Anticancer altretamine recognition by bovine serum albumin and its role as inhibitor of fibril formation: Biophysical insights.

Binding of anticancer drug altretamine with bovine serum albumin (BSA) and its inhibitory effect on fibrillation of the protein has been studied by using a combination of spectroscopic and calorimetric methods. Altretamine is observed to bind with BSA with a moderate binding affinity of the order of 105, which is weakly temperature dependent. Circular dichroism, fluorescence spectroscopic and dynamic light scattering methods have been employed to monitor the conformational change in the protein. Time correlated single photon counting measurements have confirmed ground state complexation of the drug with the protein. Docking studies have led to identification of binding sites on BSA at site III in domain IB. Thioflavin T (ThT) fluorescence emission has been used as a tool to monitor the formation of fibrils/aggregates in BSA. It is observed that anticancer drug altretamine can also act as an inhibitor of fibrillation in BSA and hence can be useful in the treatment of neuro-degenerative diseases. Differential scanning calorimetry has been employed to study the thermal transitions of BSA at different stages of the fibrillation process with and without altretamine to obtain insights into the extent of stabilisation provided by the drug to the protein in native, nucleation/elongation and matured state in the fibrillation process.

The human gut chemical landscape predicts microbe-mediated biotransformation of foods and drugs.

Microbes are nature's chemists, capable of producing and metabolizing a diverse array of compounds. In the human gut, microbial biochemistry can be beneficial, for example vitamin production and complex carbohydrate breakdown; or detrimental, such as the reactivation of an inactive drug metabolite leading to patient toxicity. Identifying clinically relevant microbiome metabolism requires linking microbial biochemistry and ecology with patient outcomes. Here we present MicrobeFDT, a resource which clusters chemically similar drug and food compounds and links these compounds to microbial enzymes and known toxicities. We demonstrate that compound structural similarity can serve as a proxy for toxicity, enzyme sharing, and coarse-grained functional similarity. MicrobeFDT allows users to flexibly interrogate microbial metabolism, compounds of interest, and toxicity profiles to generate novel hypotheses of microbe-diet-drug-phenotype interactions that influence patient outcomes. We validate one such hypothesis experimentally, using MicrobeFDT to reveal unrecognized gut microbiome metabolism of the ovarian cancer drug altretamine.

Prodrugs of hydroxymethylpentamethylmelamine: a principal active metabolite of the antineoplastic agent hexamethylmelamine.

Hexamethylmelamine is in clinical use as an antineoplastic agent. Derivatives and prodrugs of two of its biologically-active metabolites were prepared in an effort to alter its solubility and to enhance bioavailability. In this study prodrugs of pentamethylmelamine and hydroxymethylpentamethylmelamine were synthesized. Among the compounds prepared were N-(methoxymethyl)pentamethylmelamine, N-(ethylthiomethyl)pentamethylmelamine, and several N'-alkyl-N'-methyl-N-(aminomethyl)pentamethylmelamine and N'-aryl-N'-methyl-N-(aminomethyl)pentamethylmelamine derivatives. The aqueous solubility of these prodrugs relative to hexamethylmelamine was compared. The half-lives of these prodrugs were also determined at pH 7.4. The more stable derivatives were assayed at pH 6.4. These prodrugs represent a novel approach for the delivery of the suspect active metabolite of hexamethylmelamine, hydroxymethylpentamethylmelamine.

Hexamethylmelamine: pharmacology and mechanism of action.

Several conclusions can be drawn from a review of HMM preclinical and clinical pharmacology data. The drug is extensively metabolized by animals and by man. The drug is well absorbed following oral administration to animals, but oral bioavailability is low due to first pass metabolism. Based on limited human data and more complete animal data, absorption of HMM following oral administration may be quite high in man. We do not yet know the oral bioavailability of HMM in patients, but again based primarily on animal studies, oral bioavailability is most likely low and variable due to extensive first pass metabolism. Systemic exposure to HMM and demethylated metabolites following oral administration varies greatly from patient to patient and is sometimes quite low. Most patients are, however, exposed to a substantial fraction of the administered dose when determined by urinary recovery of the total dose (based on parent drug and metabolites or total radioactivity) or by the total plasma AUC of parent drug and all metabolites. Systemic exposure to HMM following intravenous administration is clearly greater and less variable than following oral administration. An unresolved question is whether the highly variable and often low systemic exposure after oral administration compromise antitumor activity when compared to intravenous administration. A key issue is whether or not one accepts the hypothesis that metabolism is a prerequisite for antitumor activity. The metabolic activation studies do not rule out other mechanisms of HMM antitumor activity. Modest activity of HMM was observed after prolonged exposure to cells which did not metabolize the drug. However, most of the accumulated data are consistent with the metabolic activation hypothesis. Certainly HMM has clinical activity when administered by mouth. If metabolism is required, then exposure to the total dose (parent drug and metabolites) could be of significance even when exposure to HMM is low, since every demethylated metabolite must have come ultimately from the initial HMM demethylation. We do not know whether the initial metabolic reaction (occurring in the liver rather than in the tumor) provides sufficient exposure of tumor to reactive species. Specifically, does the variable HMM plasma AUC seen after oral administration lead to variable delivery of potentially reactive species to tumor (by rapid breakdown and/or further metabolism of MPMM before it leaves the gut and/or liver) or are quantities of MPMM delivered to tumor comparable to those delivered following intravenous administration. The issue of rate of MPMM formation compared to rate of breakdown and ultimate delivery to tumor has been noted by Judson and Rutty.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacokinetics and metabolism of hexamethylmelamine in mice bearing renal cell tumors.

The pharmacokinetics of hexamethylmelamine (HMM) and its main metabolites hydroxymethylpentamethylmelamine (HMPMM), pentamethylmelamine (PMM), and 2,2,4,6, tetramethylmelamine (2,2,4,6 TetrMM) were studied in renal cell (RC) tumor tissues and plasma of CDF1 mice that had received IP bolus injections of the maximally tolerated dose (200 mg/kg) of HMM. HMM, PMM, and 2,2,4,6 TetrMM concentrations determined in RC tissues were much higher than the plasma values, as indicated by the pharmacokinetic parameters (Cmax and AUC). On the other hand, very low levels of HMPMM, generally considered to be a potentially active antitumor compound, were detected in the target tissues, whereas this hydroxylated metabolite was stable and easily determined in plasma. High HMM concentrations in RC tissues could correlate with the high sensitivity of the tumor to this drug. However, the behavior of HMPMM remains unclear; related hypotheses are presented in this paper.

Pharmacokinetics of hexamethylmelamine in intralipid following hepatic regional administration in rabbits.

Hexamethylmelamine (HMM) is a cytotoxic agent demonstrated to have broad antitumor activity. Poor solubility in aqueous media has precluded significant evaluation of parenteral administration of this drug. A formulation of HMM dissolved in Intralipid has demonstrated excellent tolerance following parenteral administration. The goal of this study was to evaluate the pharmacology of HMM in Intralipid following hepatic regional administration. The routes of administration were intraarterial via the hepatic artery with and without arterial occlusion, i.v. via the portal and jugular veins, and i.p. All animals received a total dose of 10 mg HMM/kg of body weight. Hepatic extraction of HMM was most evident via the portal vein (PV) route [AUC(PV)/AUC(i.v.) = 0.5; P less than 0.05]. Lower plasma levels and areas under the curve (AUCs) were observed for the hepatic artery and hepatic artery-stop flow groups when compared to i.v., but the difference was not significant. Administration i.p. yielded low plasma levels but a very long half-life (88 min). Hepatic tissue levels were highest in the group receiving HMM by the hepatic artery-stop flow route. We conclude that the HMM-Intralipid mixture is well tolerated, that HMM is extracted to a significant degree by the liver following PV administration, and that an i.p. installation of HMM-Intralipid results in prolonged plasma drug levels. This preclinical study supports further efforts at evaluation of parenteral administration of the HMM-intralipid mixture.

Hexamethylmelamine: a critical review of an active drug.

Hexamethylmelamine is an s-triazine that began clinical trials during the 1960s based on its level of antitumor activity in murine tumor models. Phase I studies were performed using an oral formulation given in divided doses for varying numbers of days. The most frequently reported toxicities included nausea, vomiting, abdominal cramps, anorexia, weight loss and malaise. Less frequently reported toxicities were anemia, thrombocytopenia, leucopenia and peripheral neuropathy. Clinical antitumor activity was noted in the phase I studies in a variety of tumor types. Since then a large number of studies have been performed using hexamethylmelamine as a single agent and in a variety of combinations. Unfortunately, almost none of these studies sought to define the utility of this drug relative to other treatments for the diseases in which it showed activity, or to define the contribution of this drug to the activity of any given combination. Thus its role in the treatment of patients with malignancies remains undefined.

Effect of cimetidine and ranitidine on the metabolism and toxicity of hexamethylmelamine.

In a rat model, doses of 20-120 mg/kg of cimetidine prolonged in a dose-related manner the half-life of hexamethylmelamine by 29%-80%, while doses of 1-25 mg/kg of ranitidine did not. Administration of 120 mg/kg of cimetidine with a single 350-mg/kg dose of hexamethylmelamine increased toxicity from LD30 to LD75 (P = 0.005). When combined with 25 mg/kg of ranitidine, a statistically insignificant (P = 0.16) 15% increase in hexamethylmelamine toxicity was noted. These studies indicate that cimetidine but not ranitidine increases the toxicity of hexamethylmelamine through inhibition of microsomal metabolism.

Role of hexamethylmelamine in the treatment of ovarian cancer: where is the needle in the haystack?

Hexamethylmelamine (HMM) was selected for development as an antineoplastic agent because it demonstrated activity in a variety of preclinical tumor models. Its mechanism of action is unknown. It has been used in clinical trials since 1964. The clinical toxic effects have consisted of signs and symptoms involving the following systems: gastrointestinal (nausea, vomiting, anorexia), hematologic (leukopenia, mild anemia), and neurologic (critical depression, hallucinations, peripheral motor and sensory deficits). Antitumor activity against advanced ovarian cancer was demonstrated in phase I trials and the drug was quickly incorporated into trials which utilized drug combinations. The majority of these have consisted of phase II trials without an identified control population. As might be predicted, all of the HMM-containing combinations are active. However, the contribution of HMM to the antitumor activity of the combination remains conjectural. Thus, in spite of greater than 15 years of clinical trials with a drug that has single-agent activity, the questions regarding the role of HMM in the treatment of ovarian cancer remain unanswered.

Low central nervous system penetration of N2,N4,N6,-trihydroxymethyl-N2,N4,N6,-trimethylmelamine (Trimelamol): a cytotoxic s-triazine with reduced neurotoxicity.

Trimelamol (N2,N4,N6-trihydroxymethyl-N2,N4,N6-trimethylmelamine) is an analogue of pentamethylmelamine (PMM). In early clinical trials PMM failed to show significant anti-tumour activity in man which was attributed to poor metabolic activation. Trimelamol does not require activation and is therefore expected to be more active in man. PMM caused dose-limiting emesis and sedation whereas Trimelamol is much less neurotoxic in rodents. The relative penetration of PMM and Trimelamol into mouse brain has therefore been examined. Mice receiving PMM at 90 mg kg-1 i.p. were found to have high concentrations of the drug in the CNS compared to plasma (mean brain/plasma ratio 1.04) whereas animals receiving Trimelamol had consistently low CNS concentrations (mean brain/plasma ratio 0.08). This difference did not correlate with plasma protein binding which is greater for PMM (68.2%) than for Trimelamol (17.5%). However, it does appear to be related to lipophilicity. In Phase I clinical trial Trimelamol has proved much less emetic than PMM and causes no acute sedation. It is likely that this reduction in toxicity may be explained by the relatively poor ability of Trimelamol to enter the CNS.

Effect of phenobarbital pretreatment on the metabolism and antitumor activity of hexamethylmelamine.

The effect of phenobarbital (PB) pretreatment on the N-demethylation and antitumor activity of hexamethylmelamine (HMM) was investigated in mice. In mice pretreated with PB, the levels of HMM in plasma, liver, and M5076 tumor were much lower than in mice treated with HMM alone. Plasma area under the curve (AUC) values were 15 +/- 4 versus 37 +/- 28 micrograms/ml X min, liver AUC values were 43 +/- 13 versus 246 +/- 5 micrograms/g X min, and tumor AUC values were 37 +/- 14 versus 109 +/- 24 micrograms/g X min. All of the N-demethylated metabolites except N2N4 dimethylmelamine in plasma, liver, and tumor also reached much lower levels in mice pretreated with PB. The antitumor activity of HMM was significantly antagonized by PB treatment in both M5076 reticular cell sarcoma and PC6 plasmocytoma of the mouse.

Pentamethylmelamine: review of an aqueous analog of hexamethylmelamine.

The s-triazine derivatives have shown preclinical antitumor activity against several histologic types. The most widely used compound of this class in the clinic, hexamethylmelamine, has been largely restricted to oral use because of its low solubility and lack of stability in solutions suitable for parenteral administration. New analogs were sought which were soluble and stable and retained antitumor activity. Pentamethylmelamine (PMM), the monodemethylated derivative, showed these promising characteristics. Preclinical toxicology studies of PMM in mice, dogs, and monkeys showed toxic manifestations that involved the hematopoietic, lymphatic, renal, male reproductive, gastrointestinal, and nervous systems; these changes were both infusion-rate- and dose-dependent. Clinical phase I trials of PMM were performed using a variety of infusion durations and frequency schedules. The dose-limiting toxic effect common to all of these trials was protracted nausea and vomiting. In addition, some studies reported dose-limiting central nervous system manifestations in the form of agitation, drowsiness, somnolence, and even coma. Mild to moderate hematologic changes were noted. Because of the severity and frequency of the gastrointestinal and central nervous system toxic effects observed in the completed trials, no new clinical trials of PMM sponsored by the National Cancer Institute are planned. However, the interest in finding a clinically useful parenteral triazine continues.

Pharmacokinetics and metabolism of hexamethylmelamine in mice after IP administration.

The pharmacokinetics of hexamethylmelamine (HMM) and its first metabolite (hydroxymethylpentamethylmelamine: HMPMM) following IP bolus dose of 200 mg/kg were studied in mice. The drug concentrations were determined by a sensitive reversed-phase HPLC assay. Thus, for the first time, HMM major hydroxylated and demethylated metabolite plasma levels canbedetermined at the same time. Pharmacokinetic data were analyzed by an original method using a nonlinear cost function minimized by a simplex algorithm. An important property of this computer program is that convergence is ensured in contrast to linear or nonlinear least-square regression analysis, which leads to lack of convergence or to false convergence. Both HMM and HMPMM data fit a one-compartment open model. The parameters obtained indicate that the parent drug would probably be rapidly and completely transformed by the human body into HMPMM.

Superior efficacy of trimelamol to hexamethylmelamine in human ovarian cancer xenografts.

A series of eight human ovarian cancer lines grown in nude mice were used to compare the activity of hexamethylmelamine (HMM) and N2,N4,N6-trihydroxy methyl-N2,N4,N6-trimethylmelamine (trimelamol). The tumor lines differed in histological subtype and growth rate. The drugs were administered i.p. at the maximum tolerated dose at alternate days. Differences in volume of treated and control tumors were endpoints of the study. The tumor lines varied widely in sensitivity to HMM and in four lines a T/C% below 25% was achieved. Trimelamol appeared to be more active than HMM and achieved a T/C below 25% in seven tumor lines. Thus far, the drug has demonstrated significant activity in a phase I trial in ovarian cancer patients. Comparative clinical studies of HMM vs trimelamol have not yet been performed.

Absorption and metabolism of hexamethylmelamine and pentamethylmelamine in rat everted perfused gut segments: correlation with in-vivo data.

The intestinal oxidative metabolism of hexamethylmelamine (HMM) and pentamethylmelamine (PMM) has been studied in microsomes, isolated mucosal cells and intestinal perfused segments. (sub) Cellular systems revealed an almost equal Km (53-65 microM) and Vmax (5.6-7.0 nmol min-1 g-1 intestine) for both compounds. Detailed studies in everted intestinal perfused segments, showed that HMM is metabolized to a far greater extent than PMM (e.g. 11-times, at 80 microM substrate concentration) while PMM transport was 3 times greater than the transport of unchanged HMM. Only when perfused segments were used as an in-vitro tool was a good correlation observed between the in-vivo and in-vitro rate of intestinal metabolism of HMM and PMM. It is concluded that this is due to preservation of structural integrity of the mucosa for both absorptive and metabolic processes.

Pharmacokinetics of hexamethylmelamine administered via the Ip route in an oil emulsion vehicle.

Saline and Intralipid were compared as vehicles for the ip administration of hexamethylmelamine (HMM) in mice. The drug proved stable over at least 7 weeks in solution and 2 years in crystal form. Fiftyfold higher concentrations of HMM could be achieved in Intralipid than in saline. The peritoneal pharmacokinetics were first-order and linear over the concentration range of HMM in Intralipid from 50 to 2000 micrograms/ml. The mean peritoneal half-life of HMM in Intralipid was 12.5-fold greater than the mean half-life of HMM in saline at the same concentration. Peritoneal concentration X time drug exposure, as measured by the area under the curve in single-dose elimination experiments, was 1200-fold greater for HMM at 2000 micrograms/ml in Intralipid than at 50 micrograms/ml, near saturation, in saline. The steady-state peritoneal to plasma concentration ratios were 96-104 for HMM in Intralipid at concentrations of 300-2000 micrograms/ml, and the peritoneal concentration that could be maintained by the constant ip infusion of HMM at 2000 micrograms/ml in Intralipid was at least 1600-fold greater than that maintainable with HMM in saline. The mean peritoneal clearance calculated by two independent methods and at three different concentrations of HMM in Intralipid was 0.112 +/- 0.016 ml/minute. HMM in Intralipid is a stable formulation that can be used to increase peritoneal exposure to HMM and may potentially be used iv as well.

First-pass metabolism of pentamethylmelamine in the rat liver.

The disposition of pentamethylmelamine (PMM) was studied in the male Wistar rat. PMM (5 mg/kg) was administered intraarterially, i.v. (5 and 10 mg/kg), via the portal vein, and into the duodenum to cannulated and unanesthetized rats (n greater than or equal to 4) via infusion. Parent compound and metabolites were quantified by gas chromatography. The areas under the plasma concentration-time curves of PMM after intraarterial and i.v. administration were equal and twice as large as the areas after portal vein and intraduodenal administration. This indicated insignificant lung metabolism for PMM; the low bioavailability of PMM when given via the portal vein or intraduodenally (in both cases, some 50% of an i.v. dose) was the result of presystemic metabolism in the liver. PMM was completely absorbed after intraduodenal administration, and no intestinal metabolism was observed. Linear kinetic behavior of i.v. PMM was observed in the 5- to 10-mg/kg dose range. The area under the plasma concentration-time curve of the first metabolite N2,N2,N4,N6-tetramethylmelamine was significantly greater when PMM was given via the portal vein or intraduodenally than when given intraarterially or i.v. This indicated either extrahepatic elimination/renal excretion of PMM or the existence of an additional metabolic pathway. However, experiments with adrenalectomized rats and rats with ligated blood flow to the kidneys did not alter the area for the first metabolite. These findings may be explained by the formation of unknown metabolites and/or reactive intermediates of PMM.

The area under the curve of metabolites for drugs and metabolites cleared by the liver and extrahepatic organs. Its dependence on the administration route of precursor drug.

The venous equilibrium model (or well-stirred model) is used to determine the area under the blood concentration vs. time curve of a metabolite formed from a precursor drug. It will be shown that the AUC of a metabolite will change according to the route of precursor drug administration(whether intraarterially, intravenously, via the portal vein, or orally) when the drug and/or metabolite is eliminated by more than one organ. Elimination includes hepatic and extrahepatic metabolism and renal excretion. The validity of the model is probed by using literature data for drug and metabolite areas. Finally, the use of metabolite areas for evaluating the complete/incomplete absorption or orally administered precursor drug is discussed.