Steven J. Ford

Stabilization of bacteriophage during freeze drying

With preliminary clinical trials completed for the treatment of antibiotic resistant infections using bacteriophages, there is a need to develop pharmaceutically acceptable formulations. Lyophilization is an established technique for the storage of bacteriophage, but there is little consensus regarding drying cycles, additives and moisture content specific to phage. Here, the addition of sucrose or poly(ethylene glycol) 6000 yielded stable freeze-dried cakes only from high concentrations (0.5 M and 5%, respectively), with addition of bacteriophage otherwise causing collapse. Gelatin, which is added to storage media (a solution of salts), played no role in maintaining bacteriophage stability following lyophilization. A secondary drying cycle was most important for maintaining bacteriophage activity. The addition of high concentrations of PEG 6000 or sucrose generally caused a more rapid fall in bacteriophage stability, over the first 7-14 d, but thereafter residual activities for all phage formulations converged. There was no distinct change in the glass transition temperatures (T(g)) measured for the formulations containing the same additive. Imaging of cakes containing fluorescently labeled bacteriophage did not show gross aggregation or phase separation of bacteriophage during lyophilization. However, the moisture content of the cake did correlate with lytic activity, irrespective of the formulation, with a 4-6% moisture content proving optimal. We propose that residual moisture is followed during lyophilization of bacteriophage from minimal concentrations of bulking agent.

Lyophilized inserts for nasal administration harboring bacteriophage selective for Staphylococcus aureus: In vitro evaluation

Nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) poses an infection risk and eradication during hospitalization is recommended. Bacteriophage therapy may be effective in this scenario but suitable nasal formulations have yet to be developed. Here we show that lyophilization of bacteriophages in 1ml of a viscous solution of 1-2% (w/v) hydroxypropyl methylcellulose (HPMC) with/without the addition of 1% (w/v) mannitol, contained in Eppendorf tubes, yields nasal inserts composed of a highly porous leaflet-like matrix. Fluorescently labeled bacteriophage were observed to be homogenously distributed throughout the wafers of the dried matrix. The bacteriophage titer fell 10-fold following lyophilization to 10(8)pfu per insert, then falling a further 100- to 1000-fold over 6 to 12months storage at 4°C. This compares well with a total dose of 6×10(5)pfu in 0.2ml liquid applied into the ear during a recent clinical trial in humans. The residual water content of the lyophilized inserts was reduced upon the addition of mannitol to HPMC, but this did not have any correlation to the lytic activity. Mannitol underwent a transition from its amorphous to crystalline state during exposure of the inserts to increasing relative humidities (as would be experienced in the nose), although this transition was suppressed by higher HPMC concentrations and the presence of buffer containing gelatin and bacteriophages. Our results therefore suggest that lyophilized inserts harboring bacteriophage selective for S. aureus may be a novel means for the eradication of MRSA resident in the nose.

A cancer research UK pharmacokinetic study of BPA-mannitol in patients with high grade glioma to optimise uptake parameters for clinical trials of BNCT

This paper describes results to-date from a human pharmacokinetic study which began recruitment in December 2007. Results are presented for a single patient recruited in December 2007. A second patient was recruited in July 2008 but detailed data are not available at the time of writing. The trial is an open-label, non-comparative, non-therapeutic study of BPA-mannitol in patients with high-grade glioma, who will be undergoing stereotactic brain biopsy as part of the diagnostic process before definitive treatment. The study investigates the route of infusion (intra-venous (IV) or intra-carotid artery) and in each case will assess the effect of administration of mannitol as a blood-brain barrier disrupter. All cohorts will receive a 2 h infusion of BPA-mannitol, and for some cohorts an additional mannitol bolus will be administered at the beginning of this infusion. Measurements are made by inductively coupled plasma mass spectrometry (ICP-MS) of (10)B concentration in samples of blood, urine, extra-cellular fluid in normal brain (via a dialysis probe), brain tissue around tumour and tumour tissue. Additional analysis of the tumour tissue is performed using secondary ion mass spectrometry (SIMS). The first patient was part of the cohort having intra-venous infusion without mannitol bolus. No serious clinical problems were experienced and the assay results can be compared with available patient data from other BNCT centres. In particular we note that the peak (10)B concentration in blood was 28.1 mg/ml for a total BPA administration of 350 mg/kg which is very consistent with the previous experience with BPA-fructose reported by the Helsinki group.

Development of a lyophilised RH1 formulation: a novel DT diaphorase activated alkylating agent

RH1 is a novel aziridinylbenzoquinone alkylating agent, which is activated in tumour cells by DT diaphorase. In common with previous aziridinylbenzoquinones, RH1 exhibits limited aqueous stability and solubility. The aim of this study was to examine the pharmaceutical properties of RH1 with a view to preparing a suitable formulation for clinical trial. Stability in a neutral phosphate‐buffered solution was poor with a degradation half‐life of 50 h at 55°C, indicating that lyophilisation was preferable. The reaction kinetics indicated a similarity with previous studies for base‐catalysed degradation of aziridinylbenzoquinones. Intrinsic aqueous solubility at 0.5 mg mL−1 may be increased in solvent systems or by the use of polymers such as polyvinylpyrrolidone (PVP) or complexing agents like hydroxypropyl‐β‐cyclodextrin (HPBCD). In the latter case this increased solubility by an order of magnitude to around 5 mg mL−1. Four potential formulations based on lyophilisation of RH1 (1 mg mL−1) from buffered solution (pH 7, 0.01 M NaH2PO4) containing either 50 mg mL−1 mannitol, 40 mg mL−1 dextran, 20 mg mL−1 PVP or 50 mg mL−1 HPBCD were prepared and examined for stability characteristics. All formulations exhibited a temperature‐dependent degradation. The mannitol and dextran formulations had limited stability and degraded rapidly at all temperatures. The PVP and HPBCD formulations degraded at elevated temperatures but remained stable for up to twelve months at 4°C. Examination of the degradation kinetics in the latter systems demonstrated similarity to the solution degradation mechanism, while in the former alternative degradation pathways appeared to be occurring. The chemical stability of RH1 in lyophilised formulations is dependent upon the excipient employed and storage temperature. Either the PVP or HPBCD formulation would be suitable clinical trial formulations of RH1. The results indicate that the choice of lyophilisation excipient for aziridinylbenzoquinones cannot be based on previous literature studies of related agents.

Physicochemical investigation of the influence of saccharide‐based parenteral formulation excipients on l‐p‐boronphenylalanine solubilisation for boron neutron capture therapy

This paper investigates the physicochemical properties of possible pharmaceutical alternatives to L-p-boronphenylalanine (BPA)-fructose intravenous formulation currently employed in boron neutron capture therapy. The physicochemical properties of BPA in the absence and presence of fructose, mannitol, trehalose and hydroxypropyl-β-cyclodextrin (HPCD) was investigated by determination of pKa values, solubility, precipitation and dissolution using a Sirius T3 instrument. Complex formation was also assessed using (10) B-Nuclear magnetic resonance (NMR). The results indicate that fructose and mannitol form a complex with BPA through a reversible interaction with the boronic acid group, determined by changes in the pKa of the boronic acid group, the ultraviolet and NMR spectra, and increase in kinetic solubility. Trehalose and HPCD did not undergo this reaction and, consequently, did not affect boronphenylalanaine physicochemical properties. Although mannitol is complexed with BPA in an identical manner to fructose, it is superior because it provides increased kinetic solubility. Replacement of fructose by mannitol in the current clinical BPA formulation is, therefore, feasible with advantages of increased dosing and removal of issues related to fructose intolerance and calorific load. Results also indicated that important pharmaceutical parameters are the complexs solubility and dissociation behaviours rather than, as originally assumed, the complex formation reaction.

Pharmaceutical development of the novel arsenical based cancer therapeutic GSAO for Phase I clinical trial

The novel organoarsenical GSAO, 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid, has potential anti-angiogenic capability with application in cancer where tumour metastasis relies on neo-vascularisation. As GSAO arsenic is trivalent, the arsenoxide moiety reacts with appropriately spaced cysteine residues on adenine nucleotide translocase (ANT) mitochondrial membrane protein. Molecular oxidation of the arsenic to the pentavalent structure, as in the degradant GSAA (4-(N-(S-glutathionylacetyl)amino) phenylarsonic acid), prevents sulphydryl interaction and risks abolition of activity. We report here on formulation studies aiming to produce a parenteral product with the primary objective of restricting GSAA transformation from GSAO to protect maximal potency of the molecule. Successful anti-oxidant strategy primarily came from pH control. The presence of glycine was proposed to form a stabilising five-membered oxazarsolidinone ring with arsenoxide and this was investigated using potentiometric assays. We report on these tritration studies identifying a pK(a) of 8.2 associated with an As-OH, but not confirming ring presence. An original clinical trial pharmaceutical was successfully realised by lyophilisation of 50 mg/mL GSAO in 100 mM glycine solution, pH 7 to obtain a 48-month shelf life for the freeze-dried vials. The Phase I clinical study is ongoing in patients with solid tumours refractory to standard therapy.

Bioprocessing of bacteriophages via rapid drying onto microcrystals

We present an alternative bioprocess for bacteriophages involving room temperature coprecipitation of an aqueous mixture of phage (Siphoviridae) and a crystallizable carrier (glutamine or glycine) in excess of water miscible organic solvent (isopropanol or isobutanol). The resultant suspension of phage‐coated microcrystals can be harvested by filtration and the residual solvent removed rapidly by air‐drying at a relative humidity of 75%. Albumin or trehalose added at 5% w/w of the crystalline carrier provide for better stabilization of the phage during co‐precipitation. Free‐flowing dry powders generated from an aqueous solution of phage (∼13 log10 pfu/mL) can be reconstituted in the same aqueous volume to a phage titer of almost 10 log10 pfu/mL; high enough to permit subsequent formulation steps following bioprocessing. The phage‐coated microcrystals remain partially stable at room temperature for at least one month, which compares favorably with phage immobilized into polyester microcarriers or lyophilized with excipient (1–5% polyethylene glycol 6000 or 0.1–0.5 M sucrose). We anticipate that this bioprocessing technique will have application to other phage families as required for the development of phage therapies.

Boron phenylalanine and related impurities: HPLC analysis, stability profile and degradation pathways

Boron phenylalanine is one of the lead drug candidates in the field of Boron Neutron Capture Therapy. Its inherent low toxicity allows large doses to be administered, but this makes it important to identify, rationalise and quantify impurities. Here we report a chromatographic assay method, the conditions under which the parent compound is unstable, and the suggested degradation mechanisms.

Elimination of the antimicrobial action of the organoarsenical cancer therapeutic, 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid, before finished product sterility testing

Arsenical compounds have been used therapeutically for over 2000 years finding particular relevance as antimicrobials. After being replaced by more selective and consequently less toxic antibiotics in the last century, arsenicals have recently made a resurgence as anticancer drugs (specifically arsenic trioxide and its derivatives). Arsenical parenteral formulations require post‐manufacture sterility testing; however, their intrinsic antimicrobial activity must be neutralised before testing to eliminate the possibility of false (no‐growth) test results.

Improved pharmaceutical stability of a boronphenylalanine mannitol formulation for boron neutron capture therapy

Boron neutron capture therapy (BNCT) is a radiotherapy based cancer treatment requiring the availability of a low energy thermal neutron beam and a boron containing drug. These requirements limit BNCT availability with the latter pharmaceutical issue related to the extremely short shelf-life and clinical acceptability of the current fructose based L-boronphenylalanine (BPA) formulation. Resolution of the formulation issues would remove this factor and therefore the stability of an alternative mannitol BPA formulation has been investigated. A mannitol BPA solution formulation was prepared and either lyophilised or stored as a solution at varying temperatures. After suitable periods the formulation was analysed by HPLC for BPA and degradation products. Lyophilised and solution mannitol BPA formulations exhibited a temperature and time dependent loss of BPA with concomitant increases in degradation products. Autoclaving the solution induced and accelerated degradation. A solution or lyophilised mannitol BPA formulation has a shelf-life of between 1 and 4 years respectively, a marked improvement over the current fructose formulation. Due to temperature dependent degradation the formulation cannot be terminally sterilised by autoclaving. The enhanced stability of the mannitol formulation removes the requirement for extemporaneous aseptic preparation of BPA just prior to treatment and eliminates one of the issues complicating the delivery of BNCT.