Pharmacokinetics of O6-benzylguanine and its active metabolite 8-oxo-O6-benzylguanine in plasma and cerebrospinal fluid after intrathecal administration of O6-benzylguanine in the nonhuman primate

O6-Benzylguanine (O6BG) irreversibly inactivates the single-turnover DNA repair protein alkylguanine-alkyltransferase. Thus, O6BG increases tumor-cell sensitivity to alkylating agents such as carmustine, lomustine, procarbazine, and temozolomide. We investigated the pharmacokinetic behavior of O6BG and O6-benzyl-8-oxoguanine (8-oxo-O6BG) in cerebrospinal fluid (CSF) and plasma after intraventricular administration of O6BG in a nonhuman primate model. In our study, three animals received a single 1-mg dose of O6BG into the lateral ventricle. CSF from the 4th ventricle and plasma samples were collected after administration, and O6BG and 8-oxo-O6BG concentrations were measured by high-performance liquid chromatography. Four additional animals received 1 mg of O6BG via the intralumbar route weekly for 6 weeks to assess the feasibility and toxicity of this route of administration. The peak O6BG CSF concentration was 412+/-86 microM, the t1/2 was 0.52+/-0.02 h, the clearance was 0.22+/-0.01 ml/min, and the area under the concentration-time curve was 319+/-15 microM x h in 4th ventricular CSF. The peak CSF concentration of 8-oxo-O6BG in CSF was 1.9+/-0.4 microM, the t1/2 was 0.76+/-0.03 h, and the area under the concentration-time curve was 5.0+/-1.1 microM x h. Both O6BG and 8-oxo-O6BG were detected in the plasma 0.5-3 h after intraventricular O6BG administration. The plasma peak concentration of O6BG was 0.4 microM at 30 min, and the concentration was <0.1 microM by 3 h. The plasma concentration of 8-oxo-O6BG was 0.2 microM at 30 min and 0.6 microM at 3 h. The animals tolerated the single intraventricular dose and 6 weekly intralumbar doses of O6BG without toxicity. We concluded that intrathecal administration of O6BG is well tolerated in the nonhuman primate and seems to have a substantial pharmacokinetic advantage over systemic administration for meningeal tumors.     

Reaction of O6-benzylguanine-resistant mutants of human O6-alkylguanine-DNA alkyltransferase with O6-benzylguanine in oligodeoxyribonucleotides

Inactivation of the human DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), by O6-benzylguanine renders tumor cells susceptible to killing by alkylating agents. AGT mutants resistant to O6-benzylguanine can be made by converting Pro140 to an alanine (P140A) or Gly156 to an alanine (G156A). These mutations had a much smaller effect on the reaction with O6-benzylguanine when it was incorporated into a short single-stranded oligodeoxyribonucleotide. Such oligodeoxyribonucleotides could form the basis for the design of improved AGT inhibitors. AGT and mutants P140A and G156A preferentially reacted with O6-benzylguanine when incubated with a mixture of two 16-mer oligodeoxyribonucleotides, one containing O6-benzylguanine and the other, O6-methylguanine. When the 6 amino acids located in positions 159-164 in AGT were replaced by the equivalent sequence from the Escherichia coli Ada-C protein (mutant AGT/6ada) the preference for benzyl repair was eliminated. Further mutation incorporating the P140A change into AGT/6ada giving mutant P140A/6ada led to a protein that resembled Ada-C in preference for the repair of methyl groups, but P140A/6ada did not differ from P140A in reaction with the free base O6-benzylguanine. Changes in the AGT active site pocket can therefore affect the preference for repair of O6-benzyl or -methyl groups when present in an oligodeoxyribonucleotide without altering the reaction with free O6-benzylguanine.     

Isolation of human O6-alkylguanine-DNA alkyltransferase mutants highly resistant to inactivation by O6-benzylguanine

The activity of O6-alkylguanine-DNA alkyltransferase (AGT) protects cells from killing by methylating or chloroethylating agents. AGT is strongly inhibited by O6-benzylguanine (ED50, 0.2 microM), and this drug is presently undergoing clinical trials to enhance chemotherapy by alkylating agents. Point mutations such as P140A (ED50, 5 microM) render AGT resistant to O6-benzylguanine (BG). Selection for such mutants may prove to be a problem in the use of BG, and a better knowledge of the factors underlying resistance to BG will enable the rational design of improved inhibitors able to inactivate these mutants. BG-resistant AGT mutants may also be valuable for expression in bone marrow stem cells to reduce myelosuppression brought about by alkylating agents, to increase the therapeutic index of therapies including BG, and for use as a selectable marker to allow other genes to be expressed in such stem cells. We have therefore set up a general screen to obtain such mutants by using the ability of AGT to protect Escherichia coli GWR109 lacking endogenous AGT from killing by N-methyl-N'-nitro-N-nitrosoguanidine. When the cells were rendered permeable to BG by mutating the lipopolysaccharide membrane component forming strain TRG8, the protection by AGT expression was abolished by treating the cells with BG. The known P140A mutant was used to test the system and was highly selected for by treatment with 50 microM BG and 40 microg/ml N-methyl-N'-nitro-N-nitrosoguanidine. The sequence coding for PVP at positions 138-140 in AGT was replaced with a random nucleotide sequence, and this library was used to transform TRG8. All of the 59 colonies analyzed having AGT activity that survived the selection from the pool of 36,000 transformants were resistant to BG. Many (69%) of these mutants contained lysine at position 140, and all of these showed the highest level of resistance with <10% loss of activity when crude cell extracts were incubated with 1.2 mM BG. This result was confirmed with three mutants (P138K/V139L/P140K, P138M/V139L/P140K, and P140K), which were purified to homogeneity. The next most common residues found at position 140 were arginine (7%) and asparagine (7%). Studies carried out with purified preparations of mutants P140R and P140N revealed that these mutations also provided resistance to BG but to a lesser extent than P140K (ED50s of 190 and 7 microM, respectively). These results indicate that: (a) this screening method can be used to evaluate BG resistance of single or multiple changes throughout the AGT sequence; and (b) replacement of proline-140 with lysine is the most effective point mutation at this site causing BG resistance and is more than 200 times more effective than replacement with alanine.     

The potential role of glycine-160 of human O6-alkylguanine-DNA alkyltransferase in reaction with O6-benzylguanine as determined by site-directed mutagenesis and molecular modelling comparisons

O6-Alkylguanine DNA-alkyltransferase (ATase) repairs toxic, mutagenic and carcinogenic O6-alkylguanine (O6-alkG) lesions in DNA by a highly conserved reaction involving the stoichiometric transfer of the alkyl group to the active centre cysteine residue of the ATase protein. In the Escherichia coli Ada ATase, which is effectively refactory to inhibition by O6-benzylguanine (O6-BzG), the residue corresponding to glycine-160 (G160) for the mammalian proteins of this class is replaced by a tryptophan (W). Therefore, to investigate the potential role of the G160 of the human ATase (hAT) protein in determining sensitivity to O6-BzG, site-directed mutagenesis was used to produce a mutant protein (hATG160W) substituted at position 160 with a W residue. The hATG160W mutant was found to be stably expressed and was 3- and 5-fold more sensitive than hAT to inactivation by O6-BzG, in the absence and presence of additional calf-thymus DNA respectively. A similar, DNA dependent increased sensitivity of the hATG160W mutant relative to wild-type was also found for O6-methylguanine mediated inactivation. The potential role of the W160 residue in stabilising the binding of the O6-alkG to the protein is discussed in terms of a homology model of the structure of hAT. The region occupied by G/W-160 forms the site of a putative hinge that could be important in the conformational change that is likely to occur on DNA binding. Three sequence motifs have been identified in this region which may influence O6-BzG access to the active site; YSGG or YSGGG in mammals (YAGG in E. coli Ogt, YAGS in Dat from Bacillus subtilis), YRWG in E. coli Ada and Salmonella typhimurium (but YKWS in Saccharomyces cerevisiae) or YRGGF in AdaB from B. Subtilis. Finally,conformational and stereoelectronic analysis of the putative transition states for the alkyl transfer from a series of inactivators of hAT, including O6-BzG was undertaken to rationalise the unexpected weak inhibition shown by the alpha-pi-unsaturated electrophiles.     

A single amino acid change in human O6-alkylguanine-DNA alkyltransferase decreasing sensitivity to inactivation by O6-benzylguanine

Mammalian O6-alkylguanine-DNA alkyltransferases (AGTs) are readily inactivated by incubation with the pseudosubstrate, O6-benzylguanine, but the equivalent protein from the Escherichia coli ogt gene is much less sensitive and the Saccharomyces cerevisiae and E. coli ada gene product AGTs are completely resistant to this compound. We have expressed the normal human AGT and various point mutations (C145A, W100A, and P140A) in an ada- ogt- strain of E. coli and tested these proteins against DNA substrates containing O6-methylguanine, for inactivation by O6-benzylguanine and for the ability to produce guanine from O6-benzylguanine. The C145A mutation was inactive as expected since this residue forms the methyl acceptor site. Mutants W100A and P140A were fully active against methylated DNA substrates but the P140A mutant was much less sensitive to inactivation by O6-benzylguanine and failed to form significant amounts of [3H]guanine when incubated with O6-benzyl[8-3H]-guanine. The proline at position 140 in mammalian AGTs is replaced by alanine in the Ada and yeast AGTs and by serine in the Ogt AGT. These results suggest that this proline residue affects the configuration of the active site allowing the O6-benzylguanine to enter and react with the mammalian AGT. The production of resistance to O6-benzylguanine by a single base change raises the possibility that such resistance may arise quite readily in cells of tumors treated therapeutically with the combination of O6-benzylguanine and an alkylating agent.     

Effect of DNA on the inactivation of O6-alkylguanine-DNA alkyltransferase by 9-substituted O6-benzylguanine derivatives

Studies were carried out on the inactivation of pure human O6-alkylguanine-DNA alkyltransferase by 9-substituted O6-benzylguanine derivatives in the presence and absence of DNA. The addition of DNA increased the rate of inactivation of the alkyltransferase by O6-benzylguanine and its 9-methyl derivative but had little effect on the rate of inactivation by the 9-cyanomethyl derivative. In contrast, when O6-benzylguanine derivatives with larger 9-substituents such as ribose, 2'-deoxyribose, dihydrotestosterone, or 2-hydroxy-3-(isopropoxy)propyl were used, the addition of DNA was strongly inhibitory to the inactivation. In the case of O6-benzylguanine, O6-benzylguanosine, and O6-benzyl-2'-deoxyguanosine, these results were confirmed by directly measuring the rate of formation by the alkyltransferase of guanine, guanosine, or 2'-deoxyguanosine, respectively. The data indicated that the presence of DNA activated the alkyltransferase, rendering it more reactive with O6-benzylguanine or O6-benzyl-9-methylguanine, but that DNA interferes with the binding of inhibitors with larger 9-substituents, presumably by competing for the same binding site. Since these inactivators readily inactivate alkyltransferase in cells, the amount of cellular alkyltransferase bound to DNA must be small or readily exchangeable with the free form.     

O6-alkylguanine-DNA alkyltransferase inactivation by ester prodrugs of O6-benzylguanine derivatives and their rate of hydrolysis by cellular esterases

To modulate the bioavailability and perhaps improve the tumor cell selectivity of O6-alkylguanine-DNA alkyltransferase (AGT) inactivators, pivaloyloxymethyl ester derivatives of O6-benzylguanine (BG) were synthesized and tested as AGT inactivators and as substrates for cellular esterases. The potential prodrugs examined were the 7- and 9-pivaloyloxymethyl derivatives of O6-benzylguanine (7- and 9-esterBG), and of 8-aza-O6-benzylguanine (8-aza-7-esterBG and 8-aza-9-esterBG) and the 9-pivaloyloxymethyl derivative of 8-bromo-O6-benzylguanine (8-bromo-9-esterBG). The benzylated purines were all potent inactivators of the pure AGT and of the AGT activity in HT29 cells and cell extracts. Each ester was at least 75 times less potent than the corresponding benzylated purine against the pure human AGT. In contrast, the activities of esters and their respective benzylated purine were similar in crude cell extracts and in intact cells. The increase in potency of esters in cellular extracts could be explained by a conversion of the respective prodrug to the more potent benzylated purine in the presence of cellular esterases. The apparent catalytic activity (Vmax/Km) of liver microsomal esterase for 8-azaBG ester prodrugs was 70-130 times greater than for BG prodrugs and 10-20 times greater than for 8-bromo-9-esterBG. Tumor cell hydrolysis of the esters varied considerably as a function of cell type and prodrug structure. These data suggest that these or related prodrugs may be advantageous for selective AGT inactivation in certain tumor types.     

Phase I trial of O6-benzylguanine for patients undergoing surgery for malignant glioma

PURPOSE: The major mechanism of resistance to alkylnitrosourea therapy is the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT), which removes chlorethylation or methylation damage from the O6-position of guanine. O6-benzylguanine (O6-BG) is an AGT substrate that inhibits AGT by suicide inactivation. We conducted a phase I trial to define the presurgical dose required for depletion of tumor AGT activity in patients with malignant glioma. MATERIALS AND METHODS: Patients were to be treated 18 hours before craniotomy with intravenous doses that ranged between 40 and 100 mg/m2 given over 1 hour. Resected tumor was snap-frozen in liquid nitrogen and AGT activity analyzed by high-pressure liquid chromatography (HPLC). Up to 13 patients were treated at a specific dose of O6-BG, with a target end point of > or = 11 of 13 patients with undetectable tumor AGT levels (< 10 fmol/mg protein). RESULTS: Thirty patients with malignant gliomas were enrolled, with 11 of 11 patients treated at 100 mg/m2 O6-BG demonstrating tumor AGT levels less than 10 fmol/mg protein. No toxicity was noted in any patient treated. CONCLUSION: These results indicate that 100 mg/m2 of O6-BG can maintain tumor AGT levels less than 10 fmol/mg protein for at least 18 hours after treatment, a time interval in which bis(2-chloroethyl)nitrosourea (BCNU)-induced chloroethyl adducts are fully converted into interstrand cross-links. A 100-mg/m2 dose of O6-BG will be used in combination with BCNU in another phase I trial designed to determine the maximal-tolerated dose of BCNU.     

Simultaneous protection of G156A methylguanine DNA methyltransferase gene-transduced hematopoietic progenitors and sensitization of tumor cells using O6-benzylguanine and temozolomide

OBJECTIVE: To describe the clinical and radiologic features of superficial siderosis of the CNS after treatment of a cerebellar tumor. METHODS: Clinical assessment and MRI in four patients with superficial siderosis were performed. RESULTS: Four patients with superficial siderosis had been treated for a primary cerebellar tumor (astrocytoma in three patients, medulloblastoma in one patient) during childhood. All patients were treated with surgery and three received radiotherapy. Slowly progressive bilateral sensorineural hearing loss, gait ataxia, and limb ataxia appeared 8 to 22 years after diagnosis of the cerebellar tumor. Other clinical features were mild cognitive impairment, dysarthria, nystagmus, optic neuropathy, anosmia, and upper motor neuron signs. The CSF contained erythrocytes and increased protein. MRI with fast spin-echo T2-weighted and gradient-echo T2* sequences showed a hypointense rim of iron coating the surface of the cerebellum and brainstem. Twenty-one other patients who had survived more than 5 years after treatment of a primary cerebellar tumor did not have symptoms or signs suggestive of superficial siderosis. CONCLUSIONS: Superficial siderosis is an uncommon late complication of the treatment of a childhood cerebellar tumor, but it is probably underrecognized. The diagnosis should be suspected in patients who present with slowly progressive sensorineural hearing loss and ataxia many years after eradication of a childhood cerebellar tumor.     

High-dose ethambutol: its role in intermittent chemotherapy. A six-year study

The outcome of 81 patients receiving supervised ambulatory treatment with twice-weekly isoniazid and ethambutol is reported. The patients were divided into 2 groups, one of which received its entire outpatient treatment with isoniazid and ethambutol, the other receiving isoniazid and ethambutol after initial treatment with isoniazid and streptomycin. In both groups, there was a 100 per cent favorable response at completion of therapy and no relapes detected among those patients followed after treatment. We compared our results with other studies of intermittent-supervised therapy and concluded that twice-weekly isoniazid and ethambutol given up to 18 months after an adequate phase of daily treatment is a highly effective and well-tolerated regimen.     

The role of chemotherapy in head and neck cancer

We studied the effect of cytoreductive chemotherapy in head and neck cancer and analyzed it in terms of efficacy, remission rates, and duration, as well effect on survival. Single-agent chemotherapy, which formerly was used as a palliative therapy in recurrent and metastatic disease, had little affect on survival. More recently, multi-agent chemotherapy trials have shown significantly higher response rates, but this success has not translated into an added survival benefit. These findings led to the introduction of multi-agent chemotherapy into the induction (neoadjuvant) clinical setting. In these clinical circumstances, better objective response rates were found, particularly in the previously untreated patient. Although this therapy has resulted in better control of local disease, the impact on survival is not yet clear. Adjuvant chemotherapy is most useful in patients who have a high risk of relapse. Therapy appears to decrease its incidence, particularly at distant sites. Finally, chemoradiation trials have shown that this treatment provides a survival advantage, but at the cost of a significant increase in toxicity.     

Investigation of the role of tyrosine-114 in the activity of human O6-alkylguanine-DNA alkyltranferase

Tyrosine-114 is one of 13 totally conserved amino acids in all known sequences of O6-alkylguanine-DNA alkyltransferase (AGT). The importance of this amino acid in repair of alkylated DNA by AGT was studied by changing it to phenylalanine (F), alanine (A), threonine (T), or glutamic acid (E) in human AGT. The activities of the mutant proteins were then compared to those of the wild type with regard to abilities to do the following: (a) protect Escherichia coli from the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); (b) repair methylated DNA in vitro; (c) bind to oligodeoxynucleotides containing O6-methylguanine; and (d) react with the low molecular weight pseudosubstrate, O6-benzylguanine. When expressed at high levels in E. coli strain GWR109, lacking endogenous AGT, the wild type and the Y114F mutant were highly effective in reducing mutations and cell killing by MNNG. The Y114A mutant had a much smaller protective effect, and mutants Y114T and Y114E were inactive. Purified preparations of all four AGT mutants showed an approximately similar degree (74-120-fold) of reduction in the rate of reaction with O6-benzylguanine. In contrast, the degree of reduction in activity toward methylated DNA substrates in vitro varied according to the mutation with the more conservative Y114F producing only a 30-fold reduction and the most drastic change of Y114E abolishing activity completely. Alteration Y114A produced a 1000-fold reduction whereas Y114T reduced activity by 10000-fold. All of the mutations affected the binding of AGT to single- or double-stranded oligodeoxynucleotides containing O6-methylguanine. The extent of increase in the Kd varied according to the amino acid with 2-5-fold (F), 7-11-fold (A), 167-200-fold (T), and 600-1000-fold (E) increases. These results are consistent with tyrosine-114 playing a role both in the binding of AGT to its DNA substrate and in facilitating the transfer of the alkyl group. It is probable that AGT resembles other DNA repair proteins in bringing about a "flipping out" of the target base from the DNA helix. Tyrosine-114 is therefore an excellent candidate for a key role in the interaction with the flipped O6-methylguanine. The results also show that when large amounts of AGT are produced in the cell, substantial decreases in the efficiency with which AGT can repair methylated DNA do not prevent the ability to protect E. coli from toxic alkylating agents. Mutant Y114F, whose activity was reduced by 30-fold, was equal to wild-type AGT in bringing about this protection.     

Vaginal melanoma and the role of regional chemotherapy

Major vascular injuries complicating groin hernia repairs are very rare. Five such cases seen at the vascular unit of the Department of Surgery, UNTH, Enugu, Nigeria over a five year period are presented. The patients all of whom were adults (age range 49-65) had initial problems of hypovolaemia due to massive blood loss followed by infection and anaemia. One of the patients died from irreversible shock while still in the Casualty Unit. The vascular injuries were dealt with by either primary repair, vein graft or dacron graft depending on the prevailing conditions e.g. presence or absence of infection. The other 4 patients who had surgery did well postoperatively and on 5 to 58 months follow-up. Emphasis is laid on the underlying causes of these iatrogenic injuries which include inexperience on the part of the operator and inadequate anaesthesia (often wrongly applied local anaesthesia). If this complication however occurs, the wound should be tightly packed and patient sent immediately to a unit with vascular surgical facilities.     

Complement and its role in immune response

The complement system as a part of innate immunity is considered to provide rapid tough incomplete antimicrobial activity. However, besides providing a first-line defence, innate immunity plays additional important roles: it initiates and improves the slower, but more specific, acquired immune response. The recognition and destruction of noxious substances, as well as initiation of the acquired immune response, are accompanied by potentially hazardous inflammation. The inflammatory process has thus to be tightly regulated. In this overview, innate immunity and its interactions with acquired immunity are discussed with the main focus on the complement system. Our scientific interests are integrated into the discussion on complement.     

Apoptosis and its role in human disease

In a landmark paper published over two decades ago, Kerr et al. proposed the term apoptosis "for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations". In the ensuing years, this natural cell death process was studied at the basic science level, primarily with a view to understanding its roles in cancer and in the development and maintenance of the immune system. More recently, however, evidence has suggested a role for the failure of normal apoptosis control in many of the major diseases of the industrialized world. Though complex, apoptosis appears amenable to therapeutic intervention. The range of modern pharmaceutical strategies available to treat such disregulated gene-directed processes offers promise for advances in the control of cancer, immune system and neurodegenerative disorders, heart disease, and perhaps even the aging process itself.     

The role of chemotherapy in the management of nasopharyngeal carcinoma

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a radiosensitive tumor for which there is a high local control rate after radical radiotherapy (RT). However, for patients with locoregionally advanced disease, the rate of distant metastasis is high and the 5-year overall survival rate is poor. METHODS: A review of retrospective and prospective clinical studies was performed to assess the role of chemotherapy in three settings: metastatic disease; neoadjuvant and/or adjuvant; and concurrent chemotherapy with radiotherapy. RESULTS: Cisplatin-based combination chemotherapy results in a high response rate in patients with metastatic NPC, and a subgroup may achieve long term disease free survival. The use of neoadjuvant and adjuvant chemotherapy to treat locoregionally advanced disease has resulted in consistently high response rates, but no randomized trial to date has demonstrated an improvement in overall survival. A recent Head and Neck Intergroup study randomized patients in the United States to receive concurrent chemotherapy (cisplatin) and radiotherapy or radiotherapy only. Although this approach demonstrated significant benefit in overall survival favoring the use of concurrent chemotherapy and radiotherapy, its applicability in geographic areas of high NPC incidence remains to be proven. CONCLUSIONS: NPC is a chemosensitive tumor, and patients with metastatic disease have a high response rate. Further prospective studies will define the standard approach to treating locoregionally advanced NPC, which is likely to incorporate into the primary treatment some form of systemic chemotherapy.     

Retroviral transfer of a bacterial alkyltransferase gene (ada) into human bone marrow cells protects against O6-benzylguanine plus 1, 3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity

The antitumor activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is limited by the O6-alkylguanine-DNA alkyltransferase (ATase) in tumor cells and by delayed myelosuppression. Inactivation of neoplastic ATase by O6-benzylguanine (BG) improves the therapeutic index for BCNU. We have demonstrated previously that BG + BCNU-induced myelosuppression in mice is reduced by expression of the BG-resistant ATase ada in murine bone marrow. We have now generated an amphotropic retrovirus containing the ada gene and tested the effectiveness of ada expression in preventing BG + BCNU cytotoxicity in human hematopoietic progenitor cells. A retroviral producer clone with a biological titer of 6.5 x 10(4) colony-forming units/ml and 4.4 pmol ATase/mg protein was used for transduction of bone marrow. Cocultivation of these ada producer cells with progenitor cells from six normal individuals resulted in 1.9-3. 9-fold protection against BG + BCNU-induced cytotoxicity in committed progenitor cell assays. Furthermore, this cytoprotective effect was associated with a high transduction efficiency (40%) and a 2-fold increase of ATase activity in the surviving committed progenitor cell colonies. These data provide a basis for testing the clinical effectiveness of retroviral ada gene transfer into hematopoietic cells to increase the therapeutic index of BG + BCNU.