Inhibition of androstenediol-dependent LNCaP tumour growth by 17alpha-ethynyl-5alpha-androstane-3alpha, 17beta-diol (HE3235).

Androst-5-ene-3beta, 17beta-diol (AED) is an adrenal hormone that has been reported to sustain prostate cancer growth after androgen deprivation therapy (ADT). LNCaP cells express a mutated androgen receptor that confers the ability to respond not only to androgen but also to oestrogen and adrenal hormones such as AED, and thus provide a cell line useful for identifying compounds capable of inhibiting AED-stimulated cell growth. We sought to determine whether structurally related steroids could inhibit AED-stimulated LNCaP cell growth in vitro and tumour growth in vivo. We report here the identification of a novel androstane steroid, HE3235 (17alpha-ethynyl-5alpha-androstan-3alpha, 17beta-diol), with significant inhibitory activity for AED-stimulated LNCaP proliferation. This inhibitory activity is accompanied by an increase in the number of apoptotic cells. Animal studies have confirmed the cytoreductive activity of HE3235 on LNCaP tumours. The results suggest that this compound may be of clinical use in castration-resistant prostate cancer.

16alpha-Bromoepiandrosterone (HE2000) limits non-productive inflammation and stimulates immunity in lungs.

16alpha-Bromoepiandrosterone (HE2000) is a synthetic steroid that limits non-productive inflammation, enhances protective immunity and improves survival in clinical studies of patients with human immunodeficiency virus (HIV), malaria and tuberculosis infections. We now show that HE2000 decreased nitric oxide production by lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Treatment with HE2000 also reduced non-productive inflammation associated with carrageenan-induced pleurisy and LPS-induced lung injury in mice. In the hapten-carrier reporter antigen popliteal lymph node assay, HE2000 increased absolute numbers of lymphocytes, antigen-presenting cells, hapten-specific immunoglobulin (Ig)M antibody-forming cells and shifted the interferon (IFN)-gamma/interleukin (IL)-4 balance towards IFN-gamma production. In the cystic fibrosis transmembrane conductance regulator (CFTR(-/-)) mouse model of acute Pseudomonas aeruginosa infection, treatment with HE2000 consistently reduced bacterial burden in lungs. All HE2000 effects were dose-dependent. In H1N1 infection in mice, HE2000 was safe but not effective as a monotherapy, as treatment did not effect survival. HE2000 reduced mortality related to excessive inflammation and opportunistic lung infections in animals and patients, and this might extend to those with H1N1 influenza infection.

A new orally bioavailable synthetic androstene inhibits collagen-induced arthritis in the mouse: androstene hormones as regulators of regulatory T cells.

Dehydroepiandrosterone (DHEA) has attracted much interest because of its many antiaging, metabolic and immune-modulating effects in rodents. Synthetic derivatives, such as 5-androstene-16alpha-fluoro-17-one (HE2500) and certain natural metabolites also provide benefit in various animal models of autoimmune and metabolic diseases. But, like DHEA, low potency and low oral bioavailability suggested limited usefulness of these compounds in humans. We hypothesized that HE3286, a novel 17-ethynyl derivative would be orally bioavailable, more potent, and chemically more useful in man than its parent compound. We found that on a dose/mass basis, HE3286 demonstrated up to 25% oral bioavailability in mice. In the DBA mouse model of collagen-induced arthritis (CIA), animals receiving oral treatment with HE3286 (50 mg/kg), beginning at onset of disease, significantly decreased CIA peak scores and daily severity of arthritis scores. Benefit was associated with decreases in: (1) production of TNF-alpha, IL-6, and IL-17; and (2) decreases in joint inflammation, erosion, and synovial proliferation as judged by histological analysis. HE3286 was not found to be immune suppressive in any of the classical models tested, including mitogen-induced proliferation, delayed-type hypersensitivity, or mixed lymphocyte reaction. Instead, benefit was associated with increases in numbers and function of CD4+CD25+FOXp3+CD127- regulatory T cells (T reg). To our knowledge, this is probably the first study to report that an orally bioavailable synthetic analogue of DHEA can ameliorate ongoing disease in a CIA mouse model with relevance to rheumatoid arthritis (RA) and to correlate that finding with decreases in proinflammatory cytokines and increases in T reg cells. Hormones targeting T reg cells hold the intriguing potential to treat autoimmune, infectious, and neoplastic diseases.

Improvement in immune parameters and human immunodeficiency virus-1 viral response in individuals treated with 16alpha-bromoepiandrosterone (HE2000).

A randomised, double-blind, placebo-controlled study examined the safety, tolerance, immunological effect and anti-human immunodeficiency virus (HIV) activity of sub-cutaneously administered HE2000 (16alpha-bromoepiandrosterone) as monotherapy in treatment-naïve patients with HIV-1. Twenty-four patients received five sequential daily doses of 50 or 100 mg of HE2000 or placebo every 6 weeks for up to three courses, and were followed thereafter for 3 months. HE2000 was safe, with transient injection site reactions being the main side-effect. Peripheral blood samples, collected serially, were analysed for changes in immune cell phenotypes. Significant increases were observed in the numbers of circulating dendritic cells, early activated (CD69+ CD25-) CD8 T-cells and T-NK cells after administration of 50-mg doses of HE2000 (p < 0.05). Gene expression in peripheral blood mononuclear cells was analysed by real-time RT-PCR. Before treatment, HIV-1-infected patients had significantly elevated transcripts for a number of inflammatory mediators (p < 0.012). After 50 mg or 100 mg HE2000, but not after placebo, there were significant sustained decreases in IL-1beta, TNF-alpha, IL-6 and Cox-2 transcripts (p < 0.05). There were no significant differences in CD4 cell numbers, although patients receiving 50-mg doses demonstrated a significant decrease in viral load (- 0.6 log; p < 0.01). Anti-HIV-1 T-cell responses were analysed serially using GAG-peptides to stimulate cytoplasmic IFN-gamma responses. After three courses, the 50-mg dose group demonstrated a significant increase in CD8 T-cell response against two distinct GAG peptide pools (p < 0.03). These findings suggest that immune-based therapies may be able to impact viral load by decreasing inflammation and/or stimulating CD8 T-cells.

Dynamic interaction of 111indium-labeled monoclonal antibodies with surface antigens of solid tumors visualized in vivo by external scintigraphy.

Two 111indium-labeled murine monoclonal antibodies (MoAb), D3 and 9.2.27, directed to tumor antigens of L-10 hepatocarcinoma and human melanoma, respectively, selectively localized antigen-positive target cells in guinea pigs and nude mice. The fate of MoAb differed in the two antigen-antibody systems after reacting with their corresponding tumor antigens in vivo as reflected by patterns of distribution and turnover in vivo. The 9.2.27 localized in melanoma xenograft in nude mice after intravenous administration with slow loss from tumor but more rapid loss from normal tissues and thus demonstrated optimal imaging of small tumors (approximately equal to 5 mm) between 3 and 6 days after injection of the radiolabeled antibody. In contrast, D3 demonstrated a biphasic localization in guinea pig L-10 hepatocarcinoma with a maximal activity on the 2d day after administration and showed rapid loss from both tumor and normal tissues. Nonspecific localization of antibodies in liver and in kidney was found both in syngeneic (nude mice) and xenogeneic (guinea pig) hosts but was more pronounced in the xenogeneic species. These results indicate that the nature of the antigen-antibody interaction may be of importance in selecting MoAb for both diagnosis and therapy of malignant diseases.

Preclinical trials with combinations and conjugates of T101 monoclonal antibody and doxorubicin.

We investigated the potential for additive therapy for malignancy using an anti-human T-cell monoclonal antibody, T101, and the chemotherapy agent doxorubicin (DOX). We compared the efficacy of T101 alone, DOX alone, T101 and DOX covalently linked to dextran to form an immunoconjugate, T101 plus DOX mixed together and injected, T101 and DOX injected separately, and nonspecific murine IgG2A plus DOX mixed together. Inhibition of [3H]thymidine was examined in vitro, and the clinical efficacy of each treatment was tested on human T-cell tumors growing in athymic mice. In vitro experiments confirmed retention of immunoreactivity and cytotoxicity by the immunoconjugate, but it was not superior to DOX alone. In efficacy experiments, all therapeutic arms were superior to placebo treatment (P less than 0.05). However, the best results in the animal tumor model were obtained with T101 mixed with DOX, perhaps because of formation of weak complexes via hydrophobic bonds. This mixture was superior to all other treatments, both by growth curve analysis (P less than 0.05) and by analysis of complete regression of tumor (P less than 0.01). T101 mixed with DOX was superior to a mixture of nonspecific mouse immunoglobulin and DOX and superior to a combination of T101 injected i.v. and DOX injected i.p. The antitumor effect of T101 mixed with DOX was blocked by premodulating the target antigen with T101. These data suggest that further exploration into monoclonal antibody-anthracycline complexes is warranted.

Stability, characterization, and kinetics of 111In-labeled monoclonal antitumor antibodies in normal animals and nude mouse-human tumor models.

Monoclonal antibodies (MoAbs) against carcinoembryonic antigen were successfully radiolabeled with 111In, and the radiopharmaceutical was characterized in vitro and in normal and tumor-bearing mice. The 111In-MoAb proved to be stable in vitro and in vivo under normal conditions, although instability could be induced in vitro with large quantities of iron-free transferrin. Animal distribution studies with 111In-MoAb demonstrated tumor localization superior to 67Ga and pharmacokinetics that were highly similar to those of endogenously labeled 75Se-MoAb. The 111In-MoAb followed first-order kinetics and fit a two-compartmental model when studied in nude mice bearing human colon tumors known to express carcinoembryonic antigen. Significant quantities of radiolabel appeared in tissues other than tumor, with liver and skin having the highest concentrations. Sufficient tumor/background ratios were formed for scanning purposes. The data indicate that 111In-MoAb may prove to be effective as a radiopharmaceutical for tumor imaging.

Bifunctional antibody: a binary radiopharmaceutical delivery system for imaging colorectal carcinoma.

In clinical studies we have evaluated a unique monoclonal antibody-based drug delivery system, a bifunctional antibody designed to deliver imaging or therapeutic agents, such as radioisotopes, drugs, or biologics, to tumor cells, while minimizing the dose to normal tissue. The bifunctional antibody, with one specificity to a tumor-associated antigen (carcinoembryonic antigen) and another specificity to a hapten, is injected and allowed to localize at a tumor site for 4 days. A hapten, tagged with a radioisotope, is subsequently injected for delivery to and capture by the prelocalized antibody at the tumor site. In studies reported here, the sulfhydryl groups of Fab' fragments of ZCE-025 and CHA-255 were linked with bis-maleimidomethyl ether to form an F(ab')2 bifunctional antibody coupled by a stable thioether linkage. EOTUBE, a hydroxyethylthiourido derivative of benzyl EDTA, was used as the hapten carrier of 111In. Fourteen patients 62-82 years old with recurrent or metastatic adenocarcinoma of the colon were studied. Twenty of 21 known lesions were imaged, and eight of nine new lesions were confirmed. With this fundamentally new approach to drug delivery, clearance from normal tissue is rapid, and high tumor:normal tissue ratios are expeditiously achieved.

Pharmacokinetics of 111In-labeled anti-p97 monoclonal antibody in patients with metastatic malignant melanoma.

Twenty-eight patients with metastatic malignant melanoma received anti-p97 murine monoclonal antibody (96.5) infused over 2 h at doses between 1 and 20 mg coupled to either 2.5 or 5.0 mCi of 111In by the bifunctional chelating agent diethyltriaminepentaacetic acid. Clearance of 111In from plasma closely fit an open, one-compartment mathematical model (r2 greater than 0.90). The overall half-life of 111In plasma was approximately 31 h and did not appear to be dependent on the total dose of antibody administered. The apparent volume of distribution of the 111In label approximated the total blood volume (7.8 +/- 0.7 liters) at the 1-mg dose and decreased to 3.0 +/- 0.14 liters at the 20-mg dose, suggesting saturation of antigenic or other extravascular binding sites at higher antibody doses. The clearance of the murine monoclonal antibody itself from plasma was measured by an enzyme-linked immunosorbent assay. The pharmacokinetics for the murine antibody in plasma also fit an open, one-compartment mathematical model. All pharmacokinetic parameters for unlabeled antibody closely paralleled those found for 111In-labeled antibody pharmacokinetics. This suggests that the 111In radiolabel remains complexed to the monoclonal antibody after in vivo administration. The cumulative urinary excretion of the 111In label over 48 h was between 12 and 23% of the total administered dose and is assumed to represent 111In-labeled chelate complex unattached to antibody. Analysis of the 111In label in spleen, liver, heart, and kidney showed that the concentration of label in liver tissue was reduced with increasing antibody doses and coincided with changes in the apparent volume of distribution. These studies show that murine monoclonal antibodies are cleared slowly from the circulation in humans and that early, rapid distribution of labeled antibody to the liver can be reduced by increasing the dose of unlabeled antibody. This may be particularly important in limiting hepatic toxicity when administering antibody coupled to drugs, radionuclides, or toxins.

Modification of human leukocyte interferon pharmacology with a monoclonal antibody.

The antitumor and antiviral properties of the interferons have been well established. However, the usefulness of the interferons may be limited, in part, because of rapid clearance from the plasma and degradation by plasma or tissue enzymes. A monoclonal antibody (IFG-252.2) was developed which binds to recombinant DNA-produced human alpha-interferon (rIFN-alpha A) without measurably reducing its in vitro antiviral or antiproliferative properties. Pharmacokinetic studies of rIFN-alpha A:antibody complex in the intact, anesthetized rat showed that rIFN-alpha A activity cleared from plasma 3-fold slower than found after injection of free rIFN-alpha A. This resulted in a 15-fold increase in its calculated area under concentration curve compared to that of free rIFN-alpha A. These studies suggest that interferon bound to a monoclonal antibody may provide a means to prevent the normal clearance and degradation of free interferon and may result in prolonged antitumor and antiviral plasma activity in vivo. Furthermore, it suggests that monoclonal antibodies to various biologically active agents may be used to favorably alter their pharmacokinetics while leaving their biological activity unaltered.

Yttrium-90 and iodine-131 radioimmunoglobulin therapy of an experimental human hepatoma.

Therapeutic trials were performed on the HepG2 human hepatoblastoma implanted s.c. in the athymic nude mouse. Animals were treated with polyclonal and monoclonal antiferritin and control antibodies labeled with either iodine-131 (131I) or yttrium-90 (90Y). Administration of 400 muCi of 131I-labeled polyclonal antiferritin or 300 muCi of 90Y-labeled polyclonal antiferritin significantly increased survival (P less than 0.001). There were no tumor cures with radiolabeled polyclonal antibody therapy. Animals treated with 200 or 300 muCi of 131I-labeled monoclonal antiferritin (QCI054) did not show increased survival compared to controls. Although 400 muCi of 131I-labeled QCI significantly prolonged survival, treatment resulted in no long-term survivors. Monoclonal antiferritin labeled with 90Y significantly prolonged survival of animals (P less than 0.001) at doses of 100, 200, or 300 muCi compared with untreated controls. Fifty % of the animals treated with 200 muCi and 75% of the animals treated with 300 muCi showed no evidence of disease at 140 days following treatment. Four hundred muCi of 90Y-labeled QCI proved toxic to the animals. Increased survival was accompanied by a decrease in tumor mitotic rate and increase in cellular polymorphism as determined by pathological examination. The radiation dose absorbed in the tumor correlated directly with tumor response following treatment. The absorbed dose in tumors for complete decay of the isotope ranged from 165 and 330 cGy at the periphery and center of small tumors for an administered activity of 200 muCi of 131I-labeled polyclonal antiferritin, to 7,573 and 12,400 cGy for 300 muCi of 90Y-labeled monoclonal antiferritin QCI.

Localization of 111In- and 125I-labeled monoclonal antibody in guinea pigs bearing line 10 hepatocarcinoma tumors.

A murine monoclonal antibody (D3) with demonstrated specificity for the guinea pig line 10 hepatocarcinoma (L10) was radiolabeled with either 125I or 111In and used to image dermal tumors in vivo. In one set of experiments, L10 tumors were established middorsally in one group of animals, and the similarly derived, antigenically distinct line 1 tumor was established in another group of animals. In spite of background imaging of liver, kidney, and spleen, L10 tumors were visualized clearly. Incorporation of radiolabel was demonstrated to predominate in the L10 tumor. In a separate set of experiments, L10 and line 1 tumors were established in contralateral thighs in the same animals. L10 tumors were visualized clearly, and tissue uptake of radiolabel was demonstrated to reside predominantly in the L10 tumor.

Anti-inflammatory and immune regulatory properties of 5-androsten-3beta, 17beta-diol (HE2100), and synthetic analogue HE3204: implications for treatment of autoimmune diseases.

5-Androsten-3beta, 17beta-diol (HE2100), and a synthetic analogue HE3204 are regarded as immune-regulating hormones, because both induce changes in the reporter antigen-popliteal lymph node assay (RA-PLNA). Mice were injected in the footpad with either HE2100 or HE3204 (0.01-3 mg), and a nonsensitizing dose of trinitrophenyl ovalbumin (TNP-OVA) was used as bystander reporter antigen. Seven days later, nodes were removed and numbers of cells (CD3, CD4, CD8, CD19; flow cytometry), TNP-specific IgM, IgG1, and IgG2a antibody-forming cells (AFCs; ELISPOT assay), and cytokines (interleukin-4 [IL-4], interferon-gamma [IFN-gamma]; ELISA) were measured. HE2100 and HE3204 increased cell numbers in a dose-dependent fashion. T (helper and suppressor) cells and B cells were increased (>5-fold). HE3204 was apparently twice as potent as HE2100. Both increased the B/T ratio (fivefold), increased TNP-specific IgM and IgG1 ( approximately 50-fold), and induced IgG2a AFCs. Both increased IL-4 and IFN-gamma secretion (up to threefold). Both displayed anti-inflammatory activity in the murine model of carrageenan-induced pleurisy, as evidenced by reduced neutrophil numbers and exudate volumes. Our observations suggest that both HE2100 and HE3204 are immune-regulating steroid hormones that exhibit anti-inflammatory properties. HE2100 (1 mg/mouse per day) provided significant benefit when given at disease onset in the SJL/J female mouse model of experimental autoimmune encephalomyelitis. These compounds and their analogues are candidates for further testing in autoimmune diseases.

Phase I study of the pharmacokinetics of a radioimmunoconjugate, 90Y-T101, in patients with CD5-expressing leukemia and lymphoma.

Ten patients with advanced or refractory CD5-expressing hematologic neoplasms [two with chronic lymphocytic leukemia and eight with cutaneous T-cell lymphoma (CTCL)] were treated in a Phase I study with the radioimmunoconjugate 90Y-T101, which targets CD5+ lymphocytes. Prior imaging studies using 111In-T101 demonstrated uptake in involved lymph nodes and skin in patients with CTCL, and Phase I studies with unmodified T101 demonstrated transient responses. In this study, patients were treated with 5 or 10 mCi of 90Y chelated to T101 via isothiocyanatobenzyl diethylenetriamine pentaacetic acid, along with tracer doses of 111In-T101 for imaging. The biodistribution of the radioimmunoconjugate was determined by measuring 90Y and 111In blood clearance, urine excretion, and accumulation in bone marrow and in involved skin lesions. The intravascular pharmacokinetics of 90Y were predicted by 111In-labeled T101. The greatest differences in biodistribution between 111In and 90Y were in the higher bone accumulation of 90Y and its lower urinary excretion. Imaging studies demonstrated targeting of skin lesions and involved lymph nodes in CTCL patients. The predominant toxicity was bone marrow suppression. Rapid antigenic modulation of CD5 on circulating T and B cells was observed. Recovery of T-cell populations occurred within 2-3 weeks; however, suppression of B-cell populations persisted after 5+ weeks. All CTCL patients developed human antimouse antibody after one cycle and thus were not retreated; one patient with chronic lymphocytic leukemia received a second cycle of therapy. Partial responses occurred in five patients, two with chronic lymphocytic leukemia and three with CTCL. The median response duration was 23 weeks. One CTCL patient who subsequently received electron beam irradiation to a residual lesion is disease-free after 6 years.

90Yttrium antiferritin--a new therapeutic radiolabeled antibody.

A new radiolabel 90Yttrium has been chelated to antiferritin antibodies for the treatment of hepatocellular cancer. The isotope 90Yttrium has the advantage of no significant external radiation to other individuals, that is, outpatient therapy and potentially more therapeutic power with an increase from 0.3 Mev 131I beta radiation to 0.9 Mev 90Yttrium pure beta radiation. Six patients treated in the Phase I study have had modest hematologic toxicity and two have had partial remissions of their primary tumors. One of these patients has had complete remission of a pulmonary metastasis. The use of external radiation (900 rad) to the primary tumor in advance of radiolabeled antibody administration has increased antibody uptake and increased tumor dose rate and total dose. An extensive study of 90Yttrium antiferritin is planned.

Synthesis and analgesic activity of some long-acting piperidinospiro derivatives of methadone.

A congener of methadone, in which the metabolically labile C-6 dimethylamino moiety was replaced with a piperidinospiro derivative, was reduced and acetylated. This conversion produced a marked increase in the duration of analgesia, a trend similar to that found for methadone.

In vivo kinetics of radiolabeled monoclonal anti-CEA antibodies in animal models.

Studies were performed to determine the effect of the radiolabel and circulating carcinoembryonic antigen (CEA) on the pharmacodynamics of monoclonal anti-CEA antibodies (MoAbs). The studies were performed in normal BALB/c mice and in nude mice bearing human colon tumors. Three different tumors were used, each of which produced CEA levels characteristic of that particular tumor's secretory rate. The CEJ-326 MoAb labeled with either 111In or 125I was used in all studies. Circulating CEA induced the removal of 125I and 111In MoAbs from the vascular compartment. Liver concentrations of 111In increased and 125I levels decreased as the CEA secretory rate of the tumor rose. This indicates that circulating CEA complexes form in the vascular compartment which, in an animal model, are removed by the liver and spleen. This results in decreased tumor uptake of the labeled MoAb. The iodinated MoAb complexes are dehalogenated while the 111In is retained by the liver. This dehalogenation may account for the relatively low liver activity observed in radioimmunoimaging with intact radioiodinated anti-CEA MoAbs, provided the CEA complexes are similarly removed from the vascular compartment by the human liver.

Tumor size: effect on monoclonal antibody uptake in tumor models.

Studies were performed to determine the effect of tumor size on the incorporation of radiolabeled monoclonal antitumor antibodies (MoAbs) into human tumors growing in nude mice. The colon tumors ranged in size from 0.03-1.6 g, the melanoma from 0.1 to 6.7 g, and the lymphoma from 0.06 to 10.2 g. Indium-111 was primarily used as the radiolabel, however, both 125I and 111In were used as tracers for the MoAb in one experiment. The per g radiopharmaceutical uptake by tumors was inversely proportional to tumor size when tumor specific MoAb was administered. This finding was independent of the radiolabel and was demonstrable when the mice bore two tumors of differing size. When the MoAb was not specific for the tumor, the data were less well defined and a statistically significant correlation with size did not occur. These data are strong evidence for a decrease in per g uptake of labeled tumor specific antibodies as tumors increase in size.

Detection of occult tumor using indium 111-labeled anticarcinoembryonic antigen antibodies.

Even with the advancement of radiologic techniques, metastatic cancers can still be difficult to detect. In this study, 48 patients suspected of having occult metastases were studied by radioimmunodetection following the administration of 92.5 to 181.3 MBq of indium 111-labeled monoclonal anticarcinoembryonic antigen antibody. All but seven patients were thought to have metastatic colorectal carcinoma. In the majority of cases, physical examinations and computed tomographic scans had failed to detect a lesion. At least one lesion that was later proved to exist was detected in 34 of the 50 studies performed on these patients. Seven of eight patients with normal radioimmunodetection scans remain free of disease. One hundred one sites were detected overall; 60 were considered true-positive sites and 27 false-positive sites. Fourteen sites remained in question. Nineteen false-negative sites occurred. Radioimmunoimaging appears valuable for the detection of occult cancer where standard, noninterventional techniques have failed to detect the suspected disease.

16alpha-bromoepiandrosterone, a dehydroepiandrosterone (DHEA) analogue, inhibits Plasmodium falciparum and Plasmodium berghei growth.

Dehydroepiandrosterone (DHEA) and its analogue, 16alpha-bromoepiandrosterone (alpha-epi-Br), may have activity against viral and parasitic infections, including human immunodeficiency virus (HIV) and Cryptosporidium parvum. Therefore, we evaluated its antimalarial effects on Plasmodium falciparum and Plasmodium berghei. In vitro, chloroquine (CQ)-sensitive and resistant strains of P. falciparum parasitized red blood cells were incubated with escalating doses of alpha-epi-Br or CQ. In vivo, 62 rats were infected with P. berghei and treated with CQ or alpha-epi-Br. At the highest doses tested against a CQ-sensitive strain, parasitemias decreased from 25.4% in the saline control group to 4.3% and 4.8% in the alpha-epi-Br and CQ groups, respectively (P < 0.05). Against two CQ-resistant strains, parasitemias decreased from 22.3-28.8% and 24.8-30% in the CQ and saline groups, respectively, to 2.5-2.7% in the alpha-epi-Br groups (P = 0.003). In vivo, on Day 4, parasitemias decreased from 23% in the saline group to 9-12% and 12% in the in alpha-epi-Br and CQ groups, respectively (P < 0.05). These data demonstrate that alpha-epi-Br shows activity against CQ-sensitive and resistant strains of P. falciparum in vitro. At the doses tested against P. berghei in vivo in rats, alpha-epi-Br is comparable to CQ.