (22 40) 117,000 (7,000 1,033,000) 7.0 ( ) 102 (53 234) 55 (7 421) 8 (47%) 9 (53%) 6 (35%) 9 (53%) 7 (41%) 3 (18%) 6 (35%) 2 (12%) 1 (6%) 0 0 Hyperparasitemia (13 of 28) and circulatory shock (11 of 28) were the most common inclusion criteria. Seven patients were admitted with unrousable coma and three patients had hematocrit values 15%, and 10 required an average of two units of blood. Two patients in the control group had renal dysfunction (creatinine 265 mol/l), one had hypoglycemia, and one had had repeated generalized convulsions. Six patients in the antibody-treated group had splenomegaly but this was not observed in the controls. On admission, all patients complained of weakness, headache, and anorexia. Most complained of myalgia (95%), dizziness (95%), and nausea (90%) and 81% had vomiting, chills, and rigors and were dehydrated. Clinical response. All patients in this study showed a prompt response to antimalarial chemotherapy. Three patients left the study after 11, 21, and 22 days, respectively, for reasons unrelated to their treatment. All had negative TABLE 2 Recovery and resolution of clinical manifestations (median values and range in parentheses) Parameter Control group (n 11) Treated groups (n 17) P* Parasite clearance time (hr) 44 (31 75) 52 (29 86) 0.42 Fever clearance time (hr) 72 (34 104) 36 (4 128) 0.31 Coma recovery time (hr) Weakness (days) Headache (days) Dehydration (days) Chills and rigors (days) Vomiting (days) Hepatomegaly (days) 69 and (3 21) 4.0 (1 5) 5.0 (1 7) 4.0 (1 7) 4.0 (1 7) 9.5 (1 28) 26, 48, and (2 14) 3.0 (1 14) 2.0 (1 5) 2.5 (1 5) 3.0 (1 14) 4.0 (1 14) * By Mann-Whitney U test. Time to Glasgow Coma score of 15 (individual values for five patients). blood films and were well. The remainder had no recurrence of parasitemia during the full 28-day follow-up. The median time to clear 10% of the parasites in the control group was 3 hr and the time to reduce the parasite number by 50%, 90% and 100% was 9, 16, and 44 hr, respectively. Patients given Fab had slightly longer parasite clearance times with median values of 6, 11, 18, and 52 hr for 10%, 50%, 90%, and 100% clearance, respectively. There were no dose-related increases and these differences were not statistically significant. Most clinical manifestations present on admission gradually disappeared during the first week (Table 2). In the groups given anti-tnf- Fab, clinical manifestations resolved more quickly compared with the controls. Hepatomegaly resolved in a median of four days in the antibodytreated group compared with 10 days in the controls (P 0.15). Seven patients with cerebral malaria were enrolled and the two given 1,000 units/kg of Fab had coma recovery times (CRTs) of 26 and 48 hr, the one given 2,000 units/kg required 56 hr whereas two control patients had higher values of 69 and 67 hr. One of the control patients with cerebral malaria died and the CRT for one patient was not recorded. Most patients had repeated, transient increases in temperature during the first few days (Figure 1). Patients in the three highest dose groups showed shorter FCTs than those in the lowest dose group and the controls. However, the FCT could not be calculated for eight patients due to concomitant infections (mostly urinary tract infections successfully treated with antibiotics). The median FCT for all patients given antibody was 36 hr compared with 72 hr in the control group. The AUC and the fever index during 72 hr (AUC divided by time) calculated for individual patients also showed lower temperatures in the antibody-treated groups,

4 ANTI-TNF- Fab TREATMENT FOR SEVERE MALARIA 29 FIGURE 1. Mean SEM four hourly temperatures in the control group (, n 11) and combined antibody dose groups (O, n 17). Patients received either only saline or an antibody infusion 30 min before the first dose of artesunate at zero time. with the difference being nearly statistically significant (P 0.068, by the Kruskal-Wallis test). Adverse events. One patient in the control group died, giving an overall case fatality rate of approximately 4%. All patients tolerated the antibody and none had anaphylactic reactions or serum sickness. For the purpose of assessing safety of Fab, adverse events were defined as the worsening or appearance of additional clinical symptoms after treatment. The most common events were abdominal pain, nausea, weakness, vomiting, chills, and rigors occurring 1 5 days after starting treatment. Four of 17 patients treated with the antibody developed adverse events compared with 7 of 11 in the control group (P 0.08). Comparison of laboratory data collected before treatment and after 21 days showed no differences in measurement of hepatic and renal function between the control and Fab-treated groups. Cytokine and soluble receptor levels. On admission all patients had high TNF- concentrations, with an overall median value of 320 ng/l; the seven patients with cerebral malaria had even higher concentrations (median 650 ng/l) (Table 3). The control group showed a slight decrease in TNF- concentrations during the first 8 hr after treatment, which then decreased faster during the following 48-hr period (Figure 2A). The TNF- concentrations in the groups given antibody immediately became undetectable and remained so in all dose groups for 48 hr. Previous studies have shown that the Fab bound to TNF- is no longer recognized by the immunoassay. 11 Most patients had mildly elevated IFN- concentrations on admission, although one patient had a very high concentration (310 IU/ml). After treatment, concentrations of IFN- gradually decreased for 48 hr, but two patients had a transient reoccurrence. There was a dose-dependent reduction in IFN- concentrations in the different treatment groups (Figure 2B), but this was not statistically significant. Only 21% of patients had slightly elevated concentrations of IL-1 on admission but a higher proportion (75%) had elevated IL-2 concentrations. Both the IL-1 and IL-2 concentrations either remained constant or appeared in minor peaks during the two days following treatment and the Fab appeared to have no effect. All except one patient had increased IL-6 concentrations on admission and patients with cerebral malaria generally had higher values (median 3,500 ng/l compared with 494 ng/l for remainder). The IL- 6 values gradually decreased to normal levels in all patients during the following 48-hr period and it was difficult to judge whether the Fab had any effect since these groups had higher admission concentrations than the control group. More patients in the antibody-treated group had abnormal IL-8 concentrations on admission than the control group (47% compared with 18%), which gradually decreased during the next 48 hr. The majority (75%) of the patients had slightly elevated soluble E-selectin concentrations on admission, which gradually decreased in all groups during the first two days. Only 43% of patients had abnormal admission concentrations of ICAM-1, whereas all patients had elevated VCAM-1 levels with average values some three times above the upper normal limit. The VCAM-1 concentrations had decreased slightly after two days but remained high for all patients. Values for all three soluble receptors after 48 hr correlated Cytokine/receptor* TNF-, ng/liter (20) IFN-, IU/ml (0.8) IL-1, ng/liter (15) IL-2, IU/ml (0.4) IL-6, ng/liter (31) IL-8, ng/liter (47) E-selectin, g/liter (80) ICAM-1, g/liter (604) VCAM-1, g/liter (875) TABLE 3 Median (range) plasma cytokine and receptor concentrations on admission Control group (n 11) 340 (96 1,532) [100%] 3.1 (0.7 23) [91%] 5 (5 6.5) [0] 0.7 ( ) [91%] 494 (35 10,470) [100%] 3.9 ( ) [18%] 157 (49 295) [82%] 791 (419 1,384) [64%] 2,686 (1,596 4,195) [100%] Treated group (n 17) 300 (85 900) [100%] 3.0 ( ) [82%] 10 (5 35) [35%] 0.5 ( ) [65%] 1,100 (30 27,500) [94%] 40 ( ) [47%] 112 (33 222) [71%] 354 (159 1,174) [29%] 2,399 (1,529 3,337) [100%] * TNF- tumor necrosis factor- ; IFN- interferon- ; IL-1 interleukin-1 ; E-selectin endothelial leukocyte adhesion molecule-1; ICAM-1 intercellular adhesion molecule-1; VCAM-1 vascular cell adhesion molecule-1. Values in parentheses are the normal upper limit quoted by the manufacturer or the mean 2 SD of the quoted normal values. Values in brackets are % values above the normal limit.

5 30 LOOAREESUWAN AND OTHERS FIGURE 3. Cytokine (solid lines) and soluble receptor (dotted lines) levels in an exceptionally severe case of malaria enrolled in the control group. The patients died 5 hr after starting antimalarial treatment. Note the logarithmic scale for cytokines and receptor levels. VCAM-1 vascular cell adhesion molecule-1; IL-6 interleukin-6; TNF- tumor necrosis factor- ; ICAM-1 intercellular adhesion molecule-1; E-selectin endothelial leukocyte adhesion molecule-1. FIGURE 2. A, median tumor necrosis factor- (TNF- ) levels in the different antibody treatment groups: 250 (O), 500 ( ), 1,000 ( ), and 2,000 ( ) units/kg and the control group ( ). The upper level of a normal TNF- range is 20 ng/l and the minimum detectable concentration of the assay is 5 ng/l. B, median interferon- (IFN- ) levels following treatment. The broken line shows the upper level of the normal IFN- range (0.8 IU/ml). The minimum detectable IFN- concentration of the assay was 0.2 IU/ml. with admission concentrations and there was no suggestion of antibody dose-related reductions. A single patient enrolled in the control group showed the most dramatic increases in cytokine and soluble receptor concentrations (Figure 3). On admission, this 16-year-old male had cerebral malaria, hyperparasitemia, acute renal failure requiring hemodialyis, and respiratory failure needing mechanical ventilation. He had a temperature of 38.6 C, a blood pressure of 100/50 mm of Hg, and a pulse of 112/ min. Four hours after the first dose of artesunate, IL-1, IL- 6, IL-8, TNF-, and IFN- concentrations increased from baseline some 31-, 30-, 24-, 3-, and 2-fold, respectively. The level of soluble E-selectin remained stable but the ICAM-1 level doubled and the VCAM-1 level increased almost 3- fold before the patient died 5 hr after starting the antimalarial treatment. Levels of Fab. Pharmacokinetic parameters were calculated from individual patient data (Table 4). The peak concentrations and AUCs were dose dependent and showed good dose proportionality over the dosing range. The mean SD distribution half-life (t 1/2 ) was min (range min) and the mean SD elimination half-life (t 1/2 ) was hr (range hr). The mean SD volume of the central compartment (V c ) was ml/kg and the mean SD volume at steady state (V ss ) was ml/kg. The total body clearance of the drug from plasma (CL) was very similar in the dose groups (mean SD 14 2 ml/hr/kg). The mean SD renal clearance (CL R ) was ml/hr/kg and accounted for approximately 20% of the total body clearance. Therefore,

6 ANTI-TNF- Fab TREATMENT FOR SEVERE MALARIA 31 Dose (mg/kg) t ½ (min) TABLE 4 Pharmacokinetic parameters of ovine Fab used in severe malaria (n 17)* t ½ (hr) AUC 0 (mg hr/liter) CL (ml/hr/kg) CL R (ml/hr/kg) (22) 79 (33) 84 (16) 117 (51) 11.2 (1.4) 11.7 (1.1) 11.8 (2.0) 13.2 (1.1) 381 (81) 712 (165) 1,736 (263) 2,968 (940) 14 (3) 14 (3) 12 (2) 15 (5) 2.0 (0.8) 3.1 (0.7) 3.1 (2.1) 4.0 (3.6) 86 (9) 90 (40) 90 (20) 75 (5) 119 (19) 138 (28) 126 (13) 129 (47) Overall 93 (33) 12.0 (1.3) 14 (2) 3.0 (2.1) 85 (10) 127 (21) * Values are the mean (SD). t ½ distribution half-life; t ½ elimination half-life; AUC area under the plasma concentration-time curve; CL systemic clearance; CL R renal clearance; V C volume of plasma compartment; V ss volume of distribution at steady state. Protein determination showed that 50 units of Fab equaled 1 mg. V C (ml/kg) V ss (ml/kg) approximately 80% of the Fab was not cleared by the kidney. About 75% of the renal excretion occurred during the first 8 hr after administration of the drug. Three patients had an elevated AUC, partly due to a lower renal clearance of the drug. DISCUSSION The 28 patients enrolled in this study were representative of severe falciparum malaria as seen in Thailand. Seven had cerebral malaria and recovered consciousness in hr, which is similar to values observed in an earlier study in which quinine was used (mean SEM hr) 2 and in a recent large Vietnamese study of adults (median 66 hr, range hr). 12 The initial parasite counts include some very high values (8 more than 400,000/ l) but were again in the same range observed in previous studies. Treatment with intravenous artesunate resulted in parasite clearance in hr and combined with the longer-acting mefloquine prevented any reoccurrence of parasitemia. The TNF- concentrations in patients with malaria are much higher than in most other types of infection and may play a role in helping to clear the parasites. 13 It is, therefore, important to show that neutralizing TNF- does not delay parasite clearance. Three of the groups given Fab had longer parasite clearance values than the control group, but the increases were not dose-related. The variation in a small study is large and the effect on parasite clearance time needs to be carefully evaluated in a larger study. Whereas temperature in the Gambian children with cerebral malaria gradually decreased during the first 12 hr after treatment, 7 all patients in this study had transient, repeated temperature peaks during the first few days, which may reflect differences in the clinical manifestations of childhood and adult malaria. The three highest doses of the antibody lowered the FCT compared with the control group and a similar anti-pyretic effect was previously demonstrated by a monoclonal antibody against TNF- in cerebral malaria. 7 It is difficult to compare the present criteria for recovery with published values because there are many confounding variables; clinical symptoms in adults differ from those in children; antimalarial drugs vary in their speed and mode of action and in route of administration; the severity of malaria varies (acute, uncomplicated, severe, or cerebral malaria); and definitions of clinical variables differ between studies. For example, there was a large difference in FCTs reported in two large recent studies in Vietnam and in The Gambia that used artemether (with median values of 127 and 30 hr, respectively). 11,14 A further important endpoint used in malaria studies is mortality, but this is so low in the unit where this study was conducted (4% in the present study) that a very large study would be required. However, the time course for clinical improvement was encouragingly short and is reassuring that the antibody is not prolonging the natural history of the infection. Since an important aim of this study was to assess the safety of Fab in patients with severe malaria, a group of adverse events was defined (see Results). In fact, these adverse events were more frequent in control patients and so can not be attributed to the Fab. Artemisinin derivatives have proved safe in humans, but high doses produce neurotoxicity in several mammalian species. 15 Previous studies with this regimen of artesunate have shown that nausea (21%), vomiting (13%), diarrhea (8%), and rash (4%) may develop during the first two days after treatment, but subside without intervention. 16 Two patients also developed dizziness after receiving mefloquine. 16 In this study, there were no early anaphylactic reactions associated with the infusion of Fab and no late serum reactions were recorded. The levels of all cytokines measured in this study have been previously reported to be elevated in malaria. 17,18 On admission, all patients had markedly increased levels of TNF- and IL-6, most had elevated levels of IFN- (86%) and IL-2 (75%), but less had increased levels of IL-8 (36%) and IL-1 (21%). The TNF- and IL-6 values on admission were similar to those found in patients with louse-borne relapsing fever (LBRF) before treatment. 11 However, whereas antibiotic treatment of LBRF induces an explosive release of these cytokines, artesunate treatment did not. In the control group, concentrations of most cytokines remained consistently very high or gradually decreased over the following 2-day period. The patient who died showed a large and rapid increase in IL-1, IL-6, and IL-8 values and was an exception to this general pattern. The reduction seen in TNF- levels after antibody treatment is, at least in part, due to the Fab binding the TNF- so that the resulting complex can no longer be bound by the antibodies used in the immunoassay. 11 To detect significant reductions in IFN- and IL-6 concentrations would require a larger study. Severe falciparum malaria may affect many tissues and organs through impairment of their blood flow. Thus sticking of parasitized erythrocytes to the venular endothelium may initiate cerebral, retinal, renal, gastrointestinal, and bone marrow pathology. 19 Electron-dense knob-like protuberances on the parasitized erythrocyte membrane make contact with

7 32 LOOAREESUWAN AND OTHERS the vascular endothelium resulting in a mechanical obstruction and the parasites thereby also avoid clearance in the spleen and liver. Whereas membrane bound adhesion receptors promote the binding of infected red blood cells to the microvascular endothelium, soluble forms are thought to reverse this adhesion. 20 Tumor necrosis factor- promotes production or facilitates expression of some of these adhesion receptors. However, in this study the antibodies to TNF- did not affect the concentrations of E-selectin, ICAM-1, or VCAM-1. In the severely ill patient who died, the dramatic increase in TNF- concentrations was accompanied by increases in levels of VCAM-1. Whereas E-selectin and ICAM-1 concentrations were mostly normal after 48 hr, concentrations of VCAM-1 remained elevated and sampling should be extended in future studies. The redistribution of Fab from the vascular compartment into the interstitial fluid was relatively fast and, once equilibrium was established, the drug was eliminated partly through renal mechanism (20%). There are few published studies on pharmacokinetic parameters of Fab but the values in this study are similar to those reported for ovine affinity purified Fab used in digoxin intoxication. 21,22 However, the renal clearance reported for anti-digoxin Fab is higher (56% compared with 20%). 21 Since it is the antigen:antibody complex that is excreted, this difference is probably attributable to the relative sizes of digoxin and TNF- molecules (780 and 17,500 Da, respectively). Thus, ovine Fab bound to TNF- is less likely to pass through the glomerular membranes of the kidneys then when bound to digoxin. Since Fab was shown to have a relatively fast elimination half-life, an initial loading dose followed by multiple maintenance doses will be used in future studies to maintain high antibody concentrations during the critical early phase of the diseases. This pilot study confirmed the safety of the polyclonal antibody to TNF- used, suggesting repeated dosing as an appropriate regimen based on the pharmacokinetic data, and encourages a larger, double-blind, placebo-controlled trial of this ancillary treatment in patients with cerebral malaria. Acknowledgments: We are grateful to the nursing, medical, and laboratory staff of the Hospital for Tropical Diseases (Bangkok, Thailand) for dedicated care and help. Financial support: This study was supported by Therapeutic Antibodies, Inc. No author has an undeclared conflict of interest. Authors addresses: S. Looareesuwan, S. Krudsood, and P. Wilairatana, Department of Clinical Tropical Medicine and Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajavithi Road, Bangkok 10400, Thailand. L. Sjostrom, R. S. Porter, and F. Hills, Therapeutic Antibodies, Inc., Medical College of St. Bartholomew s Hospital, Charterhouse Square, London EC1M 6BQ, United Kingdom. D. A. Warrell, Centre for Tropical Medicine, University of Oxford, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom. Reprint requests: L. Sjostrom, Therapeutic Antibodies, Inc., Medical College of St. Bartholomew s Hospital, Charterhouse Square, London EC1M 6BQ, United Kingdom. REFERENCES 1. Ketrangsee S, Status report of malaria in Thailand. Southeast Asian J Trop Med Public Health 23 (suppl 4): Warrell DA, Looareesuwan S, Warrell MJ, Kasemsarn P, Intaraprasert R, Bunnag D, Harinasuta T, Dexamethasone proves deleterious in cerebral malaria. 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