Derek Law

Bioassay for serum itraconazole concentrations using hydroxyitraconazole standards.

Low concentrations of itraconazole in serum have been associated with therapeutic failure. Variable interpatient bioavailability and detrimental drug interactions with p450 enzyme-inducing agents are well documented. Thus, routine monitoring of serum itraconazole concentrations in patients with life-threatening mycoses is essential for patient care. Present high-performance liquid chromatography (HPLC) methods measure only concentrations of itraconazole and not its active metabolite hydroxyitraconazole. Bioassay methods using itraconazole standards overestimate concentrations in serum as measured by HPLC. We have developed a bioassay for total serum itraconazole and hydroxyitraconazole concentrations using hydroxyitraconazole standards. Itraconazole and hydroxyitraconazole concentrations in 40 clinical samples were assayed by HPLC. Total drug concentrations were measured in the same samples by bioassay with itraconazole or hydroxyitraconazole standards. The correlation of concentrations measured by the last bioassay method with HPLC determinations of both compounds was excellent (r = 0.98, slope = 0.5), with acceptable reproducibility. Small errors were seen at extremes of concentrations. The ratio of hydroxyitraconazole to itraconazole in serum varied from 0.76 to 3.2. The use of hydroxyitraconazole standards rather than itraconazole standards for determination of total itraconazole and hydroxyitraconazole concentrations in serum by bioassay gives accurate and reproducible results that correlate well with total itraconazole and hydroxyitraconazole concentrations as measured by HPLC. Our data show that although hydroxyitraconazole gives larger inhibition zones than itraconazole in bioassay standards, this is not true of patient samples, in which the two compounds make equivalent contributions.

Detection by ELISA of low numbers of Shiga-like toxin-producing Escherichia coli in mixed cultures after growth in the presence of mitomycin C.

Techniques currently available to detect Shiga-like toxin (SLT)-producing Escherichia coli lack sensitivity or require specialised equipment and facilities, and in some cases detect only strains belonging to serotype O157. We have used an ELISA technique, capable of detecting both SLTI and SLTII with crude P1 glycoprotein from hydatid cysts, in combination with enhancement of toxin production by culture with mitomycin C. Supernates of Tryptone Soya Broth cultures containing mitomycin C 200 ng/ml were tested for SLTII. For SLTI, cell lysates pre-treated with polymyxin B were tested. In tests with E. coli O157:H7 in mixed culture with E. coli strain C600 alone, or with E. coli C600, Proteus mirabilis and Enterococcus faecalis, SLTI could be detected when the proportion of toxigenic organisms represented 1% of the mixture, and SLTII when the proportion was 0.025%. When faecal samples with added E. coli O157:H7 were examined in this system, SLTII-producing strains were detected when they comprised less than 0.1% of the coliform population. This technique is a sensitive and specific assay for detecting low numbers of SLT-producing organisms in mixed culture such as occurs in cases of haemolytic uraemic syndrome and haemorrhagic colitis.

F901318 represents a novel class of antifungal drug that inhibits dihydroorotate dehydrogenase

Significance New antifungal drugs that act via novel mechanisms are urgently needed to combat the high mortality of invasive fungal disease and the emergence of resistance to existing therapies. We describe the discovery, structure, activity, and mechanism of action of F901318, a new antifungal agent. A member of a novel class of antifungals, the orotomides, F901318 acts via inhibition of dihydroorotate dehydrogenase, an enzyme of de novo pyrimidine biosynthesis. F901318 is currently in clinical development for the treatment of invasive aspergillosis. There is an important medical need for new antifungal agents with novel mechanisms of action to treat the increasing number of patients with life-threatening systemic fungal disease and to overcome the growing problem of resistance to current therapies. F901318, the leading representative of a novel class of drug, the orotomides, is an antifungal drug in clinical development that demonstrates excellent potency against a broad range of dimorphic and filamentous fungi. In vitro susceptibility testing of F901318 against more than 100 strains from the four main pathogenic Aspergillus spp. revealed minimal inhibitory concentrations of ≤0.06 µg/mL—greater potency than the leading antifungal classes. An investigation into the mechanism of action of F901318 found that it acts via inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) in a fungal-specific manner. Homology modeling of Aspergillus fumigatus DHODH has identified a predicted binding mode of the inhibitor and important interacting amino acid residues. In a murine pulmonary model of aspergillosis, F901318 displays in vivo efficacy against a strain of A. fumigatus sensitive to the azole class of antifungals and a strain displaying an azole-resistant phenotype. F901318 is currently in late Phase 1 clinical trials, offering hope that the antifungal armamentarium can be expanded to include a class of agent with a mechanism of action distinct from currently marketed antifungals.

Dihydroorotate dehydrogenase inhibitor F901318 has potent in vitro activity against Scedosporium species and Lomentospora prolificans

BackgroundnScedosporium species and Lomentospora prolificans are increasing causes of invasive infections in immunocompromised hosts and many isolates are resistant to available antifungals. Our objective was to assess the in vitro potency of F901318, a member of the orotomide class of antifungals, against Scedosporium species and L. prolificans .nnnMethodsnThe in vitro potency of F901318 was evaluated against 66 Scedosporium and 7 L. prolificans clinical isolates using the CLSI M38-A2 reference standard. Scedosporium species included Scedosporium apiospermum ( nu2009 = u2009 43), Scedosporium aurantiacum ( nu2009 = u2009 6), Scedosporium dehoogii ( nu2009 = u2009 2) and Scedosporium boydii ( nu2009 = u2009 15). Positive comparators included amphotericin B, caspofungin, posaconazole and voriconazole.nnnResultsnAgainst S. apiospermum and S. boydii F901318 geometric mean MICs/MECs (0.079 and 0.046u2009mg/L, respectively) were significantly lower than those observed with amphotericin (3.404 and 5.595u2009mg/L), posaconazole (1.937 and 1.823u2009mg/L), voriconazole (0.784 and 0.630u2009mg/L) and caspofungin (5.703 and 7.639u2009mg/L) ( Pu2009 < u2009 0.001). Against S. aurantiacum and S. dehoogii the F901318 MIC range (0.12-0.5u2009mg/L) was also lower than those for the other antifungals (0.5 tou2009>8u2009mg/L). F901318 also maintained activity against L. prolificans isolates (range 0.12-0.25u2009mg/L) in contrast to other antifungals, of which none demonstrated in vitro activity.nnnConclusionsnF901318 demonstrated potent in vitro activity against Scedosporium species and L. prolificans . This activity was maintained against isolates that had significantly reduced susceptibility to the other antifungals. Further studies are warranted to evaluate the in vivo efficacy of F901318 against Scedosporium species and L. prolificans .

Variation in morphotype, karyotype and DNA type of fluconazole resistant Candida albicans from an AIDS patient

Azole-resistant oropharyngeal and oesophageal candidiasis is a recent phenomenon observed in patients with AIDS usually previously treated with fluconazole. Some variation has been observed in antifungal susceptibility testing among separate colonies of Candida albicans from the same patient. This raises the question of whether there are multiple clones present or simply phenotypic variation in expression of azole resistance. To address this question we took 18 isolates grown from multiple swabs taken before and after experimental azole therapy from a single HIV-positive individual with fluconazole-resistant oral candidiasis and compared morphotype, karyotype, PCR-based DNA typing and azole susceptibility. Ten of the isolates were from a single 2-day period. Amongst these 10 there were seven morphotypes, five karyotypes and four polymerase chain reaction (PCR) types. Three further morphotypes, one karyotype and two PCR types were found amongst the eight isolates obtained during the subsequent 4 months. Limited variation in susceptibility to two azoles--fluconazole and D0870--was also seen. This work emphasizes both the large genotype and phenotypic variability of C. albicans isolates in the mouth of AIDS patients with fluconazole resistance, and the difficulties in interpretation of present typing methods.

Diagnosis of infections with Shiga-like toxin-producing Escherichia coli by use of enzyme-linked immunosorbent assays for Shiga-like toxins on cultured stool samples.

Shiga-like toxin-producing (SLT) Escherichia coli, particularly those belonging to serogroup O157, are responsible for haemorrhagic colitis, haemolytic uraemic syndrome and some cases of gastro-enteritis. The rapid and reliable diagnosis of all these infections is necessary for correct patient management and for epidemiological reasons, but is rarely possible with present methods. We compared the efficacy of two methods, (i) the culture of faeces in broth that contained mitomycin C followed by enzyme-linked immunosorbent assay (ELISA) for SLTs, and (ii) the culture of faeces on sorbitol MacConkey agar (SMA), in the detection of infections caused by SLT-producing E. coli. SLT-producing E. coli O157 strains were isolated on SMA from 42 of 475 faecal samples, but SLTs were detected by ELISA in culture supernates or lysates of 54 of 475 samples. SLT-producing E. coli strains were isolated subsequently from 11 of 12 ELISA-positive, SMA culture-negative samples by a colony blot technique. In four cases, SLT-producing E. coli of serogroups other than O157 were isolated and in seven cases E. coli O157 was isolated in small numbers. The ELISA is a rapid and sensitive technique for the diagnosis of SLT-producing E. coli infection, especially where low numbers of the organism are present in faeces and when the infection is caused by a serogroup other than O157.

Pharmacodynamics of the Orotomides against Aspergillus fumigatus: New Opportunities for Treatment of Multidrug-Resistant Fungal Disease

ABSTRACT F901318 is an antifungal agent with a novel mechanism of action and potent activity against Aspergillus spp. An understanding of the pharmacodynamics (PD) of F901318 is required for selection of effective regimens for study in phase II and III clinical trials. Neutropenic murine and rabbit models of invasive pulmonary aspergillosis were used. The primary PD endpoint was serum galactomannan. The relationships between drug exposure and the impacts of dose fractionation on galactomannan, survival, and histopathology were determined. The results were benchmarked against a clinically relevant exposure of posaconazole. In the murine model, administration of a total daily dose of 24 mg/kg of body weight produced consistently better responses with increasingly fractionated regimens. The ratio of the minimum total plasma concentration/MIC (Cmin/MIC) was the PD index that best linked drug exposure with observed effect. An average Cmin (mg/liter) and Cmin/MIC of 0.3 and 9.1, respectively, resulted in antifungal effects equivalent to the effect of posaconazole at the upper boundary of its expected human exposures. This pattern was confirmed in a rabbit model, where Cmin and Cmin/MIC targets of 0.1 and 3.3, respectively, produced effects previously reported for expected human exposures of isavuconazole. These targets were independent of triazole susceptibility. The pattern of maximal effect evident with these drug exposure targets was also apparent when survival and histopathological clearance were used as study endpoints. F901318 exhibits time-dependent antifungal activity. The PD targets can now be used to select regimens for phase II and III clinical trials. IMPORTANCE Invasive fungal infections are common and often lethal. There are relatively few antifungal agents licensed for clinical use. Antifungal drug toxicity and the emergence of drug resistance make the treatment of these infections very challenging. F901318 is the first in a new class of antifungal agents called the orotomides. This class has a novel mechanism of action that involves the inhibition of the fungal enzyme dihydroorotate dehydrogenase. F901318 is being developed for clinical use. A deep understanding of the relationship between dosages, drug concentrations in the body, and the antifungal effect is fundamental to the identification of the regimens to administer to patients with invasive fungal infections. This study provides the necessary information to ensure that the right dose of F901318 is used the first time. Such an approach considerably reduces the risks in drug development programs and ensures that patients with few therapeutic options can receive potentially life-saving antifungal therapy at the earliest opportunity. Invasive fungal infections are common and often lethal. There are relatively few antifungal agents licensed for clinical use. Antifungal drug toxicity and the emergence of drug resistance make the treatment of these infections very challenging. F901318 is the first in a new class of antifungal agents called the orotomides. This class has a novel mechanism of action that involves the inhibition of the fungal enzyme dihydroorotate dehydrogenase. F901318 is being developed for clinical use. A deep understanding of the relationship between dosages, drug concentrations in the body, and the antifungal effect is fundamental to the identification of the regimens to administer to patients with invasive fungal infections. This study provides the necessary information to ensure that the right dose of F901318 is used the first time. Such an approach considerably reduces the risks in drug development programs and ensures that patients with few therapeutic options can receive potentially life-saving antifungal therapy at the earliest opportunity.

In vitro activity of the novel antifungal compound F901318 against Australian Scedosporium and Lomentospora fungi

We determined the in vitro activity of the novel orotomide antifungal, F901318, against 30 Lomentospora prolificans, 20 Scedosporium apiospermum, 7 S. aurantiacum, and 3 S. boydii, isolates in comparison with standard antifungals. Against L. prolificans, F901318 was the most potent compound (MIC90 0.25xa0μg/ml); the geometric mean MIC (0.26xa0μg/ml) was significantly lower (23-80-fold) than those of itraconazole, voriconazole, posaconazole, and isavuconazole (all P < .001), and amphotericin B (P < .05). F901318 also had good activity against S. apiospermum, S. aurantiacum, and S. boydii, comparable to that of voriconazole and posaconazole but was more active than isavuconazole for all three species.

Dihydroorotate dehydrogenase inhibitor olorofim exhibits promising activity against all clinically relevant species within Aspergillus section Terrei

ObjectivesnIn vitro and in vivo activity of the dihydroorotate dehydrogenase inhibitor olorofim (formerly F901318) (F2G Limited, UK) against clinically relevant species of the Aspergillus section Terrei was evaluated.nnnMethodsnA total of 92 clinical Aspergillus section Terrei isolates [42 Aspergillus terreus sensu stricto and 50 cryptic species: Aspergillus alabamensis (nu2009=u20098), Aspergillus citrinoterreus (nu2009=u200927), Aspergillus floccosus (nu2009=u20091), Aspergillus hortai (nu2009=u200913) and Aspergillus neoafricanus (nu2009=u20091)] were evaluated. MICs were determined using the CLSI M38-A2 method. MICs of olorofim were compared with those of posaconazole, voriconazole, itraconazole and amphotericin B. The in vivo efficacy of olorofim was determined in an immunosuppressed murine model of disseminated aspergillosis.nnnResultsnOlorofim was highly active against all tested Aspergillus section Terrei isolates, exhibiting an MIC range of 0.002-0.063u2009mg/L. Slightly higher MICs were observed for A. terreus cryptic species. Olorofim MICs were lower than those observed for the azoles. Selected strains with elevated MICs of azoles were highly susceptible to olorofim. Olorofim administered by oral and intravenous routes produced survival rates of 90%-100% in A. terreus-infected mice.nnnConclusionsnOlorofim showed potent and consistent in vitro activity against all A. terreus strains tested, including those with elevated MICs of other antifungal substances. Overall, growth inhibition by olorofim was superior to that of azoles. In vivo data showed that olorofim was highly efficacious in prolonging survival of mice with disseminated aspergillosis due to A. terreus sensu stricto.