Federico Spreafico

Polymer Conjugates Pharmacokinetic Considerations for Design and Development

SummarySoluble polymer conjugates have only recently been introduced into clinical practice. They can be subdivided into 2 main categories: polymer-protein conjugates, so far the most widely studied; and polymer-drug conjugates, particularly those containing conventional antitumour agents, that are still at an early stage of development. Polymer conjugation can be used to alter the biodistribution, elimination and rate of metabolism of covalently bound drugs. In the case of protein adducts, polymer conjugation prolongs the protein plasma elimination half-life (5- to 500-fold increases in elimination half-life have been reported), reduces proteolytic degradation and has the added benefit of reducing immunogenicity. Cellular uptake of low molecular weight drugs covalently bound to polymeric carriers is restricted to the endocytic route. Thus, the organ and subcellular distribution of the drug can be modified. Cellular uptake has been used to facilitate drug targeting and decreased toxicity.In this review, the theoretical rationale for polymer conjugation is described, as is the limited clinical pharmacokinetic experience with polymer conjugates. As an alteration of the pharmacokinetic profile of a drug was one of the underlying arguments for creation of polymeric conjugates, more clinical pharmacokinetic studies are urgently needed to permit the validation of appropriate pharmacokinetic models that can be used in the future to assist in the optimisation of clinical protocols, and improved conjugate design.

Reduced cardiotoxicity of doxorubicin given in the form ofN-(2-hydroxypropyl) methacrylamide conjugates: an experimental study in the rat

SummaryA rat model was used to evaluate the general acute toxicity and the late cardiotoxicity of 4 mg/kg doxorubicin (DOX) given either as free drug or in the form of threeN-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates. In these HPMA copolymers, DOX was covalently bound via peptide linkages that were either non-biodegradable (Gly-Gly) or degradable by lysosomal proteinases (Gly-Phe-Leu-Gly). In addition, one biodegradable conjugate containing galactosamine was used; this residue was targeted to the liver. Over the first 3 weeks after the i.v. administration of free and polymer-bound DOX, all animals showed a transient reduction in body weight. However, the maximal reduction in body weight seen in animals that received polymer-bound DOX (4 mg/kg) was significantly lower than that observed in those that received free DOX (4 mg/kg) or a mixture of the unmodified parent HPMA copolymer and free DOX (4 mg/kg;P<0.01). Throughout the study (20 weeks), deaths related to cardiotoxicity were observed only in animals that received either free DOX or the mixture of HPMA copolymer and free DOX; in these cases, histological investigations revealed marked changes in the heart that were consistent with DOX-induced cardiotoxicity. Sequential measurements of cardiac output in surviving animals that received either free DOX or the mixture of HPMA copolymer and free DOX showed a reduction of ≈30% in function beginning at the 4th week after drug administration. The heart rate in these animals was ≈12% lower than that measured in age-matched control rats (P<0.05). Animals that were given the HPMA copolymer conjugates containing DOX exhibited no significant change in cardiac output throughout the study (P<0.05). In addition, no significant histological change was observed in the hearts of animals that received DOX in the form of HPMA copolymer conjugates and were killed at the end of the study. However, these animals had shown a significant increase in heart rate beginning at 8 weeks after drug administration (P<0.01). This study demonstrates that covalent binding of DOX to HPMA copolymer conjugates via both stable and biodegradable peptidyl linkages considerably reduces both the general acute toxicity and the late cardiotoxicity of DOX in the rat and could offer the potential for improving the therapeutic index in the clinical application of DOX.

Enhanced xanthine oxidase activity in mice treated with interferon and interferon inducers

Administration to mice of either interferon (IFN) or IFN inducers resulted in a marked increase of xanthine oxidase (XO) activity in different organs. Dose response studies revealed that serum XO was increased by administration of polyinosylic-polycyticylic acid (poly I-C) at doses as low as 0.1 mg/kg. In view of the well known ability of XO to generate superoxide radicals it is suggested that its induction might play a role in several biological effects of IFN.

Inhibition of human natural killer activity by cyclosporin A

SUMMARY The effect of in vitro exposure to cyclosporin A on human natural killer (NK) activity was investigated using peripheral blood lymphocytes as effectors and 51Cr-labeled K562 cells as targets. After preincubation of the effectors with the drug for 20 hr, a dose-dependent reduction of NK activity was observed at concentrations ranging from 0.1 to 10 jug/ml. Exposure to the drug during the 20-hr preincubation and the 4-hr 51Cr-release assay resulted in greater impairment of NK cytotoxicity than preincubation alone, whereas the drug, present only during the assay, had no effect on cytolytic activity. Inhibition of NK activity required a minimal exposure time to the drug of 8 hr. The inhibitory effect of cyclosporin A on NK activity was rapidly reversible as, after a 20-hr incubation with 10 μg/ml, complete recovery of cytotoxicity was already observed after a further 4-hr culture in medium. Partially purified human fibro-blast interferon augmented NK activity even in the presence of high concentrations (10 μg/ml) of cyclosporin A.

Characterization of tumor lines derived from spontaneous metastases of a transplanted murine sarcoma

Abstract Cell lines were obtained from nine individual spontaneous metastases of the murine mFS6 sarcoma by s.c. inoculation of lung deposits in syngeneic mice. When inoculated i.m., two lines (M4 and M7) from metastases had greater, and two (M8 and M9) had less metastasizing capacity than the primary mFS6 sarcoma. The remaining cell lines gave spontaneous metastases similar in incidence, number and weight to the primary mFS6 tumor. When inoculated i.v., M2 and M4 gave consistently more lung deposits than mFS6 cells, whereas cells from M1, M5, M6 and M8 produced fewer artificial metastases than the primary tumor. Eight individual lung metastases were disaggregated and tested for metastasizing capacity immediately after isolation from the lung parenchyma: spontaneous and artificial metastases from metastatic cells were similar to the primary tumor, except for one secondary which was less metastatic. The immunological characteristics of cell lines from metastases were studied by in vivo immunization and challenge. Seven lines from metastases and the primary tumor had weak cross-reacting transplantation antigens whereas two lines (M3 and M4) had no demonstrable antigenicity. Thus, cell lines from individual spontaneous metastases are heterogeneous in many respects, including metastasizing capacity. However in this model metastasis does not appear to be the expression of strong selection of variant cells with increased metastatic potential.

Characterization of the immunostimulants levamisole and tetramisole.

Abstract The immunostimulatory activity of tetramisole and its levo isomer levamisole, has been investigated in tumorous and non-tumorous experimental animals. These compounds were shown to raise the anti-SRBC response in normal and aged mice and to increase the incidence, severity and duration of allergic encephalomyelitis in rats. Treatments with levamisole were capable of increasing survival and inducing cures in L1210 leukemia-bearing mice when administered in conjunction with irradiated tumor cells. Increased anti-leukemia resistance to subsequent tumor challenges were also observed in animals primed with inactivated tumor cells and injected with levamisole. Single doses of these chemicals could also reduce the number of lung metastases in Lewis lung carcinoma bearing C57B1 6 mice whereas significant life-span prolongation and cures were seen in this system when these agents were employed in a combined chemoimmunotherapeutic approach.

Enantiomeric cisplatin analogues: an investigation on their activity towards tumors in mice

Abstract A series of enantiomeric cisplatin analogues of formula [diamPtCl2] (diam = chiral chelating diamine) and the corresponding sulfato derivatives was prepared and tested for activity against tumors in mice, particularly P 388 leukemia. The configuration of the diamines has practically no influence on the antitumor activity. The effects of the leaving group and of the nature of the diamines are briefly discussed.

The immunomodulatory activity of the plant proteins Momordica Charantia inhibitor and Pokeweed antiviral protein

The immunological activity of Momordica Charantia inhibitor (MCI) and of Pokeweed antiviral protein (PAP-S), 30,000 daltons plant proteins possessing close similarity to Ricin A chain as inhibitor of protein synthesis, was investigated in mice. In vivo, single nontoxic injections of microgram amount of these substances delayed H2-incompatible skin allograft rejection, splenocyte responsiveness to ConA and PHA, but not to LPS, and abrogated the PFC response to a T-dependent (SRBC) antigen while totally sparing that to a T-independent (S III) stimulus. Injection of these substances could also reduce NK cell activity while increasing macrophage-mediated spontaneous cytotoxicity. In vitro, MCI and PAP-S at non-cytotoxic concentrations inhibited lymphoid cell responsiveness to PHA and ConA, but not to LPS, and markedly enhanced macrophage-dependent cytotoxicity.

Attempts at the production of more selective antitumourals: Part I. The antineoplastic activity of cyclophosphazenes linked to the polyamines 1,3-diaminopropane and 1,4-diaminobutane (putrescine)

Abstract In an attempt to design antitumour cyclophosphazenes of improved specificity by linking them to some natural tumour finders, we studied the binding of gem-N 3 P 3 Az 4 Cl 2 to 1,3-diaminopropane and 1,4-diaminobutane (putrescine). Synthesis, mass spectrum and NMR as well as X-ray crystal structures of the two spirocyclic N 3 P 3 Az 4 [HN(CH 2 ) 3,4 NH] derivatives (in which the N 3 P 3 Az 4 active principle is linked to the diamine in a spiro configuration) are described. Results obtained with these compounds in 3 murine tumour systems (L1210 and P388 leukaemias and P815 mastocytoma), showing their potent antineoplastic activity in vivo obtainable at well-tolerated doses, are also described.

The effects of adriamycin and daunomycin on antitumoral immune effector mechanisms in an allogeneic system.

Abstract C3H mice pretreated with adriamycin (AM) 24 hr before transplantation with the allogeneic L1210 leukemia had a significantly longer survival than mice given an equitoxic dose of its analog daunomycin (DM). The effect of the two drugs on cellular and humoral immune effector mechanisms was thus investigated. The longer surviving AM-pretreated tumor allografted mice were found to have higher levels of cell-mediated cytotoxicity (CMC) in the peritoneal cavity, e.g., at the site where tumor growth was actually occurring. On the contrary when spleen CMC was investigated, either no differences were seen or DM-hosts had higher cytotoxic activity. DM was also found to cause a faster impairment than AM of the spleen capacity to mediate antibodydependent cellular cytotoxicity (ADCC). As regards humoral responses, serum complement-dependent and arming activity were absent in both test groups, whereas higher levels of potentiating activity were found in AM pretreated hosts. The possible significance of the lower impairment of antitumoral immune effector mechanisms in giving AM its superior antineoplastic effectiveness is discussed.