First-in-human trial of the safety, pharmacokinetics and immunogenicity of a PEGylated anti-CD40L antibody fragment (CDP7657) in healthy individuals and patients with systemic lupus erythematosus.

OBJECTIVE: The objective of this paper is to investigate the safety, pharmacokinetics (PK) and immunogenicity of CDP7657, a PEGylated anti-CD40L antibody fragment, in healthy individuals and patients with systemic lupus erythematosus (SLE). METHODS: This randomized, double-blind, single-dose, dose-escalation phase I study consisted of two parts. In part 1, 28 healthy individuals received CDP7657 IV (0.004-5 mg/kg) or placebo. In part 2, 17 patients with SLE received CDP7657 IV (5-60 mg/kg) or placebo. The CDP7657:placebo ratio was 3:1. RESULTS: Adverse events (AEs) were reported by 76% of healthy individuals and 100% of patients with SLE treated with CDP7657; most were mild or moderate in intensity. Two healthy individuals reported serious AEs (SAEs), one of which was considered treatment related (infusion-related reaction; 5 mg/kg cohort). One patient with SLE (60 mg/kg cohort) experienced three SAEs, one of which was considered treatment related (herpes zoster infection). No thromboembolic events were reported. CPD7657 exposure increased in a dose-proportional manner. Low anti-CDP7657 antibody titres were detected in the majority of CDP7657-treated participants with no apparent impact on the PK of CDP7657. CONCLUSION: Single doses of CDP7657 showed predictable PK in healthy individuals and patients with SLE and were well tolerated, with no safety signals of concern. These findings support further investigation of CDP7657 as a therapy for SLE.

Phase I evaluation of CDP791, a PEGylated di-Fab' conjugate that binds vascular endothelial growth factor receptor 2.

PURPOSE: Specific blocking of vascular endothelial growth factor receptor 2 (VEGFR-2) is a novel therapeutic approach. Here, we report the first phase I clinical trial evaluation of CDP791, a PEGylated di-Fab' conjugate that binds VEGFR-2. EXPERIMENTAL DESIGN: Cohorts of patients received CDP791 at doses between 0.3 and 30 mg/kg every 3 weeks for the initial two doses. RESULTS: The compound was well tolerated with no dose-limiting toxicity. Dose-related hypertension was observed in patients receiving CDP791 10 mg/kg or more and several patients on the higher doses developed infusion-related cutaneous hemangiomata arising 28 to 106 days after the first drug administration and resolving 3 weeks after cessation. Biopsy and histologic evaluation showed that CDP791-bound VEGFR-2 is non-phosphorylated, suggesting that the drug is biologically active. Concentrations of CDP791 considered biologically relevant were sustained for 3 weeks when doses of 10 mg/kg or more were administered. Although no reductions in vascular permeability were recorded using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), there was a significant dose level-related reduction in tumor growth. While challenging the recent dogma that active VEGF inhibitors should modulate DCE-MRI measurements of vascular permeability, this highlights the potential of serial three-dimensional tumor measurements to detect tumor growth arrest. Twelve patients received drug for more than two treatments, although no partial or complete responses were seen. CONCLUSION: The data show that CDP791 is biologically active and well tolerated, achieving appropriate plasma concentrations when administered at 10 mg/kg or more every 3 weeks.

Radiation-induced changes in glomerular and tubular cell kinetics and morphology following irradiation of a single kidney in the pig.

PURPOSE: Radiation-induced changes in glomerular and tubular cell kinetics and morphology following irradiation of a single pig kidney were assessed. METHODS AND MATERIALS: The right kidney of 13 adult female Large White pigs was irradiated with a single dose of 9.8 Gy gamma rays. Animals were serially killed between 2 and 24 weeks postirradiation (PI); 1 h prior to postmortem each pig received 500 mg bromodeoxyuridine (BrdUrd). At postmortem, both kidneys were removed and tissue taken to prepare cell suspensions. The labeling index (LI) of these suspensions was measured using flow cytometry; in vivo BrdUrd incorporation in glomerular and tubular cells was determined immunohistochemically. The kidneys were also assessed histologically. RESULTS: Irradiation of the right kidney alone resulted in a significant increase in renal cell LI in both the irradiated and the contralateral unirradiated kidney within 2 weeks of irradiation; peak values of 1.57 +/- 0.32% and 1.04 +/- 0.13%, respectively, were seen 4 weeks PI, significantly greater (P < 0.001) than the preirradiation value of 0.18 +/- 0.01%. The LI values then declined with time, but remained greater than those seen prior to irradiation. A similar pattern of response was determined from counts of labeled glomerular and tubular cells identified immunohistochemically. The increase in labeled glomerular cells was seen 2 weeks PI, whereas that for the tubular cells did not occur until 4 weeks PI. The irradiated kidney exhibited diffuse, progressive glomerular alterations. In contrast, tubular damage was focal; the irradiated kidney also exhibited a prominent vasculopathy, involving arteriolar and peripheral interlobular artery thickening. The contralateral unirradiated kidney appeared unchanged. CONCLUSION: These findings confirm the hypothesis that the morphologic and kinetic responses observed after irradiation of a single kidney are similar to those observed after irradiation of both kidneys. Renal irradiation results in significant alterations in glomerular and tubular cell proliferation and morphology within 2-4 weeks of irradiation; glomerular changes appear predominant.

Radiation-induced changes in the kinetics of glomerular and tubular cells in the pig kidney.

Both kidneys of 13 mature female Large White pigs were irradiated with a single dose of 9.8 Gy 60Co gamma rays. The pigs were killed serially between 2 to 24 weeks after irradiation. One hour prior to sacrifice bromodeoxyuridine (BrdU) (500 mg/pig) was injected intravenously. At postmortem the kidneys were removed and tissue was taken to prepare cell suspensions. The labeling index (LI) of these suspensions was determined using flow cytometry. In vivo BrdU incorporation in tubular and glomerular cells was determined immunohistochemically. The kinetics of glomerular and tubular cells was evaluated by counting the number of labeled cells/glomerulus and the number of labeled tubular cells/field of view. An average of 1200 glomeruli and 1500 fields of view/time were counted. Similar analyses were performed on renal tissue from unirradiated control animals. Flow cytometry revealed rapid and significant increases in the LI of kidney cells; 2 weeks after irradiation the LI increased from a control value of 0.18 +/- 0.01 to 1.23 +/- 0.22% (P < 0.001). By 4 weeks the maximal value of 2.45 +/- 0.36% was seen; the LI then declined progressively but at 24 weeks after irradiation still remained significantly above control values (P < 0.001). A similar pattern of response was determined by counting the labeled glomerular and tubular cells identified immunohistochemically. However, the increase in labeled glomerular cells occurred 2 weeks after irradiation, whereas that for the tubules occurred 4 weeks after irradiation. These findings indicate that irradiation of the kidney, classically regarded as a "late-responding" organ, is associated with rapid and significant changes in the kinetics of both tubular and glomerular cells.

Radiation-induced cell cycle delay measured in two mouse tumors in vivo using bromodeoxyuridine.

The magnitude of the delay of cells in the phases of the cell cycle after irradiation may be related to the radioresponsiveness of tumor cell populations. In this study we have quantified division delay in two mouse tumors in vivo after single and fractionated doses of X rays and single doses of neutrons. The incorporation of bromodeoxyuridine and flow cytometry provided a sensitive and quantitative method to detect cell cycle perturbations after radiation treatment. The more rapidly growing SAF tumor showed less G2-phase delay per gray than a more slowly proliferating tumor, the Rh (0.9 vs 1.8 h). In addition, the SAF tumor failed to show any G1/S-phase delay while the Rh tumor experienced a longer G1-phase delay than that measured for G2 phase (3.1 vs 1.8 h). There was a trend in both tumors for lower doses to be more effective in producing cell cycle delays. Neutrons caused longer G2-phase delays on a unit dose basis, 2.5 and 5.4 h for the SAF and Rh tumors, respectively. The RBE for neutrons for division delay was found to be 2.9 and 2.8 for the SAF and Rh tumors, while the RBE for growth delay was 3.4 and 3.5. Fractionation of the X-ray dose caused a reduction in division delay at higher total doses (10 or 12 Gy) but was without effect at the lower dose studied (6 Gy). These studies show the feasibility of measuring cell cycle delays in vivo, and future developments are suggested for a possible predictive test in patients receiving radiotherapy.

Proliferative changes in two murine tumours in response to single or fractionated doses of X-rays.

Studies were carried out to investigate proliferative changes in two murine experimental tumours in response to radiation. Results were generated using bromodeoxyuridine labelling and flow cytometry. This study demonstrates the possible ambiguity of previous studies using tritiated thymidine in which inability to discriminate normal and tumour cell components in murine tumours may lead to different values for cell kinetic parameters. In particular, the sarcoma F appeared to have a growth fraction of 0.62 when all cells were considered; in reality the growth fraction of the tumour cells only (based on DNA content discrimination) was close to unity. Radiation, administered either as single or fractionated doses, caused little change in the proliferative characteristics of the sarcoma F tumour but had profound effects on the adenocarcinoma Rhodesia tumour. Major changes were the accumulation of cells in G2 for several days after the end of the radiation treatment in both tumours and a dramatic drop in labelling index of the Rhodesia tumour. In neither tumour was there any evidence to suggest an increase in tumour cell proliferation during or after the irradiations. The diploid cells within the sarcoma F tumour showed an initial depression of labelling index followed by a rapid increase overshooting the control labelling index at higher radiation doses. Much of the effects could be attributed to cell cycle delays.

The effect of post-irradiation anisotonic treatment on cell survival and repair of DNA damage.

V79 379A cells were irradiated and then exposed to anisotonic PBS for 20 min. This enhanced the radiation effect resulting from the fixation of potentially lethal damage. The induction of DNA single- and double-strand breaks is not increased by this treatment. Anisotonic treatment delayed the onset of repair of DNA damage. However when cells were returned to normal medium, they repaired the damage to a similar extent as cells not exposed to the anisotonic treatment. We suggest that the fixation of damage by post-irradiation anisotonic treatment is mediated through an increased probability of misrepair of DNA damage due to the delay in the onset of repair. This is supported by the observation that there is a reduced effect of post-irradiation anisotonic treatment on cells that have a markedly reduced ability to repair double-strand breaks.

Radiosensitization by misonidazole during recovery of cellular thiols following depletion by BSO or DEM.

V79 cells have been depleted of their endogenous thiols by treatment with 100 microM BSO for 16-18 hr, or 0.5 mM DEM for 1 hr. The recovery of cellular thiols after removal of the drugs was determined by h.p.l.c. or flow cytometry and the sensitizer enhancement ratio for 100 microM misonidazole was measured as a function of time after removal of the drugs. The SER of 1.2 for control (hypoxic) cells increased to 1.8 for BSO treated (hypoxic) cells and 2.2 for DEM treated ones, when thiol levels were below 10% of controls. The SER and thiol levels returned to control values within 5 hr of removing DEM. After BSO there was little change during the first 5 hr and then a gradual return to normal values by 24 hrs. The major fall in the SER after removal of the drugs occurred as the cellular thiols increased to 60% of control values.

Effect of anemia on tumor radiosensitivity under normo and hyperbaric conditions.

The effect of chronic anemia on tumor radiosensitivity in a murine tumor has been investigated. Anemia was induced by bilateral kidney irradiation given several months before tumor implantation. Anemic, anemic transfused, and normal non-anemic age-matched tumor bearing animals were irradiated with X rays (2 F/24 hr) either in air, air plus misonidazole, or under hyperbaric oxygen. The most resistant response was that of tumors grown in normal mice treated in air. Anemia produced an increase in radiosensitivity which was further enhanced by red blood cell replacement. The most sensitive overall response was seen in the anemic-transfused group treated with HBO.

Cell kinetics of mouse kidney using bromodeoxyuridine incorporation and flow cytometry: preparation and staining.

The kidney is recognized as a dose-limiting tissue by certain radiation treatments. The relationship between the onset of compensatory proliferation in response to irradiation and the expression of functional damage is difficult to study because of the low cell turnover in slowly proliferating tissues. We report on a method to obtain a suitable cell preparation from mouse kidney for study by flow cytometry using the recently developed staining techniques for bromodeoxyuridine incorporation. The labelling index of 0.3% in untreated mouse kidney was easily measured because large numbers of cells could be analysed rapidly. We show that compensatory proliferation after unilateral nephrectomy remains elevated for up to 3 weeks after surgery. Using the BrdU/FCM technique we were able to measure the duration of the S phase in normal and nephrectomized kidneys which we found to be 8.5 hr in both cases. The estimates of potential doubling time were similar to the time scale observed to elapse before functional damage is observed in normal kidneys and those in which damage is precipitated by surgery.

Fractionation studies with WR-2721: normal tissues and tumour.

We have studied the ability of WR-2721 to protect skin, kidney and an anaplastic murine tumour against single or fractionated X-ray treatments. Skin reactions, four different kidney assays, regrowth delay and local control of tumours have been used to construct dose-response curves from which the degree of radioprotection can be quantified as a protection factor. Low doses of WR-2721 (0.2-0.3 mg X g-1) were used before each of 1, 5 or 10 fractions. The degree of protection was similar in all three systems and it did not change significantly with fractionation.

The influence of overall treatment time on renal injury after multifraction irradiation.

The influence of overall treatment time on the radiation response of the mouse kidney was studied in an experiment in which 16 fractions were administered either evenly distributed over 20, 40 or 80 days, or as a split course (8 F/3 days; 74 days rest; 8 F/3 days). Urine output and an isotope assay of glomerular filtration were used to test the mice sequentially. The data were used both to obtain dose-response curves and also to determine the latent period before a chosen level of injury was expressed functionally. Prolonging the overall time from 20 to 80 days increased the isoeffect dose by 2-5 Gy (4-9%) for the isotope assay, and by 4-9 Gy (7-18%) for the urine output assay. This additional recovery as the interval between fractions was prolonged from 1 to 5 days is consistent with slow repair and can be expressed as a small "T" exponent of 0.02-0.12. (One analysis gave a result consistent with negative repair, but the errors on this result were unusually wide.) When the radiation was given as a split course, at the rate of 2 fractions per day, with a large gap of 10.5 weeks between courses, there was no additional sparing compared with 16 fractions over 20 days. This indicates that any sparing that might have resulted from slow repair or stimulated repopulation in the gap has been counterbalanced by having less time for repair of sublethal injury when intervals of 6-12 h are used instead of 24-48 h. Clearly no great increase in the tolerance dose for mouse kidney resulted from the split course.

Enhancement of misonidazole radiosensitization by buthionine sulphoximine.

The influence of glutathione (GSH) depletion on the radiation response and on the radiosensitizing efficiency of misonidazole (miso) has been studied in two types of mouse tumour and in mouse skin. Buthionine sulphoximine (BSO) has been administered in a variety of regimes, leading to a maximal depletion of GSH to 37% of control values in one tumour (CA MT) and 61% in the other (SA FA). Pretreatment with BSO did not alter the radiosensitivity of either tumour when treated with X-rays. It had a slight effect on the sensitizing efficiency of miso, corresponding to a factor less than three, which was detectable only at the highest X-ray doses used. No enhancement of miso efficiency was seen with 5 daily fractions. Prolonged administration of BSO resulted in a slight radiosensitization of mouse skin. When combined with miso the effect was very small and was only detectable at high X-ray doses. BSO however produced a marked enhancement of the acute toxicity of miso, as judged by lethality after large single doses.

The RBE for renal damage after irradiation with 3 MeV neutrons.

Mouse kidneys were locally irradiated with single doses or up to 8 fractions of 240 kV X rays or 3 MeV neutrons. Damage was assessed from measurements of urine output, isotope clearance or haematocrit levels. All three assays gave steep dose-response curves by 4-5 months after irradiation. The repair capacity of the kidney was considerable after X-irradiation but was very small after irradiation with neutrons. Thus the RBE increased sharply with increasing fractionation. After large doses, an RBE of 2.3-2.5 was measured, rising to 4.5-5.1 after 8 fractions of 4 to 5 Gy X rays. Linear-quadratic analysis of these data has allowed RBE's to be calculated outside the measured dose range. The limiting RBE predicted at very low doses per fraction is 20 to 26, whereas at extremely high doses it would be as low as 1.2 to 1.4. This indicates that high RBE values may be seen in a slow turnover tissue after low doses per fraction (within the clinically relevant range) although this may not be evident after larger doses. Such high RBE's arise because of the shape of the underlying X-ray dose-response curve rather than the shape of the neutron curve.

Glutathione depletion in tissues after administration of buthionine sulphoximine.

Buthionine sulphoximine (BSO) an inhibitor of glutathione (GSH) biosynthesis, was administered to mice in single and repeated doses of 0.5, 1 and 5 mmol kg-1 (i.p.). The resultant pattern of GSH depletion was studied in liver, kidney, skeletal muscle and three types of murine tumor. Liver and kidney exhibited a rapid depletion to GSH levels of ca. 20% of controls after single doses of 1-5 mmol kg-1 BSO. Muscle was depleted to a similar level, but at a slower rate after a single dose. All three tumors required repeated administration of BSO over several days to obtain a similar degree of depletion to that shown in the other tissues.

Increased collagen and fluid content of mouse kidneys at 9 months after single or fractionated X irradiation.

Mouse kidneys have been analyzed at sacrifice, 9 months after single-dose and fractionated irradiation, using wet and dry weight, a biochemical determination of hydroxyproline, and quantitation of dilated renal tubules in histological preparations. Dose-response curves have been constructed to determine the sensitivity and precision of the assays and to study the influence of dose fractionation on a variety of radiation responses of the kidney. There was a marked loss of kidney weight, measured either wet or dry, with maximum changes from control values by factors of 3 and 5, respectively. The wet:dry weight ratio increased with X-ray dose, indicating that relative fluid content was increased even 9 months after irradiation. This could be partly attributed to dilated renal tubules. Total collagen content per kidney, determined by a hydroxyproline assay, showed a less marked dose dependence, with a maximum increase of a factor of 1.4. However, hydroxyproline per dry weight increased by a factor of 7, and this ratio proved to be the most sensitive and precise measure of radiation damage. The "fibrosis" that is detected in histological sections appears to be more a relative than an absolute alteration in connective tissue. The loss of parenchymal cell mass, particularly in the proximal tubules, is the predominant factor; the increase in the absolute amount of collagen per kidney contributes to a lesser degree. The influence of radiation dose fractionation was analyzed using a linear-quadratic response model. The alpha/beta ratios were between 0.9 and 2.9 Gy.

Radiation-induced renal damage: the effects of hyperfractionation.

The response of mouse kidneys to multifraction irradiation was assessed using three nondestructive functional end points. A series of schedules was investigated giving 1, 2, 4, 8, 16, 32, or 64 equal X-ray doses, using doses per fraction in the range of 0.9 to 16 Gy. The overall treatment time was kept constant at 3 weeks. Kidney function was assessed from 19 to 48 weeks after irradiation by measuring changes in isotope clearance, urine output, and hematocrit. The degree of anemia (assessed from the hematocrit measurements) is a newly developed assay which is an early indicator of the extent of renal damage after irradiation. All three assays yielded steep dose-effect curves from which the repair capacity of kidney could be estimated by comparing the isoeffective doses in different schedules. There was a marked influence of fractionation, with increasing dose being required to achieve the same level of damage for increasing fraction number, even between 32 and 64 fractions. The data are well fitted by a linear quadratic dose-response equation, and analysis of the data in this way yields low values (approximately 3.0 Gy) for the ratio alpha/beta. This would suggest that hyperfractionation , using extremely small X-ray doses per fraction, would spare kidneys relative to tumors and acutely responding tissues.