T-cell activation by treatment of cancer patients with EMD 521873 (Selectikine), an IL-2/anti-DNA fusion protein.

BACKGROUND: EMD 521873 (Selectikine or NHS-IL2LT) is a fusion protein consisting of modified human IL-2 which binds specifically to the high-affinity IL-2 receptor, and an antibody specific for both single- and double-stranded DNA, designed to facilitate the enrichment of IL-2 in tumor tissue. METHODS: An extensive analysis of pharmacodynamic (PD) markers associated with target modulation was assessed during a first-in-human phase I dose-escalation trial of Selectikine. RESULTS: Thirty-nine patients with metastatic or locally advanced tumors refractory to standard treatments were treated with increasing doses of Selectikine, and nine further patients received additional cyclophosphamide. PD analysis, assessed during the first two treatment cycles, revealed strong activation of both CD4+ and CD8+ T-cells and only weak NK cell activation. No dose response was observed. As expected, Treg cells responded actively to Selectikine but remained at lower frequency than effector CD4+ T-cells. Interestingly, patient survival correlated positively with both high lymphocyte counts and low levels of activated CD8+ T-cells at baseline, the latter of which was associated with enhanced T-cell responses to the treatment. CONCLUSIONS: The results confirm the selectivity of Selectikine with predominant T-cell and low NK cell activation, supporting follow-up studies assessing the clinical efficacy of Selectikine for cancer patients.

Efficacy and Safety of Proposed Biosimilar Natalizumab (PB006) in Patients With Relapsing-Remitting Multiple Sclerosis: The Antelope Phase 3 Randomized Clinical Trial.

Importance: Proposed biosimilar natalizumab (biosim-NTZ) PB006 is the first biosimilar monoclonal antibody therapy developed for multiple sclerosis (MS) treatment. Objective: To evaluate matching efficacy, safety, and immunogenicity between biosim-NTZ and reference natalizumab (ref-NTZ) in patients with relapsing-remitting MS (RRMS). Design, Setting, and Participants: The Antelope trial was a phase 3, parallel-group, randomized, active-controlled study, conducted between October 2019 and March 2021, with last patient follow-up visit on August 23, 2021. The study took place in 48 centers in 7 countries. Of 531 patients with RRMS aged 18 to 60 years screened, 266 were excluded before randomization in line with study criteria. Eligible participants had 1 or more documented relapse within the previous year and either 1 or more gadolinium-enhancing T1-weighted or 9 or more T2-weighted brain lesions, Kurtzke Expanded Disability Status Scale score of 0 to 5.0 (inclusive), and John Cunningham virus index of 1.5 or less at screening. One patient withdrew consent before dosing. Interventions: Intravenous infusions every 4 weeks of biosim-NTZ, 300 mg, or ref-NTZ, 300 mg (1:1 randomization), from week 0 to week 44 (end-of-study visit: week 48). At week 24, the ref-NTZ group was rerandomized and 30 patients were switched to biosim-NTZ for the remainder of the study. Main Outcomes and Measures: The primary end point was the cumulative number of new active lesions on magnetic resonance imaging (new gadolinium-enhancing T1-weighted lesions and new/enlarging T2-weighted lesions without double counting) over 24 weeks. Additional end points included further magnetic resonance imaging parameters, annualized relapse rate, and Kurtzke Expanded Disability Status Scale score. Safety, tolerability, and immunogenicity assessments included adverse events, laboratory evaluations, and positivity for anti-John Cunningham virus antibodies and antinatalizumab antibodies. Results: A total of 264 participants (mean [SD] age, 36.7 [9.38] years; 162 [61.4%] female) received treatment with biosim-NTZ (n = 131) or ref-NTZ (n = 133). At week 24, the model-based mean difference in cumulative number of new active lesions between biosim-NTZ and ref-NTZ treatment groups was 0.17 (least square means [SE]: biosim-NTZ, 0.34 [0.34]; ref-NTZ, 0.45 [0.28]; 95% CI, -0.61 to 0.94 within the prespecified margins of ±2.1). No significant differences between treatment groups were observed across secondary efficacy end points, safety, tolerability, or immunogenicity assessments. Conclusions and Relevance: Biosim-NTZ matched ref-NTZ in efficacy, safety, and immunogenicity for patients with RRMS in the tested setting. This phase 3 trial supports proposed biosim-NTZ as a biosimilar alternative to ref-NTZ for treating RRMS. Trial Registration: ClinicalTrials.gov Identifier: NCT04115488.

Phase 3 Trial of BI 695502 Plus Chemotherapy Versus Bevacizumab Reference Product Plus Chemotherapy in Patients With Advanced Nonsquamous NSCLC.

INTRODUCTION: Biological therapies such as bevacizumab have improved survival in patients with NSCLC. This study was conducted to confirm the equivalent efficacy of the biosimilar candidate BI 695502 to the bevacizumab reference product (RP). METHODS: In this phase 3, multicenter, randomized, double-blind trial of adult patients with recurrent or metastatic NSCLC received up to 18 weeks of induction treatment with BI 695502 or bevacizumab RP 15 mg/kg plus paclitaxel and carboplatin. Subsequent maintenance therapy comprised BI 695502 or bevacizumab RP monotherapy until disease progression or unacceptable toxicity. The primary end point was the best overall response rate (ORR) per Response Evaluation Criteria in Solid Tumors version 1.1 assessed by central imaging review, until 18 weeks after the start of treatment. RESULTS: In total, 671 patients were randomized at one-to-one ratio to BI 695502 or bevacizumab RP, of whom 335 and 328, respectively, received treatment. Of these, 228 (68.1%) and 256 (78.0%), respectively, proceeded to maintenance monotherapy. A manufacturing issue led to a small number of patients treated with BI 695502 switching to bevacizumab RP late in the study. The primary end point, best ORR, was 54.0% in the BI 695502 group and 63.1% in the bevacizumab RP group. The 90% confidence interval for the between-group ratio of best ORR (0.770 to 0.951) was within the prespecified range for equivalence (0.736-1.359). Adverse events were class-related and similar between the two treatment arms. CONCLUSIONS: This study revealed equivalent ORR after 18 weeks of treatment with BI 695502 or bevacizumab RP, with similar adverse event profiles.

Safety and efficacy of BI 695501 versus adalimumab reference product in patients with advanced Crohn's disease (VOLTAIRE-CD): a multicentre, randomised, double-blind, phase 3 trial.

BACKGROUND: BI 695501 is a biosimilar that has demonstrated similar efficacy, safety, and immunogenicity to adalimumab reference product in patients with rheumatoid arthritis and chronic plaque psoriasis. The VOLTAIRE-CD study aimed to compare the efficacy and safety of BI 695501 with adalimumab reference product in patients with Crohn's disease. METHODS: This phase 3, randomised, double-blind study was done at 92 centres in 12 countries across Europe and the USA in patients aged 18-80 years with moderately to severely active Crohn's disease (Crohn's Disease Activity Index [CDAI] score 220-450). Patients were randomly assigned 1:1 using an interactive response technology system to the BI 695501 group or adalimumab reference product group, stratified by previous exposure to infliximab (yes vs no) and simple endoscopic score for Crohn's disease at screening (<16 vs ≥16). All investigators involved in trial assessments or procedures and all patients were masked to treatment allocation until week 24. Patients received BI 695501 (40 mg/0·8 mL formulation) or adalimumab reference product (either 40 mg/0·4 mL citrate-free or 40 mg/0·8 mL) 160 mg on day 1 and 80 mg on day 15, followed by 40 mg every 2 weeks, via subcutaneous injection. The primary endpoint was the proportion of patients with clinical response (CDAI decrease ≥70 points) at week 4, with an exploratory non-inferiority margin of 0·76 for the lower limit of the two-sided 90% CI of the risk ratio (RR). The primary analysis was done in a modified full analysis set of all patients who received at least one dose of study medication and had a baseline and at least one post-baseline CDAI assessment. Safety was assessed in all patients who received at least one dose of study medication. After week 4, responders were treated until week 46; those randomly assigned to adalimumab reference product switched to BI 695501 at week 24. This study is registered at ClinicalTrials.gov (NCT02871635) and EudraCT (2016-000612-14). FINDINGS: Between Jan 4, 2017, and April 5, 2018, 147 patients were enrolled and randomly assigned to BI 695501 (n=72) or adalimumab reference product (n=75). At week 4, 61 (90%) of 68 patients in the BI 695501 group and 68 (94%) of 72 in the adalimumab reference product group had a clinical response (adjusted RR 0·945 [90% CI 0·870-1·028]). In the safety analysis set, 45 (63%) of 72 patients in the BI 695501 group and 42 (56%) of 75 in the adalimumab reference product group had an adverse event during weeks 0-24; 31 (43%) and 34 (45%) had adverse events during weeks 24-56. The most common drug-related treatment-emergent adverse events during weeks 0-24 were weight increase (three [4%] patients in the BI 695501 group) and injection-site erythema and upper respiratory tract infection (three [4%] patients for each event) in the adalimumab reference product group. The only drug-related TEAEs reported in two or more patients during weeks 24-56 were weight increase and increased γ-glutamyltransferase, which occured in two (3%) patients each in the BI 695501 group. No drug-related TEAEs were reported in two or more patients during weeks 24-56 in the adalimumab reference product followed by BI 699501 group. Serious adverse events occurred in six (8%) patients in the BI 695501 group and eight (11%) in the adalimumab reference group between weeks 0-24, and two (3%) and nine (12%) patients between weeks 24-56. Adverse events of special interest occurred in two (3%) patients in each treatment group during weeks 0-24 (acute sinusitis and pulmonary tuberculosis in the BI 695501 group and anal abscess and postoperative wound infection in the adalimumab reference product group) and two (3%) patients in each group during weeks 24-56 (psoas abscess and hypersensitivity in the BI 695501 group and pulmonary tuberculosis and erythematous rash in the adalimumab reference product followed by BI 699501 group). INTERPRETATION: Safety and efficacy were similar in patients with Crohn's disease treated with BI 695501 or adalimumab reference product. Treatment benefits were maintained in patients receiving adalimumab reference product who switched to BI 695501. These results further support the existing licensure of BI 695501 as an alternative to adalimumab reference product for patients with Crohn's disease, as well as the other indications for which BI 695501 is approved. FUNDING: Boehringer Ingelheim.

Comparison of the pharmacokinetics, safety and tolerability of two concentrations of a new liquid recombinant human growth hormone formulation versus the freeze-dried formulation.

BACKGROUND: Somatropin is recombinant human growth hormone (GH) used for the treatment of growth failure in children and GH deficiency in adults. Two concentrations of a liquid formulation have been developed: 5.83 and 8.0 mg/mL. This trial compared the pharmacokinetics (PK), safety and tolerability of these two liquid concentrations against the freeze-dried (FD) formulation in healthy volunteers. METHODS: In an open-label, single-centre, three-way crossover study, volunteers (aged 18-45 years) were given subcutaneous injections of the reconstituted FD and two liquid formulations in random sequential order, each at 4 mg/dose, with a 1-week wash-out period between doses. To suppress endogenous GH secretion, intravenous somatostatin was infused continuously 1 hour before to 24 hours after each dose, achieving a cumulative dose of 3 mg. Primary PK endpoints were area under the serum concentration-time curve (AUC0-t) and maximum serum concentration (Cmax). For each of the two liquid formulations, bioequivalence with the FD formulation was concluded if the 95% confidence intervals (CIs) for the estimated test/reference ratios of geometric means of AUC0-t and Cmax were within the standard pre-specified acceptance range (0.80-1.25). RESULTS: Fifteen men and 15 women enrolled (safety population, n = 30; PK population, n = 28). Bioequivalence with the FD formulation could be shown for both liquid formulations. The ratios of geometric means (95% CI) were 1.046 (0.980, 1.117) and 0.991 (0.929, 1.058) for AUC0-t and 0.954 (0.875, 1.040) and 0.955 (0.876, 1.041) for Cmax for the 5.83 and 8.0 mg/mL formulations, respectively. No significant differences between the three treatments in half-lives, time to reach Cmax, clearance or volume of distribution were observed. After injection, the most common side-effects were pain or injection-site reactions (all of mild intensity). There were no clinically significant abnormal vital signs, ECG or laboratory findings. There were 56 treatment-related adverse events (AEs): 49 mild, 6 moderate and 1 severe (vomiting). No serious AEs occurred. The pattern of AEs was as expected and all resolved by study end. CONCLUSION: Both concentrations of a new liquid multi-dose formulation are bioequivalent to the FD reference formulation and all are well tolerated. TRIAL REGISTRATION NUMBER: NCT01034735.

Erythropoietin overrides the triggering effect of DNA platination products in a mouse model of cisplatin-induced neuropathy.

BACKGROUND: Cisplatin mediates its antineoplastic activity by formation of distinct DNA intrastrand cross links. The clinical efficacy and desirable dose escalations of cisplatin are restricted by the accumulation of DNA lesions in dorsal root ganglion (DRG) cells leading to sensory polyneuropathy (PNP). We investigated in a mouse model by which mechanism recombinant erythropoietin (rhEPO) protects the peripheral nervous system from structural and functional damage caused by cisplatin treatment with special emphasis on DNA damage burden. RESULTS: A cumulative dose of 16 mg cisplatin/kg resulted in clear electrophysiological signs of neuropathy, which were significantly attenuated by concomitant erythropoietin (cisplatin 32,48 m/s +/- 1,68 m/s; cisplatin + rhEPO 49,66 m/s +/- 1,26 m/s; control 55,01 m/s +/- 1,88 m/s; p < 0,001). The co-application of rhEPO, however, did not alter the level of unrepaired cisplatin-DNA lesions accumulating in DRG target cells. Micro-morphological analyses of the sciatic nerve from cisplatin-exposed mice showed damaged myelin sheaths and mitochondria. Co-administered rhEPO inhibited myelin sheaths from structural injuries and resulted in an increased number of intact mitochondria. CONCLUSION: The protective effect of recombinant erythropoietin is not mediated by reducing the burden of DNA platination in the target cells, but it is likely to be due to a higher resistance of the target cells to the adverse effect of DNA damage. The increased frequency of intact mitochondria might also contribute to this protective role.

Toward experimental assessment of receptor occupancy: TGN1412 revisited.

In March 2006, 6 healthy volunteers experienced serious adverse reactions during a first-in-human clinical trial of the superagonistic anti-CD28 mAb TGN1412. A first investigation excluded contaminations of the drug product or protocol irregularities as the root cause. Later, an expert scientific group convened in the United Kingdom to develop recommendations pertinent to minimizing risks of first-in-human clinical trials. The expert scientific group concluded from in silico calculations that at the initial dose of 0.1 mg/kg, which was adjusted on the basis of the no observed adverse effect level, approximately 86.2% to 90.9% CD28 receptor occupancy was obtained. Here we developed a flow cytometric method that revealed receptor occupancy of approximately 45% to 80% under the above conditions. Thus we present a method to experimentally determine receptor occupancy that can be taken as one parameter to define the minimal anticipated biological effect level as the basis for calculating safer starting doses for first-in-human clinical trials for products in which a potential risk has been identified. Additional measures are being discussed that will help to significantly improve safety of first-in-human clinical trials.

Repair capacity for platinum-DNA adducts determines the severity of cisplatin-induced peripheral neuropathy.

The pronounced neurotoxicity of the potent antitumor drug cisplatin frequently results in the onset of peripheral polyneuropathy (PNP), which is assumed to be initially triggered by platination products in the nuclear DNA of affected tissues. To further elucidate the molecular mechanisms, we analyzed in a mouse model the formation and processing of the main cisplatin-induced DNA adduct (guanine-guanine intrastrand cross-link) in distinct neuronal cell types by adduct-specific monoclonal antibodies. Comparison of the adduct kinetics in cisplatin-injected mice either proficient or deficient for nucleotide excision repair (NER) functions revealed the essential role of this DNA repair pathway in protecting differentiated cells of the nervous system from excessive formation of such lesions. Hence, chronic exposure to cisplatin resulted in an accelerated accumulation of unrepaired intrastrand cross-links in neuronal cells of mice with dysfunctional NER. The augmented adduct levels in dorsal root ganglion (DRG) cells of those animals coincided with an earlier onset of PNP-like functional disturbance of their sensory nervous system. Independently from the respective repair phenotype, the amount of persisting DNA cross-links in DRG neurons at a given cumulative dose was significantly correlated to the degree of sensory impairment as measured by electroneurography. Collectively, these findings suggest a new model for the processing of cisplatin adducts in primary neuronal cells and accentuate the crucial role of effectual DNA repair capacity in the target cells for the individual risk of therapy-induced PNP.

Adduct-specific monoclonal antibodies for the measurement of cisplatin-induced DNA lesions in individual cell nuclei.

The anticancer drug cisplatin executes its cytotoxic activity via formation of intra- and interstrand crosslinks in DNA. The relative contribution of structurally defined cisplatin adducts to induce apoptosis and the cellular processing of these lesions is still poorly understood mostly due to the lack of sensitive analytical tools for in vivo studies. Here we describe a new method to establish and characterize monoclonal antibodies (Mab) for structurally defined DNA adducts. The two major reaction products of cisplatin, the guanine-guanine (Pt-[GG]) and adenine-guanine (Pt-[AG]) intrastrand crosslinks are recognized by Mab R-C18 and R-B3, respectively. Both antibodies were employed in an immuno-cytological assay allowing the quantification of drug-induced lesions in individual cell nuclei at clinically relevant doses. Analyzing various tissues of cisplatin-treated C57Bl/6 mice the accumulation of Pt-(GG) was highest in kidney tubular cells compared with 30, 50 and 90% lower levels in kidney stroma, liver and peripheral blood cells, respectively. Adduct kinetics revealed that wild type mouse cells remove up to 80% of the crosslinks in contrast to their complete persistence in nucleotide excision repair-deficient (XPC-/-) mice. The aptitude of the immunoassay for human molecular dosimetry studies was demonstrated by measuring adduct levels in tumor biopsies from patients treated with cisplatin.

Similar Pharmacokinetics of the Adalimumab (Humira®) Biosimilar BI 695501 Whether Administered via Subcutaneous Autoinjector or Prefilled Syringe (VOLTAIRE®-AI and VOLTAIRE®-TAI): Phase 1, Randomized, Open-Label, Parallel-Group Trials.

INTRODUCTION: BI 695501 has shown similar efficacy, safety, and immunogenicity to the adalimumab reference product, Humira®. We present two phase 1 studies comparing the pharmacokinetics, safety, and immunogenicity of BI 695501 delivered via autoinjector (AI) vs. prefilled syringe (PFS). METHODS: Both trials were randomized, open-label, parallel-group studies undertaken in subjects aged ≥ 18-65 years. VOLTAIRE®-AI (NCT02606903) recruited healthy, Caucasian, male, non-athletic volunteers with BMI ≥ 18 to ≤ 30 kg/m2. VOLTAIRE®-TAI (NCT02899338) recruited healthy men and women with BMI > 17.5 to < 35 kg/m2. In both studies, a single dose of BI 695501 40 mg was administered via AI or PFS to the abdomen (VOLTAIRE®-AI) or thigh (VOLTAIRE®-TAI). The observation period was 43/57 days and the safety follow-up was 70 days. Co-primary endpoints were AUC0-1032 or AUC0-1368, Cmax, and AUC0-∞. Safety and immunogenicity were assessed. RESULTS: Subjects (VOLTAIRE®-AI: N = 71; VOLTAIRE®-TAI: N = 162) were randomized to AI (n = 35; n = 81) or PFS (n = 36; n = 81). Baseline characteristics were balanced between treatment groups in each study. Total exposure of BI 695501 was similar for both groups; adjusted geometric mean ratios for AUC0-∞, AUC0-1032, and Cmax were 106.17, 104.09, and 114.83%, respectively, for VOLTAIRE®-AI; 103.19, 101.71 (AUC0-1368), and 100.11% for VOLTAIRE®-TAI. In both studies, similar immunogenicity was observed between groups in terms of frequency of binding and neutralizing anti-drug antibody-positive subjects. Incidence of adverse events was similar for both groups. CONCLUSIONS: Pharmacokinetics and immunogenicity of BI 695501 delivered via AI were similar to administration using a PFS, independent of injection site. No differences are expected between AI and PFS use in clinical practice. FUNDING: Boehringer Ingelheim.

Overexpression of cMOAT (MRP2/ABCC2) is associated with decreased formation of platinum-DNA adducts and decreased G2-arrest in melanoma cells resistant to cisplatin.

Resistance to various anti-neoplastic agents is a common observation in clinical management of melanoma. The biologic mechanisms conferring these different drug-resistant phenotypes, including resistance against the commonly used anti-cancer drug cisplatin, are unclear. In order to elucidate the role of the membrane adenosine triphosphate binding cassette-transporter cMOAT (canalicular multispecific anion transporter) (MRP2/ABCC2) in cisplatin resistance of melanoma, the expression of this protein was analyzed in the platinum drug-resistant cell line MeWo CIS 1. Cisplatin-resistant melanoma cells showed a distinct overexpression of cMOAT on mRNA and protein level. This observation was accompanied by a reduced formation of platinum-induced intrastrand cross-links in the nuclear DNA measured by an immunocytologic assay. This decrease in DNA platination was accompanied by an accelerated re-entry into the cell cycle after the typical cisplatin-induced G2 arrest, and a resistance to undergo apoptosis. Kinetics of formation and elimination of platinum-DNA adducts suggest that the DNA repair capacity for Pt-d(GpG) adducts was not elevated in platinum drug-resistant melanoma cells. The decrease in platinum-DNA adduct formation in cisplatin-resistant melanoma cells was rather a reflection of the protecting activity of the transporter cMOAT. In conclusion, the functional inhibition of cMOAT might be a promising strategy in the reversal of resistance to platinum-based anti-cancer drugs in human melanoma.

A phase I dose-escalation study of the immunocytokine EMD 521873 (Selectikine) in patients with advanced solid tumours.

BACKGROUND: EMD 521873 (Selectikine), an immunocytokine comprising a DNA-targeting antibody, aimed at tumour necrosis, fused with a genetically modified interleukin-2 (IL-2) moiety, was investigated in this first-in-human phase I study. METHODS: Patients had metastatic or locally advanced solid tumours failing previous standard therapy. Selectikine was administered as a 1-hour intravenous infusion on 3 consecutive days, every 3 weeks. A subgroup of patients also received 300 mg/m(2) cyclophosphamide on day 1 of each cycle. Escalating doses of Selectikine were investigated with the primary objective of determining the maximum tolerated dose (MTD). RESULTS: Thirty-nine patients were treated with Selectikine alone at dose levels from 0.075 to 0.9 mg/kg, and nine were treated at doses of 0.45 and 0.6 mg/kg in combination with cyclophosphamide. A dose-dependent linear increase of peak serum concentrations and area under curve was found. The dose-limiting toxicity was grade 3 skin rash at the 0.9 mg/kg dose-level; the MTD was 0.6 mg/kg. Rash and flu-like symptoms were the most frequent side-effects. No severe cardiovascular side-effects (hypotension or vascular leak) were observed. At all dose-levels, transient increases in total lymphocyte, eosinophil and monocyte counts were recorded. No objective tumour responses, but long periods of disease stabilisation were observed. Transient and non-neutralising Selectikine antibodies were detected in 69% of patients. CONCLUSIONS: The MTD of Selectikine with or without cyclophosphamide administered under this schedule was 0.6 mg/kg. The recommended phase II dose was 0.45-0.6 mg/kg. Selectikine had a favourable safety profile and induced biological effects typical for IL-2.

High accumulation of platinum-DNA adducts in strial marginal cells of the cochlea is an early event in cisplatin but not carboplatin ototoxicity.

Ototoxicity is a typical dose-limiting side effect of cancer chemotherapy with cisplatin but much less so with carboplatin. To elucidate the underlying molecular pathological mechanisms, we have measured the formation and persistence of drug-induced DNA adducts in the nuclei of inner ear cells of guinea pigs after short-term exposure to either cisplatin or carboplatin using immunofluorescence staining and quantitative image analysis. After application of carboplatin, all cells of the cochlea exhibited a similar burden of guanine-guanine intrastrand cross-links in DNA. In contrast, we observed a pronounced 3- to 5-fold accumulation of this cytotoxic adduct exclusively in the marginal cells of the stria vascularis between 8 and 48 h after treatment with cisplatin. In the kidney, the other critical target tissue of cisplatin toxicity, a similar high preferential formation of cytotoxic DNA adducts was measured in the tubular epithelial cells but not in other renal cell types. As for the ear, this excessive formation of DNA damage in a particular cell type was seen in animals treated with cisplatin but not those treated with carboplatin. Because cisplatin ototoxicity is often attributed to oxidative stress mediated by the generation of radical oxygen species (ROS), we have measured in parallel the levels of the lead DNA oxidation product 8-oxoguanine (8-oxoG) in cochlear cryosections. Compared with basal levels in untreated control cochleas, no additional formation of 8-oxoG was detectable up to 48 h after cisplatin treatment in the DNA of either inner-ear cell type. This suggests that the generation of ROS may be a secondary event in cisplatin ototoxicity.

Protection of platinum-DNA adduct formation and reversal of cisplatin resistance by anti-MRP2 hammerhead ribozymes in human cancer cells.

Resistance to platinum-containing antineoplastic drugs is the major limitation in their clinical use. To elucidate the role of the ABC transporter MRP2 in platinum drug resistance, its expression was analyzed in human cisplatin-resistant cell lines: the ovarian carcinoma line A2780RCIS, the adrenocortical carcinoma line D43/86RCIS and the melanoma line MeWoCIS1. All these cells showed overexpression of MRP2. For reversal of platinum resistance, 2 anti-MRP2 hammerhead ribozymes were introduced into A2780RCIS cells. Both ribozymes showed gene-silencing activities and reversed the drug-resistant phenotype. Moreover, formation of platinum-induced intrastrand cross-links was measured in DNA. The level of DNA platination corresponded inversely to the level of MRP2 expression and was accompanied by increased caspase-3-dependent apoptosis. Kinetics of formation and elimination of platinum-DNA adducts suggest that the DNA repair capacity was not altered; the decrease in platinum-DNA adduct formation was rather a reflection of the protecting activity of MRP2. In conclusion, functional inhibition of MRP2 might be a promising strategy in the reversal of resistance to platinum-based anticancer drugs. This was reflected by the specific inhibition of MRP2 by ribozyme technology, indicating that this gene therapeutic approach may be applicable as a specific means to overcome platinum resistance in human neoplasms.

Down-regulation of DNA repair in human CD34(+) progenitor cells corresponds to increased drug sensitivity and apoptotic response.

Although DNA repair processes have been shown to considerably modulate the cytotoxic effects of alkylating agents, little information is available on the role of these mechanisms in chemotherapy-induced myelosuppression. Therefore, we have analyzed in detail the DNA repair capacity of primary human hematopoietic cells from cord blood (CB) or bone marrow (BM) by 2 functional assays, the immunocytologic assay (ICA) and single-cell gel electrophoresis (comet assay). Besides substantial interindividual differences, we consistently observed significantly lower repair capacity of CD34(+) cells in comparison to CD34(-), CD19(+), or CD33(+) cells of the same donor. After exposure to the alkylating agent ethylnitrosourea (EtNU), the comet assay displayed on average twice as many DNA single-strand breaks (SSBs) in CD34(+) cells and a tripled half-life of these lesions in comparison to corresponding CD34(-) cells. Similarly, reduced SSB repair activity in CD34(+) cells was detected following melphalan or cisplatin application. When specific antibodies were used to monitor DNA reaction products of these drugs, adduct levels were significantly higher and lesions persisted longer in the CD34(+) fraction. To assess the contribution of individual pathways to overall DNA repair, modulators blocking defined steps in repair processes were coapplied with alkylating drugs. Similar "modulation pattern" in corresponding CD34(+) and CD34(-) cell fractions indicated a generalized reduction in DNA repair capacity of CD34(+) cells, rather than deficiencies in a specific pathway. Because CD34(+) cells also displayed higher frequencies of apoptosis in response to melphalan or cisplatin, these findings may help to explain the myelosuppression after exposure to alkylating agents.