A humanised mouse model of cytokine release: comparison of CD3-specific antibody fragments.

CD3-specific antibodies have shown clinical efficacy in both transplantation and autoimmunity. However, targeting CD3 in this way can lead to T-cell activation and a serious cytokine release syndrome mediated by Fcγ receptor binding. An in vivo mouse model has been developed using severe combined immunodeficient (SCID) mice to detect human T-cell depletion and cytokine release into the circulation after administration of OKT3. This system has been used to evaluate OKT3 antibody fragments lacking the entire Fc region alongside whole antibody constructs. These data clearly show that cytokine release is detected with all OKT3 antibody constructs and fragments tested and these can be ranked from highest to lowest as follows: mIgG2a>hIgG1 (Ala-Ala)>hIgG1 diFab' maleimide (DFM)>hIgG1 F(ab')2>mIgG2a F(ab')2>hIgG1 Fab'. Furthermore, the monovalent hIgG1 Fab' fragment gives the least cytokine release but it does not deplete human T-cells in this assay format. This suggests that T-cell activation may be playing a role in the mechanism of action of anti-CD3 antibodies and consequently the unwanted cytokine release is potentially unavoidable for this class of molecules. This model system provides a useful tool to aid in understanding and reducing the potential risks of cytokine release following antibody therapy.

Thermospray mass spectrometer as a quantitative specific, sensitive, detector for liquid chromatography. Its application to the analysis of pyridostigmine in human plasma.

A thermospray interface between a HPLC system and a mass spectrometer has been used to develop an assay for pyridostigmine bromide measuring plasma concentrations down to 1 ng ml-1 on a routine basis. Plasma is loaded onto an AASP reversed-phase cartridge, injected onto a HPLC system connected to a Finnigan 4500 mass spectrometer via a thermospray interface and the molecular ion monitored. A deuterated internal standard is used, but calculations with and without the internal standard show its use does not materially improve the quantitation. The precision and accuracy at the limit of quantitation is less than +/- 5% and 95-105%, respectively. The method is used to analyse samples from a bioavailability study by fully automated unattended overnight sample analysis.

Single-dose pharmacokinetics of Madopar HBS in patients and effect of food and antacid on the absorption of Madopar HBS in volunteers.

Pharmacokinetic studies in parkinsonian patients and healthy volunteers have shown that Madopar HBS behaves as a slow-release formulation of L-dopa and benserazide. In comparison with standard Madopar the rate of absorption is reduced, providing lower peak concentrations of L-dopa. The drug is released and absorbed over a period of 4-5 h, thus maintaining substantial plasma concentrations for 6-8 h after dosing. Although the bioavailability after oral dosing is reduced as compared with standard Madopar (60-70%), this difference seems to be due to incomplete absorption rather than altered disposition of the drug. The presence or absence of food in the stomach has no effect on the absorption of L-dopa from Madopar HBS, but administration of antacids further reduces the bioavailability (45%).

Single-dose studies of a slow-release preparation of levodopa and benserazide (Madopar HBS) in Parkinson's disease.

The clinical effects and pharmacokinetic profiles of single doses of Madopar HBS were compared with those of standard Madopar in two studies in patients with Parkinson's disease and 'on-off' fluctuations. In the first study, 10 fasting patients received equivalent doses (200 mg levodopa plus 50 mg benserazide) of each preparation. The clinical response to Madopar HBS was delayed and brief; the relative bioavailability was only 50%. In the second study in 7 non-fasted patients, the effects of 3 capsules of Madopar HBS 125 were compared with those of 2 capsules of standard Madopar 125. Delay to turn on was longer with HBS, but duration of time on, and delay to turn off, were longer with this preparation. The area under the concentration-time curve for plasma levodopa was greater with HBS, and the maximum levodopa concentration was similar to, but achieved later than standard Madopar.

The pharmacokinetics of intravenous and oral levodopa in patients with Parkinson's disease who exhibit on-off fluctuations.

We have studied the clinical effects and pharmacokinetics of levodopa infusions and oral therapy in seven patients with Parkinson's disease. They all showed on-off fluctuations whilst receiving long-term treatment with levodopa in combination with a peripheral decarboxylase inhibitor. Intravenous infusion at a constant rate for up to 16 h resulted in a smoother clinical response, and maintained plasma levodopa concentrations within narrower limits compared with conventional oral therapy. Following infusion rates of 32-80 mg h-1 (0.5-1.3 mg kg-1 h-1) the plasma concentration associated with optimum therapeutic response lay between 0.3 and 1.6 mg l-1. There was considerable variation in the oral absorption and elimination of levodopa, both within and between subjects. The concentration of 3-OMe dopa in plasma hardly increased during each day's levodopa therapy. In all cases levels were greater than the maximum concentrations of levodopa, sometimes by as much as a factor of 10. In contrast to most previous reports on the pharmacokinetics of levodopa, the data presented here are consistent with a two-compartment kinetic model. It is not known whether the difference in pharmacokinetics is due to chronic therapy or whether it is specific to those patients who show on-off phenomena, but such changes might be related in some way to the development of fluctuations in clinical response.

The pharmacokinetics of a new radiosensitiser, Ro 03-8799 in humans.

A new hypoxic cell radiosensitiser, Ro 03-8799 has been administered intravenously to human volunteers and its kinetic parameters derived from plasma and urine data. Good penetration of drug into tumour tissue is found, consistent with its large volume of distribution. The plasma clearance of this compound is rapid due to high metabolic and renal clearances. These parameters combine to produce an elimination half-life of 5.6 h, approximately half that of misonidazole, a well studied radiosensitiser. It is hoped that this decrease in total body exposure will also reduce the cumulative toxicity seen when misonidazole is administered repeatedly.

High-performance liquid chromatographic analysis of the new hypoxic cell radiosensitiser, Ro 03-8799, in biological samples.

A high-performance liquid chromatographic method of analysis with UV detection has been developed to measure levels of a new radiosensitiser, Ro 03-8799 and its N-oxide metabolite, in biological fluids and tissues. The accuracy and precision of the method have been determined in both plasma and urine, where the limits of quantitation are 100 and 500 ng/ml, respectively. Typical results are presented from a human volunteer study where samples were analysed by this method. Important aspects of the method, involving both sample handling techniques and chromatographic conditions are discussed.

Species variation in the metabolism of a substituted cinnamate ester anti-inflammatory pro-drug.

1. The metabolic fate of the substituted cinnamic acid ester, Ro 03-6037, has been examined in rat, mouse, baboon, dog, marmoset, rabbit and man. 2. All species are capable of reducing the cinnamate double bond, but the subsequent one-carbon fragment loss can be carried out only by rat, dog, rabbit and marmoset. 3. The inability of man, as well as baboon and mouse, to perform this terminal metabolic step, which results in formation of the active anti-inflammatory agent, renders the compound unsuitable as a drug for humans. 4. Reduction of the double bond is not carried out by gut flora. 5. An h.p.l.c. analytical method is described for estimation of the metabolites in biological fluids.

The radiosensitizer Ro 03-8799 and the concentrations which may be achieved in human tumours: a preliminary study.

A new hypoxic cell radiosensitizer, Ro 03-8799, has been administered i.v. to 2 normal and 6 patient volunteers. Generally in non-necrotic tumours the concentrations obtained were 3 times greater than in plasma sampled at the same time. These observations added to the reports concerning toxicology in monkeys and rats and radiosensitizing efficiency in the laboratory, suggest that Ro 03-8799 may prove to be much more effective sensitizer than misonidazole in man.

Some aspects of the metabolism of misonidazole.

The metabolism of the 2-nitroimidazole hypoxic cell radiosensitizer, misonidazole, has been studied in patients receiving radiotherapy. Results are presented which show that the compound reaches adequate levels in tumour tissue and that, in animal systems, reduction products of the nitro group may also be present. Ah h.p.l.c method is described which has been used to quantify unchanged misonidazole and its nitroimidazole metabolites.

Pharmacokinetic and metabolic studies of the hypoxic cell radiosensitizer misonidazole.

1. The metabolism of the radiosensitizing 2-nitroimidazole, misonidazole, has been investigated in mice, rats, baboons, human volunteers, and in patients receiving radiotherapy for advanced malignant disease. 2. Plasma levels of unchanged drug and its desmethylated metabolite have been measured, and in humans there is good correlation of peak plasma concn. with drug dose. All drug-related material in plasma was accounted for as unchanged misonidazole or its desmethylated metabolite, both compounds being radiosensitizers in vitro. 3. Extensive faecal excretion of material not containing any nitro group occurred in mice, rats, and baboons dosed with radiolabelled drug. 4. Renal excretion is the preferred route of elimination in man, baboon and mouse. Nitroimidazole metabolites accounting for over half the urinary excretion in all species were identified. 5. The compound penetrates solid murine tumours in concentrations sufficient to achieve radiosensitization.

Why hypertensive patients vary in their response to oral debrisoquine.

The relation between dose, systemic availability, and response to oral debrisoquine was studied in 13 hypertensive patients receiving no other treatment. In 11 who received the same daily dose (40 mg) the fall in mean standing systolic blood pressure varied between 0-3 and 44-4 mm Hg. There was a ninefold difference in the daily urinary excretion and pre-dose plasma concentration of unchanged drug but an inverse correlation between daily urinary excretion of debrisoquine and its 4-hydroxy metabolite (r= -0-86), suggesting that a low recovery of debrisoquine occurs because of extensive metabolism. There was a significant correlation between the fall in standing systolic blood pressure and the mean daily urinary excretion (r= +0-82) and pre-dose plasma concentration (r= +0-82) of unchanged debrisoquine. In contrast, there was a significant inverse correlation between the urinary recovery of the metabolite and the fall in blood pressure (r= -0-82). The availability of debrisoquine is the major determinant of response to this drug. In the absence of side effects a poor response may be an indication to increase the daily dose rather than add another hypotensive agent.