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This paper presents in situ observations of the epitaxial nucleation and growth of the stable polymorph of a steroid, 7alphaMna, on a specific face of the metastable form at low supersaturation, using optical microscopy and in situ Raman spectroscopy. The presence of the metastable polymorph is essential for the nucleation and growth of the stable one. The order of the metastable zones of the stable and metastable polymorphs is reversed for the epitaxial growth process as compared to the case of 3D nucleation. The rate of transformation of the metastable polymorph to the stable one can be controlled by the supersaturation.

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This paper investigated the pharmacokinetics and biotransformation of mirtazapine in healthy human volunteers. The results showed that the area under the plasma drug concentration-time curve (AUC) of mirtazapine in human plasma appeared to be three times higher than the AUC of demethylmirtazapine. As mirtazapine is marketed as a racemic mixture and both enantiomers possess pharmacological properties essential for the overall activity of the racemate, the pharmacokinetics of mirtazapine were examined and appeared to be enantioselective. The R(-)-enantiomer showed the longest elimination half-life from plasma. This was ascribed to the preferred formation of a quaternary ammonium glucuronide of the R(-)-enantiomer. This glucuronide may be deconjugated, leading to a further circulation of the parent compound, thus causing a prolongation in the elimination half-life. The S(+)-enantiomer was preferentially metabolised into an 8-hydroxy glucuronide. Other metabolic transformation pathways found for mirtazapine were demethylation and N-oxidation. Mirtazapine was extensively metabolised and almost completely excreted in the urine (over 80%) and faeces within a few days after oral administration.

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This paper describes in vitro cytotoxicity experiments with 213Bi- and 225Ac-immunoconjugates on the human epidermoid tumour cell line A431 using a blood group A-reactive murine IgG (2D11) as the specific antibody and MOPC 21 as the control antibody. With both radionuclides, specific cell-killing was achieved. The observed cytotoxicity of 213Bi (T1/2 - 47 min) indicates that this radionuclide is a useful alternative for the alpha-emitter 212Bi in the treatment of blood-borne malignancies. 225Ac-immunoconjugates (T1/2 of 225Ac is 10 days) may be applicable for the treatment of solid tumours, since the daughter radionuclides of 225Ac contribute to the cytotoxic efficacy by a field effect (i.e. toxicity in an area distal from the antibody-binding site). The lack of an adequate chelator for 225Ac is a major drawback.

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A simple electrophoretic IEF procedure was developed for the quantitation of bifunctional DTPA ligand molecules in DTPA-protein conjugates. From a calibration plot of pI versus substitution ratios of reference conjugates, the concentrations of DTPA conjugated to protein were determined. Molar ratios of DTPA to protein agreed satisfactorily with the ratios obtained by a spectrophotometric technique using a colored yttrium(III) complex of arsenazo III. The IEF method was successfully applied on preparations of benzyl-DTPA to mAbs MOPC-21, SC-20 (aCEA), and human serum albumin.

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The specific delivery of radioisotopes to a tumor at minimal radiation of normal tissue is the ultimate aim of radioimmunotherapy. In this respect a two-step pretargeting regimen generally leads to an improved tumor to normal tissue uptake ratio compared to direct administration of radioimmunoconjugates. In this paper, in vitro studies are described in which the specific hybridization of complementary DNA fragments is the recognition mechanism in a pretargeting regimen comprising tumor cell saturation with a monoclonal antibody (MoAb)-oligonucleotide conjugate, followed by administration of the radiolabeled complementary oligonucleotide. Complementary oligodeoxynucleotides (15-mers; melting temperature, 68 degrees C) were prepared on a DNA synthesizer. The 5'-end was derivatized with a functional group for labeling with iodine, and the 3'-end was substituted with an amino function suitable for conjugation to an antibody (or attachment of a biotin residue). Both terminal modifications ensure stability of the oligonucleotides against exonucleases because the unconjugated form is stable for 24 h and the conjugated form is stable for several days when incubated in human plasma at 37 degrees C. Antibody-DNA conjugates were prepared by introduction of sulfhydryl groups into the oligonucleotide, followed by conjugation to maleimide-substituted MoAbs. Typically, 3 oligonucleotides were conjugated to an IgG, and 4-6 were conjugated to an IgM with preservation of immunoreactivity. Histochemistry on fresh frozen sections of breast cancer tissue demonstrated qualitatively the specificity of our two-step procedure. In vitro experiments with human tumor cell lines and tumor-specific MoAbs showed that, after saturation with tumor-specific MoAb-DNA conjugates, quantitative hybridization of the tumor cell-bound oligonucleotides occurred at a 30-fold excess of the labeled complementary oligonucleotide: hybridization was complete after 30 min of incubation. No reaction was observed with an irrelevant MoAb-DNA conjugate. The oligonucleotide was neither taken up by tumor cells or endothelial cells nor hybridized to a significant extent with human genomic DNA. These data indicate the feasibility of this two-step approach in radioimmunotherapy.

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After separate administration of mianserin (CAS 24219-97-4) enantiomers to rats, the hydroxy-metabolites of S-mianserin were excreted mainly as conjugates whereas the amount of free phenolic metabolites was greater after administration of the R-enantiomer. The sulphate-conjugate of 8-hydroxy-mianserin was stereoselectively found in faeces only after administration of S-mianserin. Based on in vitro experiments with sulphatase, the stability of this conjugate towards enzymic hydrolysis appears a likely explanation. In vitro experiments with liver homogenates of rats revealed little enantioselectivity with respect to oxidative metabolism. After prior enzyme induction with phenobarbital, however, enantioselectivity with regard to N-oxidation was found. The 3-oxo-mianserin metabolite, found in vitro, is most likely the product of a rearrangement of mianserin-N-oxide. N-formyl-demethyl-mianserin, identified in vitro, is considered to be an artefact.

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An in vitro autoradiographic study was performed to characterize specific rat brain binding sites for non-opioid neuroleptic-like gamma-type endorphins, using [35S]Met-des-enkephalin-gamma-endorphin ([35S]Met-DE gamma E; [35]S-beta-endorphins(5-17)) with high specific activity as radioligand. The binding sites appeared to be confined to rat forebrain regions, e.g., orbital cortex, frontal cortex, cingulate cortex, piriform cortex, nucleus accumbens, amygdala, mediodorsal nucleus of the thalamus and arcuate and periventricular nuclei of the hypothalamus. These regions are part of the mesocorticolimbic feedback circuit. Densitometric analysis of the autoradiographs revealed that the density of the binding sites was highest in the mediodorsal nucleus of the thalamus and the amygdala. Concentration-dependent displacement of [35S]Met-DE gamma E (500 pM) with DE gamma E yielded an IC50 of 0.6 nM whereas DE alpha E (beta-endorphin(6-16)) had an IC50 of 210 nM. Various endorphins, sharing the gamma-endorphin C terminus, displaced [35S]Met-DE gamma E to the same extent as non-labelled DE gamma E (at 10(-6) M) whereas non-endorphin peptides did not show displacing capacity. Possible relationships of the binding sites with opioid receptors were investigated. DAMGO (mu) and DPDPE (delta) displaced [35S]Met-DE gamma E to some extent at 10(-6) M whereas U69,593 (kappa) was inactive, suggesting that the binding sites for gamma-type endorphins may resemble mu- and delta-opioid receptors in some aspects. Similarly, relationships with dopamine receptors were investigated. Haloperidol partially displaced [35S]Met-DE gamma E whereas sulpiride, SKF38,393 and 3-PPP at 10(-6) M did not induce significant displacement. Thus, binding sites are distinct from dopamine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

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This paper proposes the utilization of 225Ac for the alpha-radioimmunotherapy of cancer. The isotope decays with a radioactive half-life of 10 days into a cascade of short-lived alpha- and beta-emitting isotopes. In addition, when indicated by the pharmacokinetic requirements of particular clinical applications, 213Bi, with a radioactive half-life of 47 min, can be chosen as an alternative source of alpha-particles in radioimmunotherapy. This isotope is the last alpha emitter in the 225Ac decay-cascade and can be extracted from a 225Ac source at the bedside of the patient. 225Ac can quasi ad infinitum be obtained from one of its precursors, 229Th, which can be made available by various means. The indications for the use of alpha-particles as an alternative to more traditional classes of radiation are derived from the particle-kinetic characteristics and the radioactive half-life of their source isotope, as well as from the properties of the target-selective carrier moiety for the source isotope. It may be expected that useful applications, complementary to and/or in conjunction with other means of therapy will be identified.

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One of the major challenges in radioimmunotherapy is the specific delivery of radioisotopes to tumor cells while minimizing normal tissue radiation. In this respect, the application of two-step pretargeting schemes generally leads to more favorable tumor to normal tissue uptake ratios than direct administration of radioimmunoconjugates. In this study, we present the specific hybridization of complementary DNA fragments as a novel recognition mechanism in pretargeting. Briefly, our strategy involves first administration of antibody-DNA conjugate, followed by targeting with radiolabeled complementary DNA (antisense DNA). Complementary oligodeoxynucleotides (14-mers, Tm = 57 degrees C), in which part of the phosphodiesters has been replaced by methylphosphonates (to ensure stability against nucleases), were prepared on a DNA synthesizer. The oligonucleotides were further derivatized via a uridine moiety at their 5'-end in such a way that radiolabeling or conjugation with antibodies could be accomplished. Both a murine IgG (anti-hCG) and the human anti-tumor IgM 16.88 were conjugated with one to three oligonucleotides via the heterobifunctional cross-linker SMCC. Incubation of these immunoconjugates with the radiolabeled antisense DNA revealed specific hybridization with the antibody-linked oligonucleotides. Antigen binding studies performed with antigen-coated matrices showed that the immunoreactivity of the antibody-DNA conjugate is preserved. Moreover, it is demonstrated that the radiolabeled DNA is still capable of hybridizing selectively with the oligonucleotides of the immunoconjugate, when the latter is bound to its antigen.

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1. The biotransformation and excretion of the antidepressant mianserin were studied after oral administration of the labelled drug to rats, mice, rabbits, guinea pigs and humans. Mianserin was well absorbed and almost completely metabolized in all five species. 2. Major metabolic pathways of mianserin were p-oxidation of the N-substituted aromatic ring followed by conjugation, and oxidation and demethylation of the N-methyl moiety, followed by conjugation. Direct conjugation of the N-methyl moiety was observed as a metabolic pathway specific for man. 3. Conjugated metabolites were isolated by h.p.l.c. and identified by 1H-n.m.r. and FAB spectrometry. Novel metabolites such as an N-O-glucuronide in the guinea pig and an N-sulphonate in rat and guinea pig, were identified using these techniques. A quaternary N-glucuronide was found only in man.

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The metabolism of trans-5-chloro-2-methyl-2,3,3a,12b-tetrahydro-1 H-dibenz[2,3:6,7]oxepinol [4,5-c]pyrrolidine maleate (Org 5222) labelled with [3H] or [14C] was investigated in Wistar rats. Metabolites were identified by mass spectrometry, 13C- and 1H-NMR analysis, IR spectroscopy and, wherever possible, by comparison with authentic reference compounds. The metabolites found in plasma, bile, faeces and urine revealed the processes of metabolism in which Org 5222 underwent oxidation to yield an N-oxide existing in two diastereoisomeric forms, or N-demethylation to yield a demethyl metabolite. A novel metabolite was found in bile, viz. a carbamate glucuronide, formed from an intermediate carbamic acid, derived from the addition of CO2 to the demethyl metabolite.

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1. Methods for the synthesis of drug conjugates with sulphuric acid have been reviewed. 2. Some analytical methods are presented for the analysis of sulphate conjugates. 3. The synthesis of several types of N, O and C beta-D-glucuronides is reviewed. Different beta-coupling reactions of protected glucuronides are presented. 4. Application of n.m.r. and mass spectrometry to the analysis of beta-D-glucuronides is discussed.

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For the identification of intact underivatized drug conjugates, the mass spectrometric technique of choice is fast atom bombardment (FAB); the combined use of both positive and negative ion FAB usually provides information on the molecular mass and nature of conjugates under study, while the number of exchangeable hydrogen atoms can be determined using trideuterated glycerol as the FAB-matrix. Electron impact and desorption chemical ionization spectra can be used to study the aglycone part of the conjugated metabolites. With this approach metabolites conjugated with glucuronic acid, sulphuric acid and amino acids have been identified. The identification was supported by analysis of reference compounds, prepared by chemical synthesis. The examples given are selected from current metabolism studies on drug candidates in development within Organon's research.

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An assay has been developed and validated for the routine monitoring of vecuronium in plasma. It consists of solid-phase extraction using C18 disposables as sample pre-treatment, high-performance liquid chromatography and post-column ion-pair extraction with fluorimetric detection. The fluorescent anion 9,10-dimethoxyanthracene-2-sulphonate (DAS) is used as the counter ion. The detection limit is ca. 5 ng/ml in plasma with a signal-to-noise ratio of 10. The assay is also applicable for monitoring vecuronium and its potential metabolites in other biological media, e.g., urine, bile and tissue (liver, kidney) homogenates.

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The uptake and distribution of several 4-(substituted amino)-iodoquinolines, 131I-labelled, and of 67Ga-citrate were investigated in Syrian golden hamsters with Greene melanomas. Because of its uptake in the melanoma (tumour/eye ratio 8.9), 4-(dimethylamino-ethylamino)-7-iodoquinoline (NM113) was selected for further investigations as a possible radiopharmaceutical for ocular scintigraphy when labelled with 123I. High uptake of NM113 turned out to be incidental and could not be favourably influenced by several pharmacological pretreatments. Affinity for melanin, as shown in in vitro experiments, unfavourably influences NM113 tumour-to-eye ratios. In comparison with 67Ga-citrate, the latter, with a high tumour-to-eye ratio (44.8 after 24 h) is more promising in patient studies in which NM113 123I was not.

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The incorporation of [2-14C]-2-thiouracil and a series of [125I]-5-iodo-2-thiouracils ([125I]ISUra(s)) into cultured Greene hamster melanoma cells was determined in order to establish their properties as false precursors in the melanin-biosynthetic pathway. The cold trichloroacetic acid-precipitable incorporation of [2-14C]-2-thiouracil as well as [125I]ISUra into melanoma cells after a 24- to 48-hr labeling period proved to be completely tyrosinase dependent (more than 99.5% inhibition could be achieved by 0.5 mM phenylthiourea). [125I]ISUra incorporation was 3-fold higher than was [2-14C]-2-thiouracil incorporation and was enhanced by 1 mM theophylline treatment. [125I]ISUra incorporation into hamster, rabbit, and human melanoma cells showed a linear relationship with cell melanin content. Methylation of the sulfur completely prevented the incorporation, while propylation but not methylation at position 6 resulted in lower incorporation. [125I]ISUra proved to be a marker for melanogenesis and may be useful in studies on the differentiation of cultured melanoma cells.

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The tissue distribution of a number of 5-[131I]-iodo-2-thiouracil derivatives was measured in Syrian golden hamsters with Greene melanoma. These compounds were rapidly (in less than 1 h) distributed in all tissues, while in most tissues fast elimination (T1/2 1-3 h) was observed. Because of retention of the 131I activity in the tumour, high tumour/non-tumour tissue ratios were found (e.g. tumour/eye 2.3-10, tumour/skin 1.5-3) suggesting that some of these compounds might be used as melanoma delineating agents, when labelled with 123I.

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