Allen B. Clarkson, Ph.D.

Pneumocystis S-adenosylmethionine transport: A potential drug target
Perez-Leal O.; Moncada C.; Clarkson A.B.; Merali S.
2011 ;45(6):1142-1146, American journal of respiratory cell & molecular biology
Pneumocystis pneumonia (PCP) is a life-threatening condition in immunosuppressed patients. Current treatments are inadequate, and new drug leads are needed. This fungus depends on its host for S-adenosylmethionine (AdoMet), a critical metabolic intermediate ordinarily synthesized by individual cells as needed. Pneumocystis contains a gene coding for the AdoMet-synthesizing enzyme methionine ATP transferase (MAT), and the protein is expressed. However, the fungus lacks MAT activity, and infection causes the depletion of host plasma AdoMet.The uptake of Pneumocystis AdoMet was shown to be exquisitely specific, which suggests the transport of AdoMet as a potential drug target. Here we report on the discovery of PcPET8, a Pneumocystis gene with homology to mitochondrial AdoMet transporters. When expressed by Saccharomyces cerevisiae, it locates properly to the mitochondrion and complements a strain of S. cerevisiae lacking its native mitochondrial AdoMet transporter. The importance of AdoMet transport is demonstrated by the ability of the AdoMet analogue sinefungin to block the uptake of Pneumocystis AdoMet and inhibit growth in culture. Because PcPET8 is likely critical for Pneumocystis, the yeast construct has potential as a surrogate for testing compounds against Pneumocystis
— id: 146269, year: 2011, vol: 45, page: 1142, stat: Journal Article,

Pneumocystis pneumonia and S-adenosylmethionine plasma levels
Skelly, Michael; Merali, Salim; Clarkson, Allen B
2011 Jun;62(6):490-492, Journal of infection
— id: 140492, year: 2011, vol: 62, page: 490, stat: Journal Article,

SILAC analysis of oxidative stress-mediated proteins in human pneumocytes: new role for treacle
Duan, Xunbao; Kelsen, Steve G; Clarkson, Allen B Jr; Ji, Rong; Merali, Salim
2010 Jun;10(11):2165-2174, Proteomics
To better understand lung oxidant stress responses, we examined A549 lung cells exposed to H(2)O(2) using 'stable isotope labeling by amino acids.' We identified 466 cytosolic and 387 nuclear proteins; H(2)O(2) exposure produced >or=twofold differences in 31, all were downregulations. None were previously reported as oxidant stress response proteins, although they share common functions. One of the responders, treacle, was linked to p53, an important oxidative stress response. The Treacher Collins-Franceschetti syndrome can result from treacle mutation and insufficiency was suggested to cause increased p53 leading to the syndrome. However, results here indicate p53 and treacle responses to H(2)O(2) are independent: treacle remains suppressed after p53 recovery; the threshold for treacle reduction is well above that for p53 induction; and treacle suppression by short interfering RNA does not modify the p53 response. Evidence of treacle antioxidant activity include reduction being driven by proteasome degradation independently of mRNA, typical for oxidant-absorbing proteins, and increased sensitivity to H(2)O(2) consequent to short interfering RNA suppression. Data here show a link between oxidative stress and treacle reduction, demonstrate that treacle does not control p53, provide evidence of a treacle oxidant defense role, support the hypothesis that oxidant stress plays a role in the Treacher Collins-Franceschetti syndrome, and raise the possibility that treacle plays an anti-oxidant role in lungs
— id: 122684, year: 2010, vol: 10, page: 2165, stat: Journal Article,

Mechanism and tissue specificity of nicotine-mediated lung S-adenosylmethionine reduction
Moncada, Camilo A; Clarkson, Allen; Perez-Leal, Oscar; Merali, Salim
2008 Mar 21;283(12):7690-7696, Journal of biological chemistry
We previously reported that chronic nicotine infusion blocks development of Pneumocystis pneumonia. This discovery developed from our work demonstrating the inability of this fungal pathogen to synthesize the critical metabolic intermediate S-adenosylmethionine and work by others showing nicotine to cause lung-specific reduction of S-adenosylmethionine in guinea pigs. We had found nicotine infusion to cause increased lung ornithine decarboxylase activity (rate-controlling enzyme of polyamine synthesis) and hypothesized that S-adenosylmethionine reduction is driven by up-regulated polyamine biosynthesis. Here we report a critical test of our hypothesis; inhibition of ornithine decarboxylase blocks the effect of nicotine on lung S-adenosylmethionine. Further support is provided by metabolite analyses showing nicotine to cause a strong diversion of S-adenosylmethionine toward polyamine synthesis and away from methylation reactions; these shifts are reversed by inhibition of ornithine decarboxylase. Because the nicotine effect on Pneumocystis is so striking, we considered the possibility of tissue specificity. Using laser capture microdissection, we collected samples of lung alveolar regions (site of infection) and respiratory epithelium for controls. We found nicotine to cause increased ornithine decarboxylase protein in alveolar regions but not airway epithelium; we conclude that tissue specificity likely contributes to the effect of nicotine on Pneumocystis pneumonia. Earlier we reported that the full effect of nicotine requires 3 weeks of treatment, and here we show recovery is symmetrical, also requiring 3 weeks after treatment cessation. Because this time frame is similar to pneumocyte turnover time, the shift in polyamine metabolism may occur as new pneumocytes are produced
— id: 78756, year: 2008, vol: 283, page: 7690, stat: Journal Article,

Kupffer cells are obligatory for Plasmodium yoelii sporozoite infection of the liver
Baer, Kerstin; Roosevelt, Michael; Clarkson, Allen B Jr; van Rooijen, Nico; Schnieder, Thomas; Frevert, Ute
2007 Feb;9(2):397-412, Cellular microbiology
Previous studies suggested Plasmodium sporozoites infect hepatocytes after passing through Kupffer cells, but proof has been elusive. Here we present new information strengthening that hypothesis. We used homozygous op/op mice known to have few Kupffer cells because they lack macrophage colony stimulating factor 1 required for macrophage maturation due to a deactivating point mutation in the osteopetrosis gene. We found these mice to have 77% fewer Kupffer cells and to exhibit reduced clearance of colloidal carbon particles compared with heterozygous phenotypically normal littermates. Using a novel quantitative reverse transcription polymerase chain reaction assay for P. yoelii 18S rRNA, we found liver infection of op/op mice to be decreased by 84% compared with controls. However, using another way of limiting Kupffer cells, treatment with liposome-encapsulated clodronate, infection of normal mice was enhanced seven- to 15-fold. This was explained by electron microscopy showing temporary gaps in the sinusoidal cell layer caused by this treatment. Thus, Kupffer cell deficiency in op/op mice decreases sporozoite infection by reducing the number of portals to the liver parenchyma, whereas clodronate increases sporozoite infection by opening portals and providing direct access to hepatocytes. Together these data provide strong support for the hypothesis that Kupffer cells are the portal for sporozoites to hepatocytes and critical for the onset of a malaria infection.
— id: 73010, year: 2007, vol: 9, page: 397, stat: Journal Article,

Effect of nicotine on lung S-adenosylmethionine and development of Pneumocystis pneumonia
Shivji, Mehboob; Burger, Suzanna; Moncada, Camilo Andres; Clarkson, Allen B Jr; Merali, Salim
2005 Apr 15;280(15):15219-15228, Journal of biological chemistry
Because S-adenosylmethionine (AdoMet) is required by Pneumocystis carinii in vitro, Pneumocystis infection depletes plasma AdoMet of rats and humans, nicotine reduces AdoMet of guinea pig lungs, and smoking correlates with reduced episodes of Pneumocystis pneumonia (PCP) in AIDS patients, we tested the effect of nicotine treatment on PCP using a rat model. Intraperitoneal infusion of 400 microg of R-(+) nicotine kg(-1) h(-1) intraperitoneal for 21 days caused a 15-fold reduction in lung AdoMet although neither plasma nor liver were changed. Infusion of 4 and 400 microg kg(-1) h(-1) into immunosuppressed rats, beginning when rats were inoculated with P. carinii, caused 85 and 99.88% reductions, respectively, in P. carinii cysts at sacrifice 21 days later; P. carinii nuclei were reduced by 91.2 and >99.99%, respectively. This effect was reversed by concomitant administration of AdoMet with nicotine. Treatment with AdoMet alone increased infection intensity. We conclude that AdoMet is a critical and limiting nutrient for Pneumocystis thus can serve as a therapeutic target for PCP. Regarding the mechanism, nicotine treatment caused no change in rat lung activity of AdoMet synthesizing methionine ATP transferase activity nor was there any evidence of increased AdoMet utilization for methylation reactions. Except of a doubling of putrescine, nicotine treatment also did not change lung polyamine content. However, key polyamine anabolic and catabolic enzymes were upregulated, and there were corresponding changes in polyamine metabolic intermediates. We conclude that chronic nicotine treatment increases lung polyamine catabolic/anabolic cycling and/or excretion leading to increased AdoMet-consuming polyamine biosynthesis and depletion of lung AdoMet
— id: 55785, year: 2005, vol: 280, page: 15219, stat: Journal Article,

S-adenosylmethionine and Pneumocystis
Merali, Salim; Clarkson, Allen Boykin Jr
2004 Aug 15;237(2):179-186, FEMS microbiology letters
Pneumocystis is a parasitic fungus causing pneumonia in immunosuppressed mammals and S-adenosylmethionine a key intermediary metabolite for all cells. Other than a species of Rickettsia bacteria and an aberrant strain of the protozoan Amoeba proteus, Pneumocystis is the only cell known unable to synthesize AdoMet; it must extract this key compound from its host. This was discovered using a culture system and confirmed by observing depletion of AdoMet in the plasma of infected animals. Depletion also occurs in patients with Pneumocystis pneumonia (PcP), a phenomenon suggested as a basis for a method for diagnosis and evaluation of response to therapy. Preliminary data indicate that deliberate reduction of host lung AdoMet by nicotine treatment is therapeutic in the rat model of Pneumocystis pneumonia
— id: 47850, year: 2004, vol: 237, page: 179, stat: Journal Article,

S-adenosylmethionine concentrations in diagnosis of Pneumocystis carinii pneumonia
Skelly, Michael; Hoffman, Julie; Fabbri, Marilyn; Holzman, Robert S; Clarkson, Allen B Jr; Merali, Salim
2003 Apr 12;361(9365):1267-1268, Lancet
Pneumocystis carinii is unable to synthesise S-adenosylmethionine and thus scavenges this intermediate. We aimed to test whether measurement of concentrations of this metabolic intermediate in plasma could provide a new method for rapid diagnosis of Pneumocystis carinii pneumonia (PCP). We measured S-adenosylmethionine plasma concentrations in 12 healthy controls, 16 patients with confirmed or suspected PCP, and 36 patients with other infections. Median concentration in healthy controls was 106 nmol/L (range 86-128), but the protein was undetectable in eight patients with histologically proven and seven with suspected PCP, and was 8 nmol/L in another confirmed case (p<0.0001). In 36 patients with other infections, S-adenosylmethionine concentrations were much the same as in controls: 18 had bacterial pneumonia, two tuberculosis, five cryptococcal meningitis, three had other infections, and eight had asymptomatic HIV-1 infection. After treatment for PCP, S-adenosylmethionine concentrations rose rapidly in all but one patient who died of the disease. Measurement of plasma S-adenosylmethionine concentrations could prove useful for diagnosis of PCP and assessment of patients' response to treatment
— id: 34384, year: 2003, vol: 361, page: 1267, stat: Journal Article,

Action of deferoxamine against Pneumocystis carinii
Clarkson AB Jr; Turkel-Parrella D; Williams JH; Chen LC; Gordon T; Merali S
2001 Dec;45(12):3560-3565, Antimicrobial agents & chemotherapy
We found earlier that deferoxamine (DFO), a drug used for treatment of iron overload, is active against a rat model of Pneumocystis carinii pneumonia (PCP). We had assumed a mode of action by deprivation of nutritional iron; however, data here show that DFO penetrates P. carinii, causing irreversible damage, thus indicating a different mode of action. Penetration was demonstrated by showing DFO uptake by high-pressure liquid chromatography analysis. By using calcein-AM as an indicator, exposure to DFO was shown to cause a reduction in P. carinii cytoplasmic free iron. Exposure to >or=100 microM DFO for >or=8 h in vitro caused growth to cease and cell numbers to decline over several days. This direct and irreversible damage to P. carinii led to the prediction that infrequent delivery of DFO to the lungs via an aerosol would be an effective treatment in the animal model of PCP. This prediction was confirmed by demonstrating that a once-a-week aerosol treatment of rats was 100% effective both as a prophylactic and as a curative treatment in a rat model of PCP
— id: 34381, year: 2001, vol: 45, page: 3560, stat: Journal Article,

Changes in S-adenosylmethionine levels in the course of treatment of human Pneumocystis carinii pneumonia (PCP). A potential aid in rapid diagnosis
Skelly, M; Hoffman, J; Fabbri, M; Holzman, RS; Clarkson, AB; Merali, S
2001 OCT 1 ;33(7):1091-1091, Clinical infectious diseases
— id: 54852, year: 2001, vol: 33, page: 1091, stat: Journal Article,

Effect of a bis-benzyl polyamine analogue on Pneumocystis carinii
Merali S; Saric M; Chin K; Clarkson AB Jr
2000 Feb;44(2):337-343, Antimicrobial agents & chemotherapy
Pneumocystis carinii is the causative agent of P. carinii pneumonia (PCP), an opportunistic infection associated with AIDS and other immunosuppressed conditions. Although polyamine metabolism of this fungus has been shown to be a chemotherapeutic target, this metabolism has not been thoroughly investigated. Reported here is the effect of one polyamine analogue, N, N'-bis[3-[(phenylmethyl)amino]propyl]-1,7-diaminoheptane (BBS), on P. carinii. BBS inhibits the growth of P. carinii in culture, but at concentrations higher than those required to inhibit the growth of other pathogens. However, BBS is at least as active in an animal model of PCP as in other models of diseases studied. BBS causes some reduction in P. carinii polyamine content and polyamine biosynthetic enzyme activities, but the effect is less than that observed with other pathogens and very much less than the effect of the polyamine biosynthesis inhibitor DL-alpha-difluoromethylornithine. BBS enters P. carinii cells via a polyamine transporter, unlike all other cells that have been studied. P. carinii cells do not remove the benzyl groups of BBS, as is reported for mammalian cells. The most likely mode of action is displacement of natural polyamines. Overall, the activity of BBS provides further evidence that polyamines and polyamine metabolism are rational targets for the development of drugs to treat PCP. Because the details of BBS-P. carinii interaction differ from those of other cells studied, polyamine analogues may provide a highly specific treatment for PCP
— id: 57563, year: 2000, vol: 44, page: 337, stat: Journal Article,

S-adenosylmethionine and Pneumocystis carinii
Merali S; Vargas D; Franklin M; Clarkson AB Jr
2000 May 19;275(20):14958-14963, Journal of biological chemistry
We previously reported that S-adenosylmethionine (AdoMet), a key molecule in methylation reactions and polyamine biosynthesis, enhances axenic culture of the AIDS-associated opportunistic fungal pathogen Pneumocystis carinii. Here we report that AdoMet is absolutely required for continuous growth. Two transporters are present, one high affinity, K(m) = 4.5 microm, and one low affinity, K(m) = 333 microm. The physiologically relevant high affinity transporter has a pH optimum of 7.5 and no related natural compounds compete for uptake. Transport is 98% inhibited at 4 degrees C, 24% inhibited by 20 mm sodium azide, and 95% inhibited by the combination of 20 mm sodium azide and 1 mm salicylhydroxamic acid; thus transport is active and dependent on both a cytochrome chain and an alternative oxidase. In vitro, AdoMet is used at a rate of 1. 40 x 10(7) molecules cell(-1) min(-1). AdoMet synthetase activity was not detected by a sensitive radiolabel incorporation assay capable of detecting 0.1% of the activity in rat liver. In addition, the AdoMet plasma concentration of rats is inversely correlated with the number of P. carinii in the lungs. These findings demonstrate that P. carinii is an AdoMet auxotroph. The uptake and metabolism of this compound are rational chemotherapeutic targets
— id: 11700, year: 2000, vol: 275, page: 14958, stat: Journal Article,

Continuous axenic cultivation of Pneumocystis carinii
Merali S; Frevert U; Williams JH; Chin K; Bryan R; Clarkson AB Jr
1999 Mar 2;96(5):2402-2407, Proceedings of the National Academy of Sciences of the United States of America
Continuous axenic culture of Pneumocystis carinii has been achieved. A culture vessel is used that allows for frequent medium exchange without disturbance of organisms that grow attached to a collagen-coated porous membrane. The growth medium is based on Minimal Essential Medium with Earle's salt supplemented with S-adenosyl-L-methionine, putrescine, ferric pyrophosphate, N-acetyl glucosamine, putrescine, p-aminobenzoic acid, L-cysteine and L-glutamine, and horse serum. Incubation is in room air at 31 degrees C. The pH of the medium begins at 8.8 and rises to approximately 9 as the cells grow. Doubling times calculated from growth curves obtained from cultures inoculated at moderate densities ranged from 35 to 65 hours. With a low-density inoculum, the doubling time is reduced to 19 hours. The morphology of cultured organisms in stained smears and in transmission electron micrographs is that of P. carinii, and P. carinii-specific mAbs label the cultured material. Cultured organisms are infective for immunosuppressed rats and can be stored frozen and used to reinitiate culture
— id: 57054, year: 1999, vol: 96, page: 2402, stat: Journal Article,

Continuous infusion of DL-alpha-difluoromethylornithine and improved efficacy against a rat model of Pneumocystis carinii pneumonia
Chin K; Merali S; Saric M; Clarkson AB Jr
1996 Oct;40(10):2318-2320, Antimicrobial agents & chemotherapy
The rapid depletion of Pneumocystis carinii polyamines caused by in vitro exposure to DL-alpha-difluoromethylornithine (DFMO; also called eflornithine or Ornidyl) and the rapid repletion following removal of this drug suggested that the in vivo efficacy of DFMO against P. carinii pneumonia (PCP) may be limited by troughs in drug concentration resulting from the schedule of administration. This led to the prediction that, compared with the response to the standard animal protocol of administering DFMO in drinking water, the response of a rat model of PCP to DFMO would be lessened by bolus administration and improved by continuous infusion. These predictions were confirmed. Intraperitoneal bolus administration of up to 3 g of DFMO kg of body weight-1 was completely ineffective, although this dose has been shown to be effective when given in the drinking water. Conversely, continuous infusion improved the response against PCP seven- to ninefold over the response to drinking water administration. These findings suggest that, compared with the standard clinical investigational protocol for treatment of PCP with DFMO given in four divided daily doses, continuous infusion combined with monitoring of drug concentrations in plasma may improve efficacy and/or reduce the already low rate of adverse effects
— id: 57060, year: 1996, vol: 40, page: 2318, stat: Journal Article,

Subpopulations of Pneumocystis carinii separated by a Percoll gradient
Chin K; Merali S; Shaw MM; Bartlett MS; Clarkson AB Jr
1996 Sep-Oct;43(5):53S-53S, Journal of eukaryotic microbiology
— id: 14316, year: 1996, vol: 43, page: 53S, stat: Journal Article,

Clinically achievable plasma deferoxamine concentrations eliminate Pneumocystis carinii trophozoites in a rat model
Merali S; Chin K; Grady RW; Clarkson AB Jr
1996 Sep-Oct;43(5):52S-52S, Journal of eukaryotic microbiology
— id: 14317, year: 1996, vol: 43, page: 52S, stat: Journal Article,

Trophozoite elimination in a rat model of Pneumocystis carinii pneumonia by clinically achievable plasma deferoxamine concentrations
Merali S; Chin K; Grady RW; Clarkson AB Jr
1996 May;40(5):1298-1300, Antimicrobial agents & chemotherapy
In a rat model of Pneumocystis carinii pneumonia, a 3-week infusion of deferoxamine producing concentrations in plasma of > or = 1.5 micrograms m-1 eliminated the trophozoite life cycle stage. Since this concentration is well below that routinely achieved in patients treated for iron overload, deferoxamine has promise as a therapy for AIDS-associated P.carinii pneumonia
— id: 57497, year: 1996, vol: 40, page: 1298, stat: Journal Article,

Polyamine analysis using N-hydroxysuccinimidyl-6-aminoquinoyl carbamate for pre-column derivatization
Merali S; Clarkson AB Jr
1996 Jan 26;675(2):321-326, Journal of chromatography. Pt. B. Biomedical applications
N-Hydroxysuccinimidyl-6-aminoquinoyl carbamate (AccQ.Fluor) was used as a polyamine pre-column derivatization reagent prior to HPLC analysis using a 5-micron C8 reversed-phase column. The fluorescence detector excitation wavelength was set at 250 nm and emission at 395 nm. Quantitation, reproducibility, linearity, recovery and stability were demonstrated. The lower limit of detection was 660 fmol. This method is 45 and 61 times more sensitive than those using the pre-column derivatizing agents dansyl chloride and orthophthalaldehyde, respectively. Applicability to biological samples was demonstrated by analyses of polyamines in extracts of mouse erythrocytes and Trypanosoma brucei brucei
— id: 57360, year: 1996, vol: 675, page: 321, stat: Journal Article,

Polyamine content of Pneumocystis carinii and response to the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine
Merali S; Clarkson AB Jr
1996 Apr;40(4):973-978, Antimicrobial agents & chemotherapy
Difluoromethylornithine (DFMO; eflornithine hydrochloride [Ornidyl]), a suicide inhibitor of the key polyamine biosynthesis enzyme ornithine decarboxylase (ODC), is effective in treating Pneumocystis carinii pneumonia, a common opportunistic infection associated with AIDS. Despite DFMO's specificity for ODC, the reason for its selective toxicity against P. carinii is unknown since both host and parasite are dependent on the same enzyme for polyamine biosynthesis. A new high-performance liquid chromatography method was used with P. carinii cells isolated from infected rat lungs to measure polyamine content, to confirm the presence of ODC, and to examine the effect of DFMO on polyamine concentrations. Putrescine, spermidine, and spermine were found to be present at 2.00 +/- 0.54, 1.26 +/- 0.51, and 1.59 +/- 0.91 nmol (mg of protein)-1, respectively, neither unusually high nor low values. ODC's specific activity was 79 +/- 11 pmol (mg of protein)-1 h-1, again not a remarkable value. However, the rates of both DFMO-induced polyamine depletion and subsequent repletion upon DFMO removal were unusually high. A 3-h exposure to 1 mM DFMO in vitro caused the depletion of putrescine, spermidine, and spermine to levels 12, 29, and 16%, respectively, of that of control cells. After DFMO removal and incubation for 1 h in serum-free media, polyamine levels returned to 78, 88, and 64%, respectively, of that of the control cells not exposed to DFMO. Since such depletions and repletions usually occur over periods of days rather than hours, these rapid changes may provide a clue to the selective action of DFMO against P. carinii and may guide the development of new compounds and an optimal drug administration schedule for DFMO
— id: 57414, year: 1996, vol: 40, page: 973, stat: Journal Article,

Clinically achievable plasma deferoxamine concentrations are therapeutic in a rat model of Pneumocystis carinii pneumonia
Merali S; Chin K; Del Angel L; Grady RW; Armstrong M; Clarkson AB Jr
1995 Sep;39(9):2023-2026, Antimicrobial agents & chemotherapy
The iron-chelating drug deferoxamine (DFO) has been shown to be active in animal models of Pneumocystis carinii pneumonia (PCP), with effective daily intraperitoneal bolus dosages being 400 and 1,000 mg of DFO mesylate kg of body weight-1 in mouse and rat models, respectively. Continuous infusion produced a moderately improved response in a rat model. The data reported here demonstrate that the response achieved by continuous infusion of 195 and 335 mg of DFO mesylate kg-1 day-1 in the rat model is associated with mean concentrations in plasma of 1.3 and 2.5 micrograms of DFO ml-1 and mean concentrations in lung tissue of 4.9 and 6.0 micrograms of DFO g of lung tissue-1, respectively. Since current clinical use of DFO mesylate for the treatment of iron overload produces higher concentrations in the plasma of patients, DFO may prove to be a useful anti-PCP treatment. The 2.4- to 3.8-fold higher DFO concentration observed in lung tissue compared with that observed in plasma may be important in the response of PCP to DFO
— id: 12733, year: 1995, vol: 39, page: 2023, stat: Journal Article,

Response of rat model of Pneumocystis carinii pneumonia to continuous infusion of deferoxamine
Merali S; Chin K; Grady RW; Weissberger L; Clarkson AB Jr
1995 Jul;39(7):1442-1444, Antimicrobial agents & chemotherapy
The iron-chelating drug deferoxamine mesylate (DFO) is active against Pneumocystis carinii in vitro and in rat and mouse models of P. carinii pneumonia. Because DFO has a short half-life, daily divided or continuous dosage was expected to improve the dose response, as is the case with DFO treatment of malaria. Therefore, results of single daily intraperitoneal injections were compared with results of an evenly divided four-times-daily dosage and the efficacy of delivery with implanted infusion pumps. The highest bolus dosage (1,000 mg kg-1 of body weight day-1) was as effective as the standard combination of trimethoprim with sulfamethoxazole. Unexpectedly, very little improvement was observed with the divided or continuous dosage, and several mechanisms that could account for this are discussed
— id: 12761, year: 1995, vol: 39, page: 1442, stat: Journal Article,

Competition between inhibitors of the trypanosome alternative oxidase (TAO) and reduced coenzyme Q9
Pollakis G; Grady RW; Dieck HA; Clarkson AB Jr
1995 Oct 12;50(8):1207-1210, Biochemical pharmacology
The trypanosome alternative oxidase (TAO) is an attractive target for chemotherapy for the diseases caused by African trypanosomes because there is no equivalent enzyme in mammalian hosts. Many inhibitors of this enzyme have been described, but there have been no data on the mechanism of inhibition. In the present study, reduced 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (decyl-CoQ-H2) was used as a substitute for the natural substrate CoQ9-H2 to allow direct measurements of the TAO in crude mitochondrial preparations from Trypanosoma brucei brucei. A Km value of 3.8 microM was obtained for this substrate. The following five compounds that have alkyl side chains from 1 to 4 carbons and belong to three classes of inhibitors showed a competitive inhibition pattern with respect to decyl-CoQ-H: p-methoxybenzhydroxamic acid, p-ethoxybenzhydroxamic acid,p-n-butyloxybenzhydroxamic acid, methyl 3,4-dihydroxybenzoate and N-n-butyl-3,4-dihydroxybenzamide. The following four compounds belonging to the same chemical classes but having alkyl side chains from 10 to 12 carbons showed uncompetitive inhibition patterns: p-n-dodecyloxybenzhydroxamic acid, n-decyl 3,4-dihydroxybenzoate, n-dodecyl 3,4-dihydroxybenzoate, and N-n-decyl-3,4-dihydroxybenzamide. Clearly, the first group of inhibitors compete with CoQ-H2 for the active site of the TAO. We propose that the uncompetitive patterns produced by the second group of inhibitors are due to the greater lipophilicity of these compounds and the resulting change in the interaction of the inhibitors and the membrane containing the TAO, thus affecting the local concentration of the inhibitors at the active site
— id: 56886, year: 1995, vol: 50, page: 1207, stat: Journal Article,

Combination chemotherapy of drug-resistant Trypanosoma brucei rhodesiense infections in mice using DL-alpha-difluoromethylornithine and standard trypanocides
Bacchi CJ; Nathan HC; Yarlett N; Goldberg B; McCann PP; Sjoerdsma A; Saric M; Clarkson AB Jr
1994 Mar;38(3):563-569, Antimicrobial agents & chemotherapy
Combinations of DL-alpha-difluoromethylornithine (DFMO; eflornithine; Ornidyl) with either suramin or melarsen oxide were found to be effective against acute laboratory model infections with Trypanosoma brucei rhodesiense. We used clinical isolates known to be resistant to these drugs when used singly. An infection with a melarsen oxide-refractory isolate was cured by a combination of low-dose DFMO (0.5% in the drinking water) plus low-dose suramin (1 mg/kg of body weight given intraperitoneally). Another strain, moderately resistant to arsenical drugs, was cured with combinations of 4% DFMO with 5 mg of melarsen oxide per kg. Furthermore, a combination of DFMO (2% in the drinking water) and suramin (20 mg/kg) provided a 100% cure rate in a central nervous system model, although the same doses of these drugs used singly were completely ineffective. The synergism of DFMO and suramin against an acute infection was improved when suramin was given at the end of the DFMO administration. No adverse interactions were observed when high doses of DFMO combined with high doses of suramin were administered to uninfected mice. These results suggest that combinations of DFMO and suramin should be examined clinically for activity in arsenical-drug-refractory cases of East African sleeping sickness
— id: 14318, year: 1994, vol: 38, page: 563, stat: Journal Article,

VOLATILE ORGANIC-COMPOUNDS IN THE BREATH OF RATS WITH PNEUMOCYSTIS-CARINII PNEUMONIA
PHILLIPS, M; CLARKSON, AB; MERALI, S; PHILLIPS, A; GREENBERG, J
1994 OCT ;42(3):A459-A459, Clinical research
— id: 52383, year: 1994, vol: 42, page: A459, stat: Journal Article,

Ornithine decarboxylase in Pneumocystis carinii and implications for therapy
Saric M; Clarkson AB Jr
1994 Nov;38(11):2545-2552, Antimicrobial agents & chemotherapy
Pneumocystis carinii pneumonia (PCP) can be treated with eflornithine (difluoromethylornithine, DFMO, Ornidyl), a competitive irreversible inhibitor of ornithine decarboxylase (ODC), a key enzyme for polyamine biosynthesis. Because ODC has been reported to be absent from P. carinii, it has been assumed that eflornithine affects P. carinii only indirectly, by affecting host polyamine biosynthesis. If this is true, then improvements in the selectivity of antipolyamine therapy for PCP would be limited. Since the presence of ODC in P. carinii is an important issue, a new search for this enzyme was made. Not only were initial assays negative, but P. carinii extract reduced the background catalytic action of pyridoxal-5'-phosphate, the coenzyme required by the enzyme. This suggested the presence of an inhibitor, which was further supported by the observation that a P. carinii extract could suppress a source of known ODC activity. The inhibitory activity could be removed by a desalting column or by dialysis, allowing detection of P. carinii ODC. Indirect evidence indicates that the inhibition is only apparent and is caused by unlabeled ornithine in the extract of P. carinii which interferes with the radiolabel-based assay system. P. carinii and host ODCs respond differently to changes in pH. P. carinii ODC is much less susceptible to inhibition by eflornithine than host ODC. The presence of ODC in P. carinii suggests that P. carinii ODC is the target of eflornithine and that P. carinii ODC may have sufficiently specific properties that inhibitors with improved selectivity against P. carinii ODC could be identified
— id: 56707, year: 1994, vol: 38, page: 2545, stat: Journal Article,

Non-cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei
Bienen EJ; Maturi RK; Pollakis G; Clarkson AB Jr
1993 Aug 15;216(1):75-80, European journal of biochemistry
The life cycle of Trypanosoma brucei brucei involves a series of differentiation steps characterized by marked changes in mitochondrial development and function. The bloodstream forms of this parasite completely lack cytochromes and have not been considered to have any Krebs cycle function. It has been suggested that glycolysis is the sole source of ATP in all bloodstream forms. However, earlier results indicated that in the mitochondria of the transitional intermediate/short stumpy bloodstream forms, the biochemical pathways are present that could allow intra-mitochondrial production of ATP. Using a high mannitol buffer to enhance permeability, we confirm previous observations showing that transitional forms maintain motility and respiratory activity with 2-oxoglutarate as the sole substrate. Using a luminometer to measure intracellular ATP levels via the luciferin/luciferase chemiluminescence assay, we show that these same transitional forms, but not long slender forms, maintain high levels of intracellular ATP in the presence of 2-oxoglutarate. Further, in the presence of bongkrekic acid, an inhibitor of the mitochondrial adenine nucleotide translocase, ATP levels are reduced with subsequent death and lysis of the cells when 2-oxoglutarate, but not glucose, is used as sole substrate. These data are direct evidence of ATP production by transitional bloodstream form mitochondria
— id: 13092, year: 1993, vol: 216, page: 75, stat: Journal Article,

DNA probe for rRNA as a measurement of Pneumocystis carinii in rat lungs
Fakiri, Eleni; Pollakis, Georgios; Saric, Muhamed; Clarkson, Allen B., Jr.
1993 ;4(4):171-176, Methods in molecular & cellular biology
5' 32P-labeled-26-mer oligo-deoxyribonucleotide probe was used with a 'dot blot' apparatus to detect and measure the amount of Pneumocystis carinii rRNA in the total RNA extracted from rat lungs. The results of this DNA/RNA hybridization technique correlated well with standard cytological means of counting P. carinii while offering objectivity and superior precision
— id: 98813, year: 1993, vol: 4, page: 171, stat: Journal Article,

N-n-alkyl-3,4-dihydroxybenzamides as inhibitors of the trypanosome alternative oxidase: activity in vitro and in vivo
Grady RW; Bienen EJ; Dieck HA; Saric M; Clarkson AB Jr
1993 May;37(5):1082-1085, Antimicrobial agents & chemotherapy
On the basis of our previous demonstration of the high inhibitory activity of a series of p-n-alkyloxybenzhydroxamic acids and n-alkyl esters of 3,4-dihydroxybenzoic acid against the trypanosome alternative oxidase in a cell-free mitochondrial preparation of Trypanosoma brucei brucei, we synthesized a series of N-n-alkyl-3,4-dihydroxybenzamides for evaluation as inhibitors of this enzyme. This class of compounds was selected with the expectation of their having similar inhibitory activity to but greater solubility than the esters and hydroxamic acids noted above and greater resistance to serum hydrolases in vivo. We predicted that such properties would allow an inhibitor of the trypanosome alternative oxidase to be coadministered with glycerol as a means of providing treatment for infections by African trypanosomes. As expected, such benzamides were both more soluble and more stable, some being more active against the target enzyme than the corresponding ester. One, N-n-butyl-3,4-dihydroxybenzamide, was selected for evaluation in vivo against T. brucei brucei. When combined with glycerol, this benzamide was found to be curative. A regimen wherein 450 mg of N-n-butyl-3,4-dihydroxybenzamide per kg and 15 g of glycerol per kg were given hourly in three divided doses cured 17 of 19 mice with established T. brucei brucei infections. This combination is more active in vivo than any other designed to block simultaneously both the unique respiratory electron transport system and the anaerobic glycolytic pathways of these pathogenic protozoa
— id: 14319, year: 1993, vol: 37, page: 1082, stat: Journal Article,

Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor
Pontes de Carvalho LC; Tomlinson S; Vandekerckhove F; Bienen EJ; Clarkson AB; Jiang MS; Hart GW; Nussenzweig V
1993 Feb 1;177(2):465-474, Journal of experimental medicine
Here we report the presence of a trans-sialidase on the surface of Trypanosoma brucei culture-derived procyclic trypomastigotes. The enzyme is not detected in lysates of bloodstream trypomastigotes enriched for either stumpy or slender forms. The trans-sialidase catalyzes the transfer of alpha(2-3)-linked sialic acid residues to lactose. beta-galactopyranosyl residues are at least 100 times better acceptors for sialic acid than alpha-galactopyranosyl residues. In the absence of efficient acceptors, the purified enzyme transfers sialic acid to water, i.e., it acts as a sialidase. Although the T. cruzi and T. brucei trans-sialidases have very similar donor and acceptor specificities, they are antigenically distinct. Sodium dodecyl sulfate-polyacramide gel electrophoresis under nonreducing conditions and silver staining of the purified trans-sialidase reveals a single band of 63 kD. When the surface membrane of live procyclic trypomastigotes is trans-sialylated, using radioactive sialyllactose as the donor substrate, it appears that the only sialylated surface molecule is procyclin. Pronase treatment of live parasites removes only part of the surface sialic acid, in agreement with recent data showing that the glycosylphosphatidylinositol anchor of procyclin is sialylated (Ferguson, M. A. J., M. Murray, H. Rutherford, and M. J. McConville. 1993. Biochem. J. In press)
— id: 13271, year: 1993, vol: 177, page: 465, stat: Journal Article,

Mitochondrial development in Trypanosoma brucei brucei transitional bloodstream forms
Bienen EJ; Saric M; Pollakis G; Grady RW; Clarkson AB Jr
1991 Apr;45(2):185-192, Molecular & biochemical parasitology
Intermediate and short stumpy bloodstream forms of Trypanosoma brucei brucei are transitional stages in the differentiation of mammal-infective long slender bloodstream forms into the procyclic forms found in the midgut of the tsetse vector. Although the mitochondria of the proliferative long slender forms do not accumulate rhodamine 123, the mitochondria of the transitional forms attain this ability thus revealing the development of an electromotive force (EMF) across the inner mitochondrial membrane. The EMF is inhibited by 2,4-dinitrophenol, rotenone and salicylhydroxamic acid but not by antimycin A or cyanide. Consequently, NADH dehydrogenase, site I of oxidative phosphorylation, is the source of the EMF and the plant-like trypanosome alternative oxidase (TAO) supports the electron flow serving as the terminal oxidase of the chain. Although the TAO is present in the long slender forms as well, it serves only as the terminal oxidase for electrons from glycerol-3-phosphate dehydrogenase. The data presented here, combined with older data, lead to the conclusion that the mitochondria of transitional intermediate and short stumpy forms likely produce ATP. This putative production is either by F1F0 ATPase driven by the complex I proton pump or by mitochondrial substrate level phosphorylation, or most likely by both. These conclusions contrast with the previously held dogma that all bloodstream form mitochondria are incapable of ATP production
— id: 14094, year: 1991, vol: 45, page: 185, stat: Journal Article,

Differential susceptibility to DL-alpha-difluoromethylornithine in clinical isolates of Trypanosoma brucei rhodesiense
Bacchi CJ; Nathan HC; Livingston T; Valladares G; Saric M; Sayer PD; Njogu AR; Clarkson AB Jr
1990 Jun;34(6):1183-1188, Antimicrobial agents & chemotherapy
DL-alpha-Difluoromethylornithine is an enzyme-activated inhibitor of ornithine decarboxylase and an antagonist of polyamine metabolism that has been successful in clinical trials against West African sleeping sickness caused by Trypanosoma brucei gambiense. Its potential for use against the more virulent East African form of the disease, caused by T. brucei rhodesiense, is not certain. We examined 14 East African clinical isolates from the Kenya Trypanosomiasis Research Institute strain bank plus 2 established isolates for susceptibility to DL-alpha-difluoromethylornithine and to standard trypanocides. Seven of 16 strains were partially or totally refractory to DL-alpha-difluoromethylornithine in our test system. Four strains were also refractory to arsenical drugs, and five were refractory to diamidines. The results indicate that other novel agents or combinations of established agents may be needed for chemotherapy of East African disease
— id: 14322, year: 1990, vol: 34, page: 1183, stat: Journal Article,

Cure of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense infections in mice with an irreversible inhibitor of S-adenosylmethionine decarboxylase
Bitonti AJ; Byers TL; Bush TL; Casara PJ; Bacchi CJ; Clarkson AB Jr; McCann PP; Sjoerdsma A
1990 Aug;34(8):1485-1490, Antimicrobial agents & chemotherapy
A structural analog, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxy adenosine (MDL 73811), of decarboxy S-adenosyl-L-methionine, the product of the reaction catalyzed by S-adenosyl-L-methionine (AdoMet) decarboxylase (DC), was found to inhibit Trypanosoma brucei brucei AdoMet DC. The inhibition was time dependent (tau 50, 0.3 min), exhibited pseudo-first-order kinetics (Ki, 1.5 microM), and was apparently irreversible. The natural substrate of the reaction, AdoMet, protected the enzyme from inactivation, suggesting that MDL 73811 was directed at the enzyme active site and was probably catalytically activated. Administration of MDL 73811 to T. b. brucei-infected rats resulted in rapid inhibition of AdoMet DC activity, a decrease in spermidine, and an increase in putrescine in the trypanosomes isolated from treated rats. Treatment of T. b. brucei-infected mice with MDL 73811 (20 mg/kg of body weight intraperitoneally twice daily for 4 days) resulted in cures of the trypanosome infections. Additionally, drug-resistant T. brucei rhodesiense infections in mice were cured by either a combination of MDL 73811 (50 mg/kg intraperitoneally three times per day for 5 days) and relatively low oral doses of alpha-difluoromethylornithine or MDL 73811 (50 mg/kg per day for 7 days) administered alone in implanted miniosmotic pumps. These data suggest that MDL 73811 and, perhaps, other inhibitors of AdoMet DC have potential for therapeutic use in various forms of African trypanosomiasis
— id: 14321, year: 1990, vol: 34, page: 1485, stat: Journal Article,

Deferoxamine and eflornithine (DL-alpha-difluoromethylornithine) in a rat model of Pneumocystis carinii pneumonia
Clarkson AB Jr; Saric M; Grady RW
1990 Sep;34(9):1833-1835, Antimicrobial agents & chemotherapy
The iron chelator deferoxamine and the polyamine biosynthesis inhibitor eflornithine (DL-alpha-difluoromethylornithine) were examined for anti-Pneumocystis carinii activity in the rat model of P. carinii pneumonia. The activity of deferoxamine at 250, 500, and 1,000 mg/kg given intraperitoneally provides evidence that iron chelation is a promising novel approach to P. carinii chemotherapy. Results with eflornithine at 2, 3, and 4% in drinking water confirm and extend previously reported activity in the rat model
— id: 14320, year: 1990, vol: 34, page: 1833, stat: Journal Article,

Respiration of bloodstream forms of the parasite Trypanosoma brucei brucei is dependent on a plant-like alternative oxidase
Clarkson AB Jr; Bienen EJ; Pollakis G; Grady RW
1989 Oct 25;264(30):17770-17776, Journal of biological chemistry
CoQ links the sn-glycerol-3-phosphate dehydrogenase and oxidase components of the cyanide-insensitive, non-cytochrome-mediated respiratory system of bloodstream African trypanosomes. In this and other characteristics, their respiratory system is similar to the alternative oxidase of plants. The parasites contain 206 ng of CoQ9 mg protein-1 which co-sediments with respiratory activity. The redox state of this CoQ responds in a manner consistent with respiratory function: 60% being in the reduced form when substrate is available and the oxidase is blocked; 13% being in the reduced form when the oxidase is functioning and there is no substrate. The addition of CoQ to aceton-extracted cells stimulates salicylhydroxamic acid-sensitive respiration by 56%. After inhibition of respiration by digitonin-mediated dispersal of the electron transport components, liposomes restore 40% of respiratory activity while liposomes containing CoQ restore 66% of this activity. A less hydrophobic analogue, reduced decyl CoQ, serves as a direct substrate for the trypanosome oxidase supporting full salicylhydroxamic acid-sensitive respiration. After digitonin disruption of electron transport, the nonreduced form of this synthetic substrate can reestablish the chain by accepting electrons from dispersed sn-glycerol-3-phosphate dehydrogenase and transferring them to the dispersed oxidase. Similarities between the alternative oxidase of plants and the oxidase of the trypanosome respiratory system include: mitochondrial location, lack of oxidative phosphorylation, linkage of a dehydrogenase and an oxidase by CoQ, lack of sensitivity to a range of mitochondrial inhibitors, and sensitivity to a spectrum of inhibitors which selectively block transfer of electrons from reduced CoQ to the terminal oxidase but do not block electron transfer to the cytochrome bc1 complex of the mammalian cytochrome chain
— id: 10454, year: 1989, vol: 264, page: 17770, stat: Journal Article,

Trypanocidal CoQ analogues: their effect on other mitochondrial systems
Clarkson AB Jr; Bienen EJ; Pollakis G; Grady RW
1989 ;94(2):245-251, Comparative biochemistry & physiology. Pt. B
1. A comparative study of compounds which inhibit the respiration of the infective form of the protozoan parasite Trypanosoma brucei brucei, such as salicylhydroxamic acid, other substituted benzhydroxamic acid, esters of 2,3- and 3,4-dihydroxybenzoic acid and structurally related compounds, showed that they have a remarkable degree of selectivity for the trypanosome as compared to rat liver mitochondria even though they are putative CoQ analogues and both respiratory systems are dependent on CoQ. 2. The minimal inhibition of mammalian mitochondrial function could not be assigned to inhibition of ubiquinone function in these mitochondria. 3. CoQ-reducing mitochondrial dehydrogenases from rat liver, trypanosomes and skunk cabbage (Symplocarpus foetidus) were insensitive to these inhibitors. 4. The alternative oxidase of skunk cabbage mitochondria was sensitive to a spectrum of trypanosome respiration inhibitors suggesting a similarity to the oxidase of the trypanosome although differing degrees of sensitivity and differing responses to alterations in the molecular structure of the inhibitors indicate that the milieu of the active sites are dissimilar
— id: 10817, year: 1989, vol: 94, page: 245, stat: Journal Article,

Efficacy of DL-alpha-difluoromethylornithine in a rat model of Pneumocystis carinii pneumonia
Clarkson AB Jr; Williams DE; Rosenberg C
1988 Aug;32(8):1158-1163, Antimicrobial agents & chemotherapy
Pneumocystis carinii pneumonia is often the terminal event for patients with the acquired immunodeficiency syndrome. Eflornithine (DL-alpha-difluoromethylornithine [DFMO]; Ornidyl; Merrell Dow Research Institute, Cincinnati, Ohio) has been used successfully against this protozoan disease in limited clinical trials, although not all patients respond to therapy. In contrast, results of the only reported experiments with DFMO in an animal model were negative. We retested DFMO against P. carinii in an immunosuppressed rat model by inclusion of 3% DFMO in the drinking water, a dose rate about twice that used previously. A combination of trimethoprim and sulfamethoxazole, a proven anti-P. carinii agent, was used as a positive control. After 3 weeks of anti-P. carinii pneumonia therapy, the surviving rats were sacrificed and the degree of parasitosis was judged by examination of lung sections stained with silver methenamine to reveal cysts. In three separate experiments, DFMO showed definite anti-P. carinii pneumonia activity; the parasitosis of DFMO-treated animals was significantly less than that of control animals (P less than 0.001 for all experiments). DFMO was not as active as trimethoprim-sulfamethoxazole, however. Several other experimental therapies were tested, including dapsone and two additional antiprotozoal agents: suramin and diminazene aceturate (Berenil; Farbwerke Hoechst, Frankfurt, Federal Republic of Germany). Diminazene aceturate, a veterinary drug related to the standard anti-P. carinii pneumonia agent pentamidine, was very active (P less than 10(-10]. Suramin and dapsone were weakly active. The combinations suramin-diminazene aceturate and suramin-DFMO were tested, but they were antagonistic rather than synergistic
— id: 11012, year: 1988, vol: 32, page: 1158, stat: Journal Article,

Effects of the ornithine decarboxylase inhibitors DL-alpha-difluoromethylornithine and alpha-monofluoromethyldehydroornithine methyl ester alone and in combination with suramin against Trypanosoma brucei brucei central nervous system models
Bacchi CJ; Nathan HC; Clarkson AB Jr; Bienen EJ; Bitonti AJ; McCann PP; Sjoerdsma A
1987 Jan;36(1):46-52, American journal of tropical medicine & hygiene
Two ornithine decarboxylase inhibitors, DL-alpha-difluoromethylornithine (eflornithine; DFMO) and a-monofluoromethyldehydroornithine methyl ester (delta MFMO X CH3) were compared in their ability to cure two distinct Trypanosoma brucei brucei central nervous system murine model infections. Both inhibitors cured the TREU 667 and LUMP 1001 isolates if used in combination with a single (20 mg/kg) injection of suramin, a trypanocide in current clinical use. The curative dose of delta MFMO X CH3 in combination with suramin was 1.09 g/kg/day, administered in the drinking water for 14 days; used with suramin, the curative dose of DFMO was 5.3 g/kg/day for 14 days (5 times the delta MFMO X CH3 dose required). In host animals, delta MFMO X CH3 was not toxic and was accumulated by trypanosomes 6-8 times faster than DFMO. Since DFMO by itself has been highly effective against T. b. gambiense infections in humans (12-15 g/day for 6 weeks) the present data suggest that delta MFMO X CH3 might be effective in a shorter regimen and at lower doses
— id: 14324, year: 1987, vol: 36, page: 46, stat: Journal Article,

Use of difluoromethylornithine (DFMO, eflornithine) for late-stage African trypanosomiasis
McCann PP; Bitonti AJ; Bacchi CJ; Clarkson AB Jr
1987 ;81(4):701-702, Transactions of the Royal Society of Tropical Medicine & Hygiene
— id: 14323, year: 1987, vol: 81, page: 701, stat: Journal Article,

Esters of 3,4-dihydroxybenzoic acid, highly effective inhibitors of the sn-glycerol-3-phosphate oxidase of Trypanosoma brucei brucei
Grady RW; Bienen EJ; Clarkson AB Jr
1986 Oct;21(1):55-63, Molecular & biochemical parasitology
Alkyl esters of 3,4-dihydroxybenzoic acid are inhibitors of the sn-glycerol-3-phosphate oxidase system of Trypanosoma brucei brucei in vitro and have significant trypanocidal activity in vivo when combined with glycerol. While the parent acid has little inhibitory activity in vitro, the esters are highly active with activity increasing as the chain length of the esterifying alcohol increases. The n-dodecyl ester was more than 400 times as active as salicylhydroxamic acid and 15 times more active than the corresponding p-n-alkyloxybenzhydroxamic acid, one of the most active sn-glycerol-3-phosphate oxidase inhibitors previously reported. When combined with glycerol (to block an alternative pathway of glycolysis) and tested in vitro against intact parasites, this ester was 100 times more effective than salicylhydroxamic acid and 10 times more effective than p-n-dodecyloxybenzhydroxamic acid. It was also active against T. b. brucei in mice when combined with glycerol whereas the latter compound was not. Esters of 3,4,5-trihydroxybenzoic acid (gallic acid) were also highly active while those of 2,3-dihydroxybenzoic acid were much less inhibitory and those of 2,5-dihydroxybenzoic acid were inactive. A related compound, 2',4',5'-trihydroxybutyrophenone, was also active as predicted by its structure but was too toxic to be of interest as a drug candidate
— id: 14326, year: 1986, vol: 21, page: 55, stat: Journal Article,

p-Alkyloxybenzhydroxamic acids, effective inhibitors of the trypanosome glycerol-3-phosphate oxidase
Grady RW; Bienen EJ; Clarkson AB Jr
1986 Jun;19(3):231-240, Molecular & biochemical parasitology
Energy production in bloodstream forms of African trypanosomes of the genus Trypanosoma involves two pathways unique to the parasite and which can be blocked by a combination of salicylhydroxamic acid (SHAM) and glycerol. Although this leads to rapid parasite destruction both in vitro and in vivo, the toxicity of SHAM precludes practical use of SHAM/glycerol as a therapeutic regimen. Based on our hypothesis that SHAM operates by interfering with ubiquinone, we attempted to develop this approach by synthesizing and screening a series of hydroxamic acids which more closely resemble ubiquinone: the p-n-alkyloxybenzhydroxamic acids. We also examined a variety of mono-, di- and trisubstituted benzhydroxamic acids together with a selected group of secondary heterocyclic hydroxamic acids. We found an increase in activity of the p-n-alkyloxy compounds with increasing chain length up to 12 carbon atoms with longer chains offering little advantage. The most active compound, p-n-tetradecyloxybenzhydroxamic acid, had an apparent Ki of 0.43 microM indicating a specific activity 70 times greater than SHAM. Although this represents a vast improvement, the low water solubility of these compounds reduces their bioavailability to the point where they are not practical substitutes for SHAM. Consequently, improvement in the SHAM/glycerol approach to chemotherapy appears to lie with improving solubility by altering lipophilicity of the alkyl side chain
— id: 14327, year: 1986, vol: 19, page: 231, stat: Journal Article,

Inhibition of polyamine biosynthesis by alpha-difluoromethylornithine in African trypanosomes and Pneumocystis carinii as a basis of chemotherapy: biochemical and clinical aspects
McCann PP; Bacchi CJ; Clarkson AB Jr; Bey P; Sjoerdsma A; Schecter PJ; Walzer PD; Barlow JL
1986 Nov;35(6):1153-1156, American journal of tropical medicine & hygiene
The symposium provided dramatic evidence of the value of the use of polyamine inhibition via alpha-difluoromethylornithine (DFMO, eflornithine) for advances in chemotherapy of Trypanosoma brucei gambiense sleeping sickness and Pneumocystis carinii pneumonia in acquired immune deficiency syndrome (AIDS) and also for further understanding the metabolic importance of the ubiquitous polyamines in these organisms
— id: 14325, year: 1986, vol: 35, page: 1153, stat: Journal Article,

TRYPANOSOMIASIS RESEARCH
Bacchi, CJ; Clarkson, AB
1985 ;227(4683):118-11?, Science
— id: 30794, year: 1985, vol: 227, page: 118, stat: Journal Article,

DFMO SURAMIN COMBINATION THERAPY FOR THE TREATMENT OF A MOUSE MODEL OF AFRICAN SLEEPING SICKNESS INVOLVING THE CENTRAL NERVOUS-SYSTEM (CNS)
BIENEN, EJ; CLARKSON, AB; BACCHI, CJ; NATHAN, HC; HUTNER, SH; MCCANN, PP; SJOERDSMA, A
1984 ;31(4):A7-A8, Journal of protozoology
— id: 40762, year: 1984, vol: 31, page: A7, stat: Journal Article,

New drug combination for experimental late-stage African trypanosomiasis: DL-alpha-difluoromethylornithine (DFMO) with suramin
Clarkson AB Jr; Bienen EJ; Bacchi CJ; McCann PP; Nathan HC; Hutner SH; Sjoerdsma A
1984 Nov;33(6):1073-1077, American journal of tropical medicine & hygiene
Using a previously described mouse model of late-stage African trypanosomiasis (i.e., involvement of the central nervous system), we demonstrate that a combination of DL-alpha-difluoromethylornithine (DFMO) and suramin is curative. In the curative protocol, DFMO is given as a 2% solution in the drinking water for 14 days and suramin is administered as a single dose (20 mg/kg intravenously) on day 1 of DFMO administration. Since: 1) DFMO has very low toxicity, 2) suramin is one of the least toxic of the presently used trypanocides, and 3) suramin and DFMO act synergistically in mouse models of both acute and late stage trypanosomiasis, we conclude that this combination offers special promise in the treatment of African trypanosomiasis in man
— id: 14328, year: 1984, vol: 33, page: 1073, stat: Journal Article,

Efficacy of combinations of difluoromethylornithine and bleomycin in a mouse model of central nervous system African trypanosomiasis
Clarkson AB Jr; Bacchi CJ; Mellow GH; Nathan HC; McCann PP; Sjoerdsma A
1983 Sep;80(18):5729-5733, Proceedings of the National Academy of Sciences of the United States of America
DL-alpha-Difluoromethylornithine, a polyamine biosynthesis inhibitor, and bleomycin, a currently used antineoplastic agent, have each previously been shown to be curative for acute short-term infections of mice with Trypanosoma brucei brucei, an African trypanosome closely related to those that cause the human disease African sleeping sickness. These agents were tested singly and in combination in a previously described mouse model of sleeping sickness with demonstrable brain involvement. The original model is extended by using two additional strains of outbred mice and by demonstrating that melarsoprol, an arsenical and currently the only drug used for human African trypanosomiasis involving the brain, was also curative for these brain infections. Neither difluoromethylornithine nor bleomycin alone was curative for the brain infections; however, many combinations of the two drugs were found to be 100% curative with no evidence of immediate toxicity
— id: 14329, year: 1983, vol: 80, page: 5729, stat: Journal Article,

Rheumatoid factors and Chagas' disease
Clarkson AB Jr; Mellow GH
1983 Mar 11;219(4589):1238-1239, Science
— id: 14330, year: 1983, vol: 219, page: 1238, stat: Journal Article,

RHEUMATOID-FACTOR AND TRYPANOSOMIASIS
MELLOW, GH; CLARKSON, AB
1983 ;30(3):A7-A7, Journal of protozoology
— id: 40603, year: 1983, vol: 30, page: A7, stat: Journal Article,

Pathogenesis of anemia in Trypanosoma brucei-infected mice
Amole BO; Clarkson AB Jr; Shear HL
1982 Jun;36(3):1060-1068, Infection & immunity
The pathogenesis of anemia was studied in trypanosome-infected mice. A strain of Trypanosoma brucei, TREU 667, was used which first produces an acute phase marked by waves of parasitemia. Erythrocytes from infected animals were coated with immunoglobulin M during or just before the waves of anemia and parasitological crises. Erythrocytes from normal animals could be sensitized with "precrisis" sera presumably containing antigen and antibody. These data suggest that anemia during the acute phase is due to sensitization of erythrocytes with immunoglobulin M-antigen complexes. The anemia is partially compensated by a strong erythropoietic response. The acute phase is followed by a chronic phase marked by a constant high parasitemia and immunosuppression. The less marked anemia occurring during this latter phase is due to hemodilution and perhaps a low but significant immune response to the parasites, which causes continuing erythrocyte sensitization by immunoglobulin M-antigen complexes
— id: 14332, year: 1982, vol: 36, page: 1060, stat: Journal Article,

Role of calcium in trypanocidal drug action
Clarkson AB Jr; Amole BO
1982 Jun 18;216(4552):1321-1323, Science
The synergistic effect of serum on the drug combination of salicylhydroxamic acid plus glycerol, which is active against Trypanosoma brucei, is due to diffusible calcium ions. The synergistic activity can be removed by dialysis of the serum or by addition of calcium chelating agents. A buffer containing calcium can mimic the synergistic activity of serum. This finding may have important implications in the clinical management of African trypanosomiasis in humans. Calcium also has a synergistic effect on melarsoprol, the only drug available for treating sleeping sickness patients with central nervous system involvement, and the concentration of calcium has been reported to be depressed inthe serum of experimentally infected animals
— id: 14331, year: 1982, vol: 216, page: 1321, stat: Journal Article,

Trypanosoma lewisi: enhanced resistance in naive lactating rats and their suckling pups
Mellow GH; Clarkson AB Jr
1982 Apr;53(2):217-228, Experimental parasitology
— id: 14333, year: 1982, vol: 53, page: 217, stat: Journal Article,

Trypanosoma brucei: host parasite interaction in parasite destruction by salicylhydroxamic acid and glycerol in mice
Amole BO; Clarkson AB Jr
1981 Feb;51(1):133-140, Experimental parasitology
— id: 14337, year: 1981, vol: 51, page: 133, stat: Journal Article,

Trypanosoma brucei brucei: a systematic screening for alternatives to the salicylhydroxamic acid-glycerol combination
Clarkson AB Jr; Grady RW; Grossman SA; McCallum RJ; Brohn FH
1981 Sep;3(5):271-291, Molecular & biochemical parasitology
Salicylhydroxamic acid (SHAM) and glycerol, when administered together, cause destruction of bloodstream forms of Trypanosoma brucei brucei, both in vitro and in vivo, but the dose required is exceedingly high. In an attempt to improve the efficacy of this drug combination, we examined the ability of various polyols and hydroxamic acids to substitute for glycerol and SHAM, respectively. No satisfactory substitute for glycerol was found. The inhibition of the trypanosomal alpha-glycerophosphate oxidase system (GPO) by SHAM (Ki 21 microM) was uncompetitive. Only primary and secondary aromatic hydroxamates were inhibitory. Among a series of 19 benzhydroxamates, no correlation existed between their acidity or their affinity for iron and their inhibition of the GPO in a cell free preparation. The Ki's of most of the primary hydroxamates ranged from 10 to 24 microM, with the more lipophilic derivatives being slightly more active. The Ki's of secondary hydroxamates were more variable, the best having Ki's of about 10 microM. Several other classes of iron chelators were also evaluated. Tropolones were active with 3-bromo-4,5-benzotropolone being as active as SHAM. 3,4-Dihydroxybenzaldehyde (Ki 15 microM) also inhibited the GPO. On the other hand, diphenylamine and 8-hydroxyquinoline, known inhibitors of the GPO, were 30 to 50 times less active. The results suggest that a lipophilic aromatic iron-chelating agent may be useful as a substitute for SHAM in combination therapy
— id: 14336, year: 1981, vol: 3, page: 271, stat: Journal Article,

Rheumatoid factor-like immunoglobulin M protects previously uninfected rat pups and dams from Trypanosoma lewisi
Clarkson AB Jr; Mellow GH
1981 Oct 9;214(4517):186-188, Science
The serum of lactating rats that have never been infected with the protozoan parasite Trypanosoma lewisi contains a rheumatoid factor-like immunoglobulin M (IgM). This IgM amplifies a specific immunoglobulin G (IgG) response to the parasite and accounts for the unusual resistance of previously uninfected lactating rats and their suckling pups to infection with T. lewisi. A similar rheumatoid factor-like IgM, which is induced late in the usual course of infection with T. lewisi in nonlactating rats, amplifies an earlier IgM response and terminates the infection. To our knowledge, this is the first description of a rheumatoid factor, which is classified as an autoimmune antibody, acting in a protective manner
— id: 14335, year: 1981, vol: 214, page: 186, stat: Journal Article,

Further studies on difluoromethylornithine in African trypanosomes
McCann PP; Bacchi CJ; Clarkson AB Jr; Seed JR; Nathan HC; Amole BO; Hutner SH; Sjoerdsma A
1981 Dec;59(5-6):434-440, Medical biology
DL-alpha-Difluoromethylornithine (DFMO), a specific enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC) was previously shown to cure mice infected with Trypanosoma brucei brucei, a parasite of game and cattle in Africa and Trypanosoma brucei rhodesiense, a human African Sleeping Sickness pathogen. Our studies now indicate that DFMO blocks ornithine decarboxylase and lowers trypanosome polyamine levels in vivo. Polyamine uptake in T.b. brucei also resembles that previously described for mammalian cells. The therapeutic potential of DFMO can now also be extended to another human pathogen, Trypanosoma brucei gambiense. Finally, DFMO acts synergistically with another drug, bleomycin, to cure acute trypanosome infections, and furthermore, this same drug combination provides a new approach to the treatment of trypanosomal infections of the central nervous system
— id: 14334, year: 1981, vol: 59, page: 434, stat: Journal Article,

DEMONSTRATION OF A BLOOD-FACTOR ACTING SYNERGISTICALLY WITH SHAM-GLYCEROL IN THE DESTRUCTION OF TRYPANOSOMA-BRUCEI
Amole, BO; Clarkson, AB
1980 ;27(3):A46-A46, Journal of protozoology
— id: 28093, year: 1980, vol: 27, page: A46, stat: Journal Article,

Trypanosoma brucei brucei: patterns of glycolysis at 37 degrees C in vitro
Brohn FH; Clarkson AB Jr
1980 Sep;1(5):291-305, Molecular & biochemical parasitology
In the absence of mammalian cells, freshly isolated monomorphic bloodstream forms of Trypanosoma brucei brucei maintain a constant and high level of aerobic glycolysis in vitro for at least 4 h at 37 degrees C when suspended in RPMI medium 1640 containing 20% heat-inactivated and dialyzed fetal calf serum and 25 mM Hepes at an initial pH of 8. In the absence of nutrients other than glucose, salts and protein, some cell death and a decrease in the rate of glycolysis are observed. In the absence of protein, extensive cell death and a decrease in the rate of glycolysis are seen. These observations may be useful in the design of short-term in vitro metabolic studies with T. b. brucei
— id: 14339, year: 1980, vol: 1, page: 291, stat: Journal Article,

Trypanosoma lewisi: avidity and adsorbability of ablastin, the rat antibody inhibiting parasite reproduction
D'Alesandro PA; Clarkson AB Jr
1980 Dec;50(3):384-396, Experimental parasitology
— id: 14338, year: 1980, vol: 50, page: 384, stat: Journal Article,

Quantitative effects of salycylhydroxamic acid and glycerol on Trypanosoma brucei glycolysis in vitro and in vivo
Brohn FH; Clarkson AB Jr
1978 Mar;35(1):23-33, Acta tropica
During anaerobic glycolysis in vitro in the presence of salicylhydroxamic acid, Trypanosoma brucei brucei converts glucose to equimolar amounts of glycerol and pyruvate as end products. Glycerol, whether generated endogenously and pyruvate as end products. Glycerol, whether generated endogenously or added exogenously, can inhibit anaerobic glycolysis sufficiently in vitro to result in cell death. The concomitant administration of salicylhydroxamic acid and glycerol to rats infected with T. brucei brucei results in a rapid clearance of parasitemia. Our results clearly demonstrate a new and approachable chemotherapeutic target for African trypanosomes
— id: 14340, year: 1978, vol: 35, page: 23, stat: Journal Article,

RESISTANCE TO TRYPANOSOMA-LEWISI ASSOCIATED WITH LACTATION
Mellow, GH; Clarkson, AB
1978 ;25(3):A18-A19, Journal of protozoology
— id: 29887, year: 1978, vol: 25, page: A18, stat: Journal Article,

Trypanosomiasis: an approach to chemotherapy by the inhibition of carbohydrate catabolism
Clarkson AB Jr; Brohn FH
1976 Oct 8;194(4261):204-206, Science
When the infected mammalian host of Trypanosoma brucei brucei is injected with a solution of the iron chelator salicyl hydroxamic acid and glycerol, the aerobic and anaerobic glucose catabolism of the parasite is blocked and the parasite is rapidly destroyed
— id: 14341, year: 1976, vol: 194, page: 204, stat: Journal Article,