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Since permanently high levels of GnRH analogues are necessary to exert direct and/or indirect antitumor effect on mammary tumors, much emphasis was put on the development of retarded-release devices (e.g. microcapsules) for GnRH derivatives. Alternatively, these compounds can be covalently coupled to high-molecular mass carrier molecules for the design of bioconjugates acting as (a) prodrugs producing prolonged release or (b) macromolecular therapeutics. In order to evaluate the feasibility of this approach, a prototype construct has been prepared with a potent GnRH antagonist Ac(D-Trp1,3, D-Cpa2, D-Lys6, D-Ala10)-GnRH (MI-1544). As a carrier, a representative of a new generation of synthetic, biodegradable branched poly[Lys(Xi-DL-Alam)] (XAK) type polypeptides with poly(L-lysine) backbone has been used in which X is an acetylated derivative of glutamic acid (AcEAK). This polyanionic polypeptide with free gamma-carboxyl groups was conjugated to MI-1544, which has only a single amino group at position 6. In this paper, we describe (i) the synthesis and structure (primary structure, conformation) properties of the MI-1544-AcEAK conjugate with a 33% degree of substitution, (ii) the effect of the covalent attachment of MI-1544 to AcEAK on its blood clearance and tissue distribution, and (iii) the hormone-related indirect (ovulation inhibitory) or direct (antiproliferative) antitumor activity of the conjugate studied by in vitro assays. Data obtained with 111In- and 125I-labeled conjugates have demonstrated that in fact the body/blood survival of MI-1544 was prolonged by 1.5-3 times. The direct in vitro antitumor effect of MI-1544 was maintained or even enhanced in the MI-1544-AcEAK conjugate. Furthermore, we have shown that this conjugate was able to antagonize the effect of GnRH in vitro or to act as free MI-1544 both in short- and long-term inhibition of ovulation even after single subcutaneous injection. These data suggest that it is feasible to use a biodegradable polymeric polypeptide for development of a macromolecular therapeutic with GnRH antagonists.

Monocytes and macrophages are engaged at various levels of cellular immune reactivity. In addition to their function in the defensive mechanism directed at infective agents, they also play a basic role in immune complex elimination and antigen handling. Previous experiments revealed that systemic lupus erythematosus (SLE), the main representative of the autoimmune diseases, is associated with impaired monocyte chemotaxis. The endogenous basic tetrapeptide tuftsin and 6 of its analogues were examined in vitro for their stimulating capacity on the chemotactic responsiveness of monocytes derived from patients with SLE. The monocyte migration assay was carried out by a modified Boyden technique and quantified by the leading front distance method and by counting the total distance covered by the monocyte locomotion. Tuftsin and 3 of its analogues significantly increased the defective chemotaxis in SLE. The tetrapeptides effective on chemotaxis also stimulated random migration and phagocytosis of the monocytes, albeit to a lesser extent. Structure-activity relationships, as well as the influence of the clinical stage of the disease were also examined. Experimental evidence leads to a favourable prediction for the immunotherapeutic value of these oligopeptides for the control of infections and the progression of the disease in patients with systemic lupus erythematosus.

A comparative investigation of various polypeptides was carried out in order to elucidate structure-activity correlations. The immunoadjuvant properties of the chemically well-characterized branched polypeptides, poly[Lys-(DL-Alam)] (Lys:Ala = 1:2.95) (AK), poly[Lys-(D-Leui-DL-Alam)] (Lys:Ala:Leu = 1:3.0:0.95) (D-LAK), and poly[Lys-Hisi-DL-Alam)] (Lys:Ala:His = 1:2.95:0.85) (HAK), were investigated. D-LAK and HAK were able to augment the antibody response of BDF inbred mice to immunization with sheep red blood cells (SRBC), as assessed by the hemolytic plaque-forming cell assay, whereas AK had no similar effect. The stimulating effect of D-LAK and HAK was dependent on dose and timing of treatment relative to SRBC immunization. However, the optimal dose levels were lower and the effective dose interval more restricted as compared to the previously described poly[Lys-(Leui-DL-Alam)] (LAK). Like LAK, both HAK and D-LAK were able to compensate for the immunosuppressive effect of the cytotoxic drugs dianhydrogalactitol, vincristine, and 5-fluorouracil, which all have different mechanisms of action, provided that combined treatment by polypeptide and drug was applied repeatedly before the SRBC immunization.

The degree of human serum albumin (HSA) binding for 16 different homopyrimidazole derivatives has been studied. The positions of UV absorption maximum, the corresponding molar extinction coefficients and the binding parameters obtained by equilibrium dialysis experiments at 4 degrees C, in pH 7.35 phosphate buffer, are surveyed. In most cases compounds with high toxicity show low binding tendency. Saturation of the ring system decreases binding, while an increase of the number of double bonds enhances the binding affinity. Important requirements for HSA binding of homopyrimidazole derivatives are the presence of a carbethoxy (or carboxyl) group in position 3 and a C6-methyl group. The highest binding affinity could be demonstrated in the case of MZ 211, an acid-type metabolite of 1,6-dimethyl-3-carbethoxy-4-oxo-6,7,8,9-tetrahydro-4H-pyrido-(1,2-alpha)-pyrimidin-1-ium-methyl-sulphate (MZ 144, Probon). Binding of MZ 144 to chemically modified albumins was studied under standard conditions. Binding studies with acetylated and methylated HSA indicated that the presence of free carboxyl groups is a requirement for binding, while the amino groups play a less important role. More selective chemical modifications of HSA were realised with acetylsalicylic acid, diethylpyrocarbonate (DEP) and o-nitrophenylsulfenyl-chloride (NPS-C1). Modifications with DEP and acetylsalicylic acid result in an increase of affinity in the binding area, probably due to blocking of a particular amino group in the vicinity of the binding site. Histidine residues are believed to be of minor importance in the binding process. When the indole ring is modified with NPS-C1, affinity at the strong binding site is significantly reduced, indicating that the Trp residue must be involved in the binding of MZ 144.

A detailed investigation is reported about the biodegradation of poly[Lys(DL-Alam)], m approximately 3, (AK) the common inside area of a branched polypeptide model system developed by our group over the last decade. Enzymatic hydrolysis was carried out by the exopeptidase aminopeptidase M, or the endopeptidase trypsin, or their mixture. Ion-exchange column chromatography, paper electrophoresis and thin-layer chromatography were utilised to achieve separation of metabolites. Breakdown products were identified by the aid of synthetic oligopeptides representing the potential fragments (DL-Ala2, DL-Ala3, Lys(DL-Alam), m = 1-3). The kinetics and the degree of enzymatic degradation were determined. The ratio of peptide/amino acid amounts in the hydrolysate was found to be 1.07 after 24 h treatment with aminopeptidase M, 3.0 with trypsin and 1.3 with aminopeptidase - trypsin mixture. The overall results indicated that the proteolysis of AK by an aminopeptidase M and trypsin mixture proceeds stepwise at multiple sites on the polypeptide chain. The degradation is significantly retarded as compared to that of alpha- or epsilon-polylysine. A mechanism of degradation is suggested based on the experimental results.

The possibilities offered by a combination of the "latency" and "carrier" principles were studied by joining the phosphoramide mustard group with amino acids. The structures of the resulting derivatives are reminiscent of cyclophosphamide, but they provide feasibility for stereoisomeric modifications and for incorporation into synthetic peptides. Modlel peptides with a different ring system containing cyclic diesters of the N,N-bis(2-chloroethyl)phosphoramidic acid were also constructed. The relative intensities of antitumor activity, toxicity, and antitumor spectrum can be influenced by these molecular manipulations, indicating that the pharmacokinetics, and probably even the enzymatic activation, are markedly dependent upon the overall physiochemical properties and stereochemistry of the molecule. Another approach has been to use larger molecular weight materials as carriers either of the phosphoramide mustard group or the cyclic amino acid derivatives. Both noncovalent and covalent linkages were tried; based on stability properties, conjugates must be regarded as more adequate potential candidates for lysosomotropism.

The binding of 1,6-dimethyl-3-carbethoxy-4-oxo-6,7,8,9-tetrahydro-homopyrimidazolium-methyl-sulfate (MZ 144, Probon) to human serum albumin (HSA) has been studied in vitro. The measurements have been performed mostly by means of the classical equilibrium dialysis method. Initially several controls were run to estimate the binding of drug to Visking membrane, time required for equilibrium and to check the stability of the drug at different pH. In addition binding was studied spectrophotometrically, essentially following the method of Klotz. Treatment of binding data was based on the equation developed by Scatchard. The data obtained do not fit the single-site binding equation but could be resolved by a computer program into two sets of binding sites. At pH 7,35,4 degrees C and using a 1% HSA solution values found for binding parameters were: N1 = 0.4359, k1 = 4077 M(-1), N2 = 1.17, k2 = 424 M(-1). Protein binding is considered to have a strong effect on drug distribution only if the affinity constant for the drug, k, has a value greater than 1 times 10(4). The HSA-MZ 144 interaction is temperature dependent and pH dependent. The percentage bound as a function of total drug concentration was calculated at pH 4.96, 7.35 and 8.5; a considerable increase was observed at pH 8.5.

The immunomodulatory potential of poly-(Lys-[Leu-poly-DL-Ala]) (LAK) (Lys:Ala:Leu = 1:3:0.7) as the first prototype of a series of new branched polypeptides was studied. Circular dichroism spectra indicated a highly ordered structure of LAK at physiological pH. The ability of the polypeptide to stimulate antibody response to immunization with sheep red blood cells (SRBC) when applied in aqueous medium was assessed in BDF1 inbred normal mice by the hemolytic plaque-forming cell and rosette-forming cell (RFC) assays. LAK, similarly to levamisole (LEV), a clinically applied immunomodulator, stimulated the host's humoral immune response in a wide dose interval (0.02-20 mg/kg; optimal dose, 1 mg/kg); however, the number of RFC was not influenced considerably. LAK was not toxic in doses of up to 25 mg/kg. The varied timing of LAK treatment (1 mg/kg i.p.) relative to SRBC immunization resulted in an oscillating potentiation of the immune response. The immunoadjuvant activity elicited by LAK--similarly to LEV--was sufficient to produce effective compensation of immunosuppression induced by the cytotoxic drugs vincristine and dianhydrogalactitol when combined treatments on multiple schedules were applied before SRBC immunization.

A new group of synthetic branched polypeptides was developed to initiate a systematic study of the relationships between the chemical structure (charge, size, primary structure, configuration and conformation), the carrier potential and the antigenic properties of these biodegradable and biocompatible macromolecules. This model system has two main advantages over the previously used ones: (i) the side chains grafted to the poly(L-lysine) backbone are composed of about three DL-Ala and a single chain-terminating amino acid with different absolute configuration and/or identity, and (ii) the conformation of these polypeptides is characterized in solution. The size, charge and inside area of the four molecules selected for this study were identical; however, the identity, the absolute configuration of the chain-terminating amino acids (D-Leu, Leu, Phe or D-Phe) and, in consequence, the conformation of the macromolecules were different. The qualitative and quantitative features of the antibody response induced by the four polypeptides were characterized in inbred mouse strains by IgM and IgG type antibody levels, as well as by isotype distribution and fine specificity of antibodies produced during the primary and memory response. The intensity of the memory response and the characteristics of subclass distribution were dependent on the conformation of the branched polypeptides. These molecules carry at least two types of antigenic determinants. One is ordered to the tetrapeptide side chain, the expression of which proved to be inversely correlated with the backbone-originated helix content of the molecules. The other antigenic determinant corresponds to the common inside area of the polypeptides which is less conformation-dependent and therefore common to all four polypeptides.

Poly(Lys-(Xi-DL-Alam] polypeptides carrying hydrophilic (X = His, Glu, Lys) or hydrophobic (X = Nle, Ile, Phe) amino acid residues and their conjugates with 4-ethoxymethylene-2-phenyl-5(4H)-oxazolone were synthesized. The conformational properties of carrier polypeptides and conjugates were studied by circular dichroism (CD) spectroscopy in the wavelength regions 190-250 and 310-380 nm, with the emphasis on analysis under near physiological conditions. Based on CD studies, it could be demonstrated that the helix-forming capacity appears to be related to the hydrophobic nature of the branch-terminating amino acid of the branched polypeptides. With respect to carrier function, the presence of a coupled derivative of oxazolone at the side chain termini generally promotes the formation of helical secondary structure. The absolute configuration of the side-chain-terminating amino acids was found to be important for the local orientation of the hapten molecule in the conjugates.