Paul Griffin

Maraviroc (UK-427,857), a Potent, Orally Bioavailable, and Selective Small-Molecule Inhibitor of Chemokine Receptor CCR5 with Broad-Spectrum Anti-Human Immunodeficiency Virus Type 1 Activity

ABSTRACT Maraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades and diverse geographic origin (geometric mean 90% inhibitory concentration of 2.0 nM). Maraviroc was active against 200 clinically derived HIV-1 envelope-recombinant pseudoviruses, 100 of which were derived from viruses resistant to existing drug classes. There was little difference in the sensitivity of the 200 viruses to maraviroc, as illustrated by the biological cutoff in this assay (= geometric mean plus two standard deviations [SD] of 1.7-fold). The mechanism of action of maraviroc was established using cell-based assays, where it blocked binding of viral envelope, gp120, to CCR5 to prevent the membrane fusion events necessary for viral entry. Maraviroc did not affect CCR5 cell surface levels or associated intracellular signaling, confirming it as a functional antagonist of CCR5. Maraviroc has no detectable in vitro cytotoxicity and is highly selective for CCR5, as confirmed against a wide range of receptors and enzymes, including the hERG ion channel (50% inhibitory concentration, >10 μM), indicating potential for an excellent clinical safety profile. Studies in preclinical in vitro and in vivo models predicted maraviroc to have human pharmacokinetics consistent with once- or twice-daily dosing following oral administration. Clinical trials are ongoing to further investigate the potential of using maraviroc for the treatment of HIV-1 infection and AIDS.

Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry To Identify Vancomycin-Resistant Enterococci and Investigate the Epidemiology of an Outbreak

ABSTRACT The control of vancomycin-resistant enterococci (VRE) has become an increasing burden on health care resources since their discovery over 20 years ago. Current techniques employed for their detection include time-consuming and laborious phenotypic methods or molecular methods requiring costly equipment and consumables and highly trained staff. An accurate, rapid diagnostic test has the ability to greatly reduce the spread of this organism, which has the ability to colonize patients for long periods, potentially even lifelong. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a technology with the ability to identify organisms in seconds and has shown promise in the identification of other forms of antimicrobial resistance in other organisms. Here we show that MALDI-TOF MS is capable of rapidly and accurately identifying vanB-positive Enterococcus faecium VRE from susceptible isolates. Internal validation of the optimal model generated produced a sensitivity of 92.4% and a specificity of 85.2%. Prospective validation results, following incorporation into the routine laboratory work flow, demonstrated a greater sensitivity and specificity at 96.7% and 98.1%, respectively. In addition, the utilization of MALDI-TOF MS to determine the relatedness of isolates contributing to an outbreak is also demonstrated.

A pilot randomised trial of induced blood-stage Plasmodium falciparum infections in healthy volunteers for testing efficacy of new antimalarial drugs.

Background Critical to the development of new drugs for treatment of malaria is the capacity to safely evaluate their activity in human subjects. The approach that has been most commonly used is testing in subjects with natural malaria infection, a methodology that may expose symptomatic subjects to the risk of ineffective treatment. Here we describe the development and pilot testing of a system to undertake experimental infection using blood stage Plasmodium falciparum parasites (BSP). The objectives of the study were to assess the feasibility and safety of induced BSP infection as a method for assessment of efficacy of new drug candidates for the treatment of P. falciparum infection. Methods and Findings A prospective, unblinded, Phase IIa trial was undertaken in 19 healthy, malaria-naïve, male adult volunteers who were infected with BSP and followed with careful clinical and laboratory observation, including a sensitive, quantitative malaria PCR assay. Volunteers were randomly allocated to treatment with either of two licensed antimalarial drug combinations, artemether–lumefantrine (A/L) or atovaquone-proguanil (A/P). In the first cohort (n = 6) where volunteers received ∼360 BSP, none reached the target parasitemia of 1,000 before the day designated for antimalarial treatment (day 6). In the second and third cohorts, 13 volunteers received 1,800 BSP, with all reaching the target parasitemia before receiving treatment (A/L, n = 6; A/P, n = 7) The study demonstrated safety in the 19 volunteers tested, and a significant difference in the clearance kinetics of parasitemia between the drugs in the 13 evaluable subjects, with mean parasite reduction ratios of 759 for A/L and 17 for A/P (95% CI 120–4786 and 7–40 respectively; p<0.01). Conclusions This system offers a flexible and safe approach to testing the in vivo activity of novel antimalarials. Trial Registration: ClinicalTrials.gov NCT01055002

Adjuvant properties of non-phospholipid liposomes (Novasomes®) in experimental animals for human vaccine antigens

Non-phospholipid liposomes composed of dioxyethylene cetyl ether, cholesterol and oleic acid were evaluated as adjuvants with human vaccine antigens, tetanus toxoid (TT) and diphtheria toxoid (DT), in mice and rabbits. Antigens encapsulated in or mixed with liposomes elicited antitoxin levels similar to those elicited by antigens given with Freunds adjuvant or adsorbed onto aluminum phosphate. All liposomal antigen preparations, antigen given with Freunds adjuvant or adsorbed onto aluminum phosphate, elicited significantly higher IgG antibodies and antitoxin levels than soluble antigens in mice after a single injection and in rabbits after each of three injections. TT encapsulated in liposomes elicited sustained anti-TT IgG antibody levels in mice after a single injection as compared to TT mixed with liposomes. TT mixed with or encapsulated within liposomes containing monophosphoryl lipid A/squalene or squalene alone, as well as aluminum phosphate adsorbed TT elicited greater primary responses in mice than TT mixed with or encapsulated within plain liposomes. Liposomal TT preparations produced a slightly higher anamnestic response in mice than aluminum phosphate adsorbed TT. Subclass analysis of anti-TT antibodies showed that the majority of the antibodies belong to IgG1 subclass. Liposomal TT preparations, particularly those with encapsulated monophosphoryl lipid A/squalene or squalene alone, consistently elicited higher levels of anti-TT IgG2a and IgG2b than aluminum phosphate adsorbed or soluble TT. None of the preparations elicited IgG3 or IgM antibodies. It appears that non-phospholipid liposomes are as potent adjuvants as the currently employed adjuvant for human vaccines (aluminum phosphate) or a benchmark adjuvant for experimental immunology (Freunds adjuvant), and may be able to modulate the immune response towards the Th1 type.

In vivo distribution of radioactivity in mice after injection of biodegradable polymer microspheres containing 14C-labeled tetanus toxoid

Radiolabeled tetanus toxoid (TT) was prepared by detoxifying chromatographically purified tetanus toxin with 14C-labeled formaldehyde. 14C-TT was encapsulated inside poly (D,L-lactide-co-glycolide, 50/50) microspheres (MS) of varying average size (approximately 10 microns and approximately 50 microns). Balb/c mice were injected subcutaneously with 5 Lf (approximately 15 micrograms) of 14C-TT, encapsulated in MS, mixed with blank MS without encapsulated antigen, as soluble antigen or adsorbed onto aluminum phosphate (AlPO4) and radioactivity was monitored at the site of injection, draining lymph nodes, blood, liver, spleen, and kidneys at various intervals. At one day, approximately 95% and 90% radioactivity disappeared from site of injection for soluble TT or blank MS mixed TT and AlPO4 adsorbed TT, respectively, whereas approximately 55% and 70% radioactivity disappeared from site of injection for MS of average size approximately 50 microns and approximately 10 microns, respectively. By 7 days, 99% of radioactivity disappeared from site of injection for soluble TT or blank MS mixed TT, whereas 2-3% radioactivity persisted at the site of injection for AlPO4 adsorbed TT for 4 weeks. In contrast, approximately 20% radioactivity stayed at the site of injection for MS injected mice up to 4 weeks. At all time points, large MS (approximately 50 microns) showed more radioactivity at the site of injection than small MS (approximately 10 microns). Other organs showing radioactivity were draining lymph nodes and kidneys. Small MS with encapsulated TT showed highest level of radioactivity in lymph nodes at 4 h. In kidneys, soluble and AlPO4 adsorbed TT showed a peak of radioactivity at 4 h whereas TT encapsulated in MS showed a peak of radioactivity at 7 days. These results indicate that AlPO4 did not act as a depot for TT at the site of injection, but TT encapsulated in MS did form a depot for approximately 1 month.

Determination of protein loading in biodegradable polymer microspheres containing tetanus toxoid

Various methods to determine loading of vaccine in biodegradable polymer microspheres encapsulating tetanus toxoid were evaluated. The microspheres were composed of poly (D-lactic acid) (PLA) and poly (DL-lactic-co-glycolic acid) (PLGA). Dissolution of microspheres in organic solvents such as methylene chloride, chloroform, or dimethyl sulfoxide and extraction of vaccine antigen or total protein with phosphate buffered saline gave variable results which depended upon the characteristics of the microspheres, such as type of polymer, excipients used in the microspheres and formulation conditions. Microspheres made from low molecular weight PLGA polymer and showing a large burst release exhibited up to 25% extraction of antigen whereas microspheres made from PLA microspheres with low burst release showed < 1% extraction. Extraction of total protein with 0.1 N NaOH and 5% sodium dodecyl sulfate showed results similar to those obtained with organic solvent extraction method. Partial digestion of microspheres with 6 N HCl at 60 degrees C for 20 h resulted in approximately 30% loss in TT protein by micro-bicinchoninic acid (BCA) assay. The major problem with this method was strong reactions in the micro-BCA assay of stabilizers, particularly sugars (glucose, sucrose) used in the microsphere formulations. Complete digestion of microspheres with 6 N HCl at 110 degrees C for 20 h or with 13.5 N NaOH at 121 degrees C for 1 h and quantitation of amino acids by a modified ninhydrin assay showed reproducible results on the protein loading in the microspheres. However, this method was affected by the presence of stabilizers, such as gelatin, which contain amino acids. Further, sucrose concentrations higher than 10% caused interference in the ninhydrin assay on samples hydrolyzed with 6 N HCl. In contrast, hydrolysis with 13.5 N NaOH did not show any interference by sucrose. Stabilizers used outside the microspheres for lyophilization purposes may be removed by washing the microspheres before loading determination or by dialysis but stabilizers used inside the microspheres would still cause interference. For reliable determination of total protein in the microspheres containing vaccines, we suggest complete digestion of microspheres with acid or base followed by amino acid analysis by colorimeteric assays such as ninhydrin method or using amino acid analyzers. The method needs to be optimized for each type of formulation to eliminate interference by the excipients. Alternatively, total protein nitrogen in the microspheres may be determined by the Kjel-dahl method if no amino acids or other nitrogen containing stabilizer is used inside the microspheres.

Experimentally Induced Blood-Stage Plasmodium vivax Infection in Healthy Volunteers

BACKGROUND Major impediments to development of vaccines and drugs for Plasmodium vivax malaria are the inability to culture this species and the extreme difficulty in undertaking clinical research by experimental infection. METHODS A parasite bank was collected from a 49-year-old woman with P. vivax infection, characterized, and used in an experimental infection study. RESULTS The donor made a full recovery from malaria after collection of a parasite bank, which tested negative for agents screened for in blood donations. DNA sequence analysis of the isolate indicated that it was clonal. Two subjects inoculated with the isolate became polymerase chain reaction positive on days 8 and 9, with onset of symptoms and positive blood smears on day 14, when they were treated with artemether-lumefantrine, with rapid clinical and parasitologic response. Transcripts of the parasite gene pvs25 that is expressed in gametocytes, the life cycle stage infectious to mosquitoes, were first detected on days 11 and 12. CONCLUSIONS This experimental system results in in vivo parasite growth, probably infectious to mosquitoes. It offers the opportunity to undertake studies previously impossible in P. vivax that will facilitate a better understanding of the pathology of vivax malaria and development of antimalarial drugs and vaccines. Trial Registration. ANZCTR: 12612001096842.

Piperaquine Monotherapy of Drug-Susceptible Plasmodium falciparum Infection Results in Rapid Clearance of Parasitemia but Is Followed by the Appearance of Gametocytemia

Background. Piperaquine, coformulated with dihydroartemisinin, is a component of a widely used artemisinin combination therapy. There is a paucity of data on its antimalarial activity as a single agent. Such data, if available, would inform selection of new coformulations. Methods. We undertook a study in healthy subjects, using the induced blood stage malaria (IBSM) model to test the antimalarial activity of single doses of piperaquine (960, 640, and 480 mg) in 3 cohorts. In a pilot study in the third cohort, gametocyte clearance following administration of 15 mg, or 45 mg or no primaquine was investigated. Results. Parasite clearance over the 48-hour period after piperaquine administration was more rapid in the 960 mg cohort, compared with the 640 mg cohort (parasite reduction ratio, 2951 [95% confidence interval {CI}, 1520–5728] vs 586 [95% CI, 351–978]; P < .001). All 24 subjects developed gametocytemia as determined by pfs25 transcripts. Clearance of pfs25 was significantly faster in those receiving primaquine than in those not receiving primaquine (P < .001). Conclusions. Piperaquine possesses rapid parasite-clearing activity, but monotherapy is followed by the appearance of gametocytemia, which could facilitate the spread of malaria. This new information should be taken into account when developing future antimalarial coformulations. Clinical Trials Registration ACTRN12613000565741.

Safety, tolerability, pharmacokinetics, and activity of the novel long-acting antimalarial DSM265: a two-part first-in-human phase 1a/1b randomised study

Summary Background DSM265 is a novel antimalarial that inhibits plasmodial dihydroorotate dehydrogenase, an enzyme essential for pyrimidine biosynthesis. We investigated the safety, tolerability, and pharmacokinetics of DSM265, and tested its antimalarial activity. Methods Healthy participants aged 18–55 years were enrolled in a two-part study: part 1, a single ascending dose (25–1200 mg), double-blind, randomised, placebo-controlled study, and part 2, an open-label, randomised, active-comparator controlled study, in which participants were inoculated with Plasmodium falciparum induced blood-stage malaria (IBSM) and treated with DSM265 (150 mg) or mefloquine (10 mg/kg). Primary endpoints were DSM265 safety, tolerability, and pharmacokinetics. Randomisation lists were created using a validated, automated system. Both parts were registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12613000522718 (part 1) and number ACTRN12613000527763 (part 2). Findings In part 1, 73 participants were enrolled between April 12, 2013, and July 14, 2015 (DSM265, n=55; placebo, n=18). In part 2, nine participants were enrolled between Sept 30 and Nov 25, 2013 (150 mg DSM265, n=7; 10 mg/kg mefloquine, n=2). In part 1, 117 adverse events were reported; no drug-related serious or severe events were reported. The most common drug-related adverse event was headache. The mean DSM265 peak plasma concentration (Cmax) ranged between 1310 ng/mL and 34 800 ng/mL and was reached in a median time (tmax) between 1·5 h and 4 h, with a mean elimination half-life between 86 h and 118 h. In part 2, the log10 parasite reduction ratio at 48 h in the DSM265 (150 mg) group was 1·55 (95% CI 1·42–1·67) and in the mefloquine (10 mg/kg) group was 2·34 (2·17–2·52), corresponding to a parasite clearance half-life of 9·4 h (8·7–10·2) and 6·2 h (5·7–6·7), respectively. The median minimum inhibitory concentration of DSM265 in blood was estimated as 1040 ng/mL (range 552–1500), resulting in a predicted single efficacious dose of 340 mg. Parasite clearance was significantly faster in participants who received mefloquine than in participants who received DSM265 (p<0·0001). Interpretation The good safety profile, long elimination half-life, and antimalarial effect of DSM265 supports its development as a partner drug in a single-dose antimalarial combination treatment. Funding Wellcome Trust, UK Department for International Development, Global Health Innovative Technology Fund, Bill & Melinda Gates Foundation.

Efficacy of OZ439 (artefenomel) against early Plasmodium falciparum blood-stage malaria infection in healthy volunteers

Objectives OZ439, or artefenomel, is an investigational synthetic ozonide antimalarial with similar potency, but a significantly improved pharmacokinetic profile, compared with artemisinins. We wished to measure key pharmacokinetic and pharmacodynamic parameters and the pharmacokinetic/pharmacodynamic relationship of artefenomel in humans to guide the drugs further development as combination therapy in patients. Patients and methods We tested artefenomel in the human induced blood-stage malaria (IBSM) model. Plasmodium infection was monitored by quantitative PCR (qPCR) and upon reaching 1000 parasites/mL single doses of 100, 200 and 500 mg of artefenomel were administered orally with evaluation of drug exposure and parasitaemia until rescue treatment after 16 days or earlier, if required. Results A single 100 mg dose had only a transient effect, while the 200 mg dose resulted in a significant reduction in parasitaemia before early recrudescence. At the highest (500 mg) dose, initial clearance of parasites below the limit of detection of qPCR was observed, with a 48 h parasite reduction ratio (PRR48) >10 000 and a parasite clearance half-life of 3.6 h (95% CI 3.4–3.8 h). However, at this dose, recrudescence was seen in four of eight subjects 6–10 days after treatment. Pharmacokinetic/pharmacodynamic modelling predicted an MIC of 4.1 ng/mL. Conclusions These results confirm the antimalarial potential of artefenomel for use in a single-exposure combination therapy. The observations from this study support and will assist further clinical development of artefenomel.