Treatment of chloroquine-resistant malaria with esters of cephalotaxine: homoharringtonine.

The esters of cephalotaxine-harringtonine, homoharringtonine and deoxyharringtonine--have been reported by both Chinese and American oncologists as useful in the treatment of human nonlymphoblastic leukaemias and selected solid tumours of the head and neck. We report our results with homoharringtonine, currently a Phase II clinical trial drug with the National Cancer Institute, in the treatment of malaria. Homoharringtonine, 2.7-3.4 nM, was effective in causing 50% growth inhibition of two strains of chloroquine-resistant Plasmodium falciparum malaria in vitro. In vivo tests in mice infected with P. yoelii showed that this drug was effective in inhibiting parasite growth in this system as well. Histologically, the drug was associated with karyorrhexis. Drug-exposed cells showed decreased levels of putrescine and spermidine and increased spermine levels. Our findings not only demonstrate the potential usefulness of homoharringtonine in the treatment of chloroquine-resistant malaria, but also demonstrate the advantage of applying comparative biochemistry and an understanding of biological mechanisms in a rational approach to the development and treatment of diseases including malaria.

The effect of daunomycin on platelets in vitro.

Daunomycin (rubidomycin, daunorubicin), an anthracycline antimetabolite used in the therapy of acute leukemia, is highly toxic to both normal and malignant cells. Treatment with daunomycin may produce thrombocytopenia and bleeding which have been attributed to bone marrow toxicity. We have examined daunomycin to determine if a direct drug effect on platelet structure and function could contribute to a hemorrhagic diathesis in some patients on therapy. Normal citrated platelet-rich plasma was reacted in vitro with daunomycin and/or collagen with structural assessment by phase and electron microscopy and functional studies by platelet aggregation, [2-14C]5-hydroxytryptamine release studies and assays for released cytoplasmic marker enzyme, lactic dehydrogenase, in the supernatant fluid. High [greater than 0.04 mg/ml (greater than 0.07 mM)] concentrations of daunomycin were associated with structural changes, specifically by platelet swelling, vacuole formation and mitochondrial swelling with interruption of the trilaminar membrane. Platelets, exposed to low doses of daunomycin, 0.001 to 0.01 mg/ml (0.00177-0.0177 mM) of platelet-rich plasma, were dysfunctional with decreased aggregation with collagen and decreased [2-14C]5-hydroxytryptamine release. These studies indicate that daunomycin has a direct effect on platelets in vitro which may explain certain instances of bleeding observed in some patients undergoing therapy.

Antimalarial activity of neplanocin A with perturbations in the metabolism of purines, polyamines and S-adenosylmethionine.

Neplanocin A, a novel carbocyclic analog of adenosine, was found to be a mixed type inhibitor of S-adenosylhomocysteine hydrolase of human red blood cells with a Ki of 2 nM and an inactivating constant, Ki, of 3 nM. When tested against Plasmodium falciparum cultured in human red blood cells, neoplanocin A inhibited malarial growth with an ED50 of 3 microM. Above 2.5 microM, some red blood cells showed morphological aberrations and became echinocytes (spiculated red blood cells). In infected red blood cells, neplanocin A caused an elevation of the concentrations of S-adenosylmethionine and S-adenosylhomocysteine in a dose-dependent manner. Concurrently, a new analog of S-adenosylmethionine, S-neplanocinylmethionine, was formed. Analysis of polyamines showed that only putrescine was decreased significantly; the others were not changed. Purine analyses showed two putative neplanocinyl nucleotides, possibly the di-and the triphosphates. Neplanocin A most likely exerted its in vitro antimalarial effect via the inhibition of S-adenosylhomocysteine hydrolase and the attendant perturbation of methylation reactions.

Ornithine decarboxylase inhibition and the malaria-infected red cell: a model for polyamine metabolism and growth.

The addition of DL-alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, to human Plasmodium falciparum-infected red cells in continuous culture decreased both parasite growth and intracellular polyamine concentrations. Growth of P. falciparum in infected red cells was assessed by parasite counts and by assays for macromolecular syntheses of protein, DNA and RNA. Polyamine concentrations were measured by an automated high-performance liquid chromatography method. Concentrations of DL-alpha-difluoromethylornithine greater than 0.3 mM decreased intracellular levels of putrescine and spermidine, reduced ornithine decarboxylase activity and inhibited growth and maturation of the intracellular parasite at the trophozoite stage. There was a concomitant decrease in the synthesis of protein and nucleic acids in the infected cells. The use of this parasitized red cell model system together with polyamine inhibitors provide means of studying polyamine synthesis and cell growth in the design of new antimalarial therapy.

The effects of aspirin-containing serum in the continuous culture of Plasmodium falciparum.

In vitro culture of Plasmodium falciparum-infected human erythrocytes (RBC) has permitted systematic study of human host-parasite relations. In this study the effect of aspirin in the culture system was examined by using serum from blood of fasting, healthy male volunteers, before and after the ingestion of aspirin. The addition of aspirin-containing serum disturbed parasite growth and development: 0-1/2 dilutions of treated/control sera inhibited parasite development, with nuclear pyknosis, pyknotic extracellular parasites (trophozoites) in the media, decreased numbers and sizes of "rings" (early trophozoites), and an increased number of later trophozoites and schizonts. Paradoxically, while the incorporation of [3H]isoleucine into protein was not affected by the aspirin-containing sera, the incorporation [3H]hypoxanthine was significantly changed and did not correlate with morphological evidence of cytotoxicity. Thus, the so-called "incorporation" of a radioactive tracer is not a fully reliable index of parasite growth in the presence of certain compounds. The findings underscore the importance, in this culture system which employs human serum, of avoiding serum from donors who have recently ingested aspirin.

Rapid identification and detection of parasitized human red cells by automated flow cytometry.

Rapid and reliable identification of various human red cells parasites is important in many chemotherapeutic and immunologic studies. Because manual microscopic counting is tedious and imprecise, we have developed a simple diagnostic procedure for the automated flow cytometric detection of in vitro infected red cells, using a nucleic acid-binding fluorescent dye, acridine orange. Human malaria (Plasmodium falciparum)-infected red cells from continuous human erythrocyte culture were incubated at room temperature in acridine orange stain for 5 min after which the samples were analyzed by flow cytometry. Since mature red cells contain no DNA, infected red cells were identified with a distinct fluorescent signal. A total of 200,000 cells per sample were counted and analyzed in less than 2 min. Rings, trophozoites, and schizonts were assessed and identified in synchronized infected red cell cultures by flow cytometry. In addition, various stages of infected red cells were isolated with a cell sorter. This rapid method permits accurate and reliable assessment of data with the exclusion of anomalous data such as damaged cells, extraneous material, and contaminating particles.

Antimalarial properties of bredinin. Prediction based on identification of differences in human host-parasite purine metabolism.

Human malaria parasites (Plasmodium falciparum) grown in continuous erythrocyte culture utilize hypoxanthine for synthesis of both guanosine and adenosine nucleotides. Unlike the mature human erythrocyte, the malaria parasite depends on a constant supply of guanylates, primarily for synthesis of nucleic acids. This parasite specific requirement for guanylates led us to predict that a block in the hypoxanthine to guanosine monophosphate pathway would be selectively lethal to the parasite. Bredinin (4-carbamoyl-1-beta-D-ribofuranyosyl-imidazolium-5-olate) inhibited the synthesis of guanosine monophosphate from inosine monophosphate by parasitized erythrocytes. This block in guanylate synthesis was fatal to both a drug-sensitive (FCR-3) and a drug-resistant (VNS) strain of the malaria parasite at a bredinin concentration of 50 microM, arresting growth of the parasite at the trophozoite stage of development. These studies emphasize the essential role of guanylates and their synthesis from hypoxanthine in the metabolism of malaria parasite. They further suggest that bredinin or similar agents that selectively interfere with parasite guanylate metabolism may have potential for antimalarial chemotherapy.

Femtomolar ion-pair high-performance liquid chromatographic method for determining Dns-polyamine derivatives of red blood cell extracts utilizing an automated polyamine analyzer.

An ultra-sensitive automated method for the determination of polyamines in red blood cell extracts by ion-pair reversed-phase high-performance liquid chromatography is described. The 5-dimethylaminonaphthalene-1-sulfonyl derivatives of putrescine, 1,6-diaminohexane, spermidine, and spermine are separated on a muBondapak C18 column using 1-heptanesulfonic acid and acetonitrile as the mobile phase. All compounds are eluted within 28 min subsequent to the initial injection. The method has a lower detection limit of 25 fmoles on column. Because of the simplicity and ease of operation, the method is applicable for use in either the research or clinical laboratory.

Myeloproliferative disorder with unusual marrow chromosome constitution.

This report describes a patient referred at 14 years in 1971, after one year's surveillance by the family physician, for a persistently elevated erythrocyte sedimentation rate, leukopenia, and relative lymphocytosis. Despite the documented myeloproliferative syndrome of seven years, significant physiologic impairment precluding strenuous ranching and rodeo work appeared only in the last six to nine months. Throughout the clinical course characterized by pancytopenia, refractory and somewhat megaloblastic anemia partially responsive to oxymetholone, and subacute myeloblastic leukemia, he showed a persistent double trisomy--48, XY, +8, +21 in bone marrow. This report reiterates the value of chromosome analysis in the study of hematologic disorders and, in addition, emphasizes the need to individualize each patient's prognosis.

Application of simultaneous UV-radioactivity high-performance liquid chromatography to the study of intermediary metabolism. I. Purine nucleotides, nucleosides and bases.

Procedures are presented for the continuous-flow, simultaneous measurement of concentration and radioactivity of purine metabolites separated by high-performance liquid chromatography (HPLC). A microprocessor-controlled radioactivity flow detector connected in series to a UV-flow detector provides on-line quantitative monitoring of separated components in the post-column effluent stream. Through use of two HPLC separations--reversed-phase and anion-exchange--quantitation of all major purine nucleotides, nucleosides and bases is possible. The procedures provides a rapid, sensitive, and convenient means for the systematic study of purine metabolism.

Purine metabolism during continuous erythrocyte culture of human malaria parasites (P. falciparum).

Through use of techniques for continuous erythrocyte culture and novel chromatographic procedures we have identified the major salvage pathways for metabolism of purine bases in P. falciparum infected human erythrocytes. The malaria parasitized erythrocyte (PRBC) differs from the unparasitized mature erythrocyte (RBC) in the following ways: PRBC primarily utilize hypoxanthine for synthesis of both adenylates and guanylates; PRBC incorporate the base guanine into guanylates at a higher rate than control RBC, PRBC do not appear to use adenine effectively due to an overwhelming competition for this base by the whole erythrocyte population; although PRBC cultures show an initial increase in [ATP] this change is interpreted to reflect a generalized RBC response to malaria infection and not a response restricted to PRBC. Our observations have identified a purine pathway involving adenylosuccinate (AMPs) present only in PRBC (HYP leads to IMP leads to AMPS leads to AMP). They also demonstrate the importance of guanylate synthesis to the malaria parasite. We have shown that the purine metabolism of unparasitized erythrocytes is perturbed during malaria infection, an effect reflected primarily by an increase in erythrocyte ATP. This increase in host erythrocyte ATP not only improves metabolic conditions for parasite growth but also places a demand on available purine resources that has implications for the severe disruption of normal erythrocyte function.

Effect of prenatal drug administration on maternal and neonatal platelet aggregation and PF4 release.

Release of platelet antiheparin activity (platelet factor 4, PF4) induced by collagen and l-epinephrine, is normal in newborn infants without antenatal drug exposure. In contrast, PF4 release in platelets of infants of mothers with membrane-stabilizing drug ingestion is decreased to a greater extent than in their respective mothers. Platelet aggregation gives a qualitative indication of response. Platelets of mothers with antenatal drug exposure and those of their infants showed only a one-wave response to adenosine diphosphate, while platelets of these same mothers still showed a two-wave aggregation response to l-epinephrine. In contrast, platelets of all neonates lacked epinephrine-induced aggregation response, even when washed and resuspended in adult plasma. Our studies show that prenatal drugs have an effect on the platelet release mechanism in both mothers and their exposed infants; but the effect on the epinephrine-induced platelet response is less in mothers. Newborn infants have different aggregation and platelet release response to epinephrine which suggests that platelet aggregation and the release reaction can occur independently of each other.

Experience with disseminated intravascular coagulation in a children's hospital.

A study was initiated to determine the frequency and significance of disseminated intravascular coagulation (DIC) in the pediatric age group. With the aid of a scoring system, DIC was diagnosed in 48 patients in a period of slightly over one year in a pediatric referral centre with 7000 annual admissions. Sixty percent of all DIC occurred in infants under one month of life. Sixty-six percent of all DIC was associated with sepsis, usually from gram-negative infections. Seventy-nine percent of affected neonates were septic. Laboratory findings of diagnostic importance were anemia with red cell fragmentation, thrombocytopenia, elevated titres of fibrin split products, abnormal thrombin time, and low factor V activity. Mortality was 64% in all ages regardless of cause. Results of management of DIC by treatment of the underlying disease with or without anticoagulation were disappointing.