Formation and biologic role of polyoma virus-antibody complexes. A critical role for complement.

Interaction of polyoma virus, specific antibody, and complement has been studied. Firm evidence has been gathered that C1 through C3 and not C5 through C9 enhance neutralization of virus-antiviral antibody (V-Ab) complexes. C enhancement of neutralization occurs primarily by agglutination of V-Ab complexes and not by virion lysis or attachment of large protein molecules to the V-Ab complex. In this model, binding of C1, 4, 2, 3 to the V-Ab complex may explain why some viruses concentrate in or infect certain cells bearing C3 receptors such as B lymphocytes, macrophages, and monocytes.

Chloroxymorphamine, and opioid receptor site-directed alkylating agent having narcotic agonist activity.

Chloroxymorphamine, the 6beta-N,N-bis(2-chloroethyl) derivative of oxymorphone, is a potent nonequilibrium narcotic agonist in the longitudinal muscle preparation of guinea pig ileum. The corresponding naltrexone analog,chlornaltrexamine, is a potent nonequilibrium antagonist of morphine. These receptor sitedirected alkylating agents possess considerable potenial as pharmacologic and biochemical probes of apoid receptors.

Practical considerations in the development of a human cancer vaccine.

Factors involved in the development of a human oncornavirus vaccine are discussed. The isolation and purification of subviral gp69/71 antigenic components enhance the feasibility of developing safe vaccine. The recent isolation of a C-type virus (the HL-23) from a human leukemic patient and its similarity to the simian sarcoma virus presents us with a unique opportunity to test the safety and potency of a vaccine in nonhuman primates.

Etiology and treatment of chemotherapeutic agent extravasation injuries: a review.

While extravasation from intravenous (IV) lines is common and usually benign, leakage of certain drugs can cause severe skin ulceration. These ulcerogenic drugs can be conveniently divided into two categories, depending on whether they bind to DNA. Chemotherapeutic agents such as doxorubicin, which bind to DNA, are especially prone to cause severe extravasation skin ulcers. These ulcers are often chronic and progressive. Neither clinical nor experimental studies have shown an antidote to doxorubicin extravasation, which is best prevented by careful technique. If extravasation is suspected, the infusion should be immediately stopped. In the event of extravasation, elevation and ice are the currently recommended treatment. While small ulcerations may on occasion heal, large ulcerations require surgical excision for relief of pain and salvage of underlying tissues.

An in situ procedure for the biopsy of pressure-wrapped hypertrophic scars.

A plexiglass device can be incorporated in the therapeutic elastic wrapping of hypertrophic scars. The removal of a small access port on this device permits the exposure of the underlying tissue for full-thickness biopsy without the necessity of first removing the pressure wrapping. The ultrastructural detail of tissues biopsied in this manner more readily reflects the in situ microarchitecture of the pressure-wrapped skin and/or scars.

Inhibition of substance P release from spinal cord tissue after pretreatment with capsaicin does not mediate the antinociceptive effect of capsaicin in adult mice.

Substance P (SP) is released from primary afferent fibers in response to nociceptive stimuli. Capsaicin, which produces an initial hyperalgesic response followed by persistent antinociception, also elicits release of SP from primary afferent fibers. Capsaicin pretreatment decreases the content and release of SP from primary afferent fibers. This effect on SP has been hypothesized to mediate the antinociceptive effect of capsaicin. To test this hypothesis, mice were injected intrathecally (i.t.) with antinociceptive doses of capsaicin or SP(1-7) before superfusion of spinal cord tissue with 3 microM capsaicin 24, 48, 96 or 168 h later. N-terminal metabolic fragments of SP that accumulate after capsaicin-induced SP release and are involved in the antinociceptive effect of capsaicin, were also tested. Like capsaicin SP(1-3), SP(1-4) and SP(1-7) were each antinociceptive when injected 24 h before nociceptive testing. However, at this time there was no decrease in capsaicin-evoked release of SP in tissue from capsaicin- and SP(1-7)-pretreated animals compared to those injected with vehicle. In contrast, capsaicin-evoked SP release decreased significantly in tissue from mice pretreated with capsaicin or SP(1-7) 48 h prior to testing. D-Substance P(1-7), which prevents antinociception, blocked capsaicin- and SP(1-7)-induced decreases in SP release, indicating that these effects are mediated by SP N-terminal activity. Total spinal cord content of SP did not differ amongst treatment groups. These data indicate that antinociception does not appear to depend on decreases in SP release or content as antinociception precedes decreases in SP release.

Relationship between analgetic ED50 dose and time-course brain levels N-alkylnormeperidine homologues.

The brain levels of meperidine and three N-alkyl homologues were determined at equal analgetic iv doses in mice. The relative levels of the four compounds in brain were found to be closely proportional to their ED50 doses even though the compounds exhibit a wide range in partition coefficient and metabolic N-dealkylation. While lipid solubility and metabolism are undoubtedly important factors in the overall time-course brain levels, it appears that during the period of analgetic measurement (5-60 min after injection) these factors do not profoundly affect the relative brain levels because peak uptake occurs within the first 5 min after administration of the homologues. As a consequence, the observed ED50 potencies appear to provide a fair approximation of the relative receptor affinities of the four homologues. N-Dealkylation was observed as a major metabolic transformation by mouse liver in vivo for all four compounds, and the extent of this N-dealkylation was found to directly correspond to the rates of N-dealkylation by mouse liver homogenate seen in an earlier study.

Synthesis of 7-arylmorphinans. Probing the "address" requirements for selectivity at opioid delta receptors.

Through arylation of 6-keto opiates with diaryliodonium iodide, a series of 7-aryl opiates (3-8) have been prepared in an effort to investigate the effect of conformational mobility of the delta "address" moiety on opioid agonist and antagonist potencies. Evaluation of the ligands in the mouse vas deferens and guinea pig ileum preparations revealed that they were less potent and less selective than the conformationally constrained ligands, naltrindole (1, NTI) and 7-(spiroindanyl)oxymorphone (2, SIOM), at delta opioid receptors. It is concluded that the coplanarity of the address moiety with the C ring of the morphinan structure enhances delta antagonist potency and selectivity.

7-spirobenzocyclohexyl derivatives of naltrexone, oxymorphone, and hydromorphone as selective opioid receptor ligands.

On the basis of previous structure-activity studies of the highly potent and selective delta-opioid receptor antagonist naltrindole (1) and the spiroindanyl analogues 2 and 3, we have synthesized epimeric pairs of spirobenzocyclohexyl derivatives of naltrexone, oxymorphone, and hydromorphone (4-9). Pharmacologic evaluation in smooth muscle assays has revealed that the oxymorphone derivatives (6, 7) are delta-selective agonists and possess receptor binding profiles that are consistent with their agonist activity. It is proposed that the spirobenzocyclohexyl group of 6 and 7 orients its benzene moiety orthogonally with respect to the C ring of the opiate in a manner similar to that of the spiroindanyl analogue 3. It is suggested that this orthogonal orientation serves as an "address" to facilitate activation of delta receptors. The finding that the hydromorphone analogues (8, 9) were full mu agonists and exhibited only partial delta agonist activity suggests that the 14-hydroxyl group also contributes to the delta agonist activity. The naltrexone derivatives (4, 5) were mu-selective antagonists and exhibited relatively weak delta antagonist activity. However, the binding data indicated a very high-affinity delta-selective binding profile that was not consistent with the pharmacology. This study illustrates the differential contributions of the delta "address" to agonist and antagonist activity and supports the idea of different recognition sites for interaction of agonist and antagonist ligands with delta-opioid receptors.

Possible contribution of a glutathione conjugate to the long-duration action of beta-funaltrexamine.

The fumaramate derivative of naltrexone, beta-funaltrexamine (beta-FNA), is a highly selective long-lasting mu opioid receptor antagonist that is active both in vitro and in vivo, presumably as a result of covalent binding to a mu receptor-based sulfhydryl group. Glutathione, which occurs in significant levels in brain and liver, was found to undergo a Michael-type reaction with beta-FNA in the test tube to give a stable conjugate 3 which occurred as an isomeric mixture. When tested in the GPI and MVD smooth muscle preparations, 3 was found to possess one-tenth the agonist activity of beta-FNA is both tissues, but showed no irreversible antagonist activity. The same result was found for the cysteine conjugate 4, except for some irreversible antagonism in the MVD. Both conjugates antagonize the antinociceptive effect of morphine in the mouse radiant heat tail-flick assay on icv administration. This antagonism persisted and actually increased over 24 h and generally paralleled the duration profile of beta-FNA. On sc administration, beta-FNA and 3 showed similar duration of antagonistic effect, while 4 exhibited only marginal activity at the early time interval. When the compounds are compared by the dose to produce equivalent antagonism, beta-FNA and 3 appeared equally effective and accessible by either route, whereas 4 showed a large difference between the two routes. It is possible that the ultra-long antagonism of the conjugates may result from their enzymatic conversion to beta-FNA in the central nervous system in view of the fact that conjugate 5, which cannot be converted to beta-FNA, did not produce antagonism of long duration in vivo. Alternatively, the protracted antagonism could arise from sequestration of 3 and 4 in tissue compartments that interface with mu opioid receptors.

Allylprodine analogues as receptor probes. Evidence that phenolic and nonphenolic ligands interact with different subsites on identical opioid receptors.

The m-hydroxy analogues of allylprodine and related structures have been synthesized and tested for narcotic agonist and antagonist activity on the electrically stimulated guinea pig ileum and by the hot-plate procedure in mice. It has been found that m-hydroxyallylprodine (alpha-2) is neither an agonist nor antagonist. Other phenolic congeners similarly have little or no activity. The fact that these results are in dramatic contrast with the structure-activity profile of morphine and closely related opiates has led to the proposal that the interaction of morphine and allylprodine (alpha-1) with the mu opioid receptor differs. This difference is postulated to arise from the recognition of the aromatic groups of morphine and alpha-1 by different aromatic-binding subsites of the receptor. These subsites are suggested to be identical with those which recognize the aromatic rings of the Tyr1 and Phe4 of the enkephalins and endorphins. A receptor model consistent with these results is proposed.

Crystal structures of alpha- and beta-funaltrexamine: conformational requirement of the fumaramate moiety in the irreversible blockage of mu opioid receptors.

alpha- and beta-funaltrexamine (alpha- and beta-FNA, 1a and 1b) are naltrexone derivatives differing only in chirality at C(6). Both epimers bind to mu opioid receptors in GPI and MVD preparations, but only the beta-epimer irreversibly blocks these receptors in both preparations. In an effort to investigate the reasons for this difference, we have determined the molecular structures of 1a and 1b by X-ray diffraction techniques. The two epimers have almost identical conformations in the fused ring system except for ring C, which is observed in a twist-boat conformation in alpha-FNA and a chair in beta-FNA. As a result the electrophilic fumaramate moieties are equatorial in both structures and orthogonal to one another when the fused rings are superimposed. In the crystal structure of beta-FNA there is a close intermolecular contact between a phenolic oxygen and the fumaramate double bond that can serve as a model for nucleophilic attack on the fumaramate group. When 1a and 1b are superimposed, the fumaramate double bond of 1a is more than 2 A away from that in its epimer 1b and in the wrong orientation for nucleophilic attack from the proposed direction to take place. The results of this study are consistent with a model that postulates the involvement of two consecutive recognition steps leading to the irreversible blockage by beta-FNA (Sayre, L. M.; Larson, D. L.; Fries, D. S.; Takemori, A. E.; Portoghese, P. S. J. Med. Chem. 1983, 26, 1229) and underscores the importance of the second recognition step in conferring selectivity in the Michael addition of a nucleophile to the fumaramate group.

Synthesis and opioid antagonist potencies of naltrexamine bivalent ligands with conformationally restricted spacers.

Bivalent ligands 1-4 with naltrexamine pharmacophores and spacers of different lengths containing a fumaryl moiety were synthesized and evaluated for mu and kappa opioid antagonist activity on the electrically stimulated guinea pig ileal longitudinal muscle (GPI). The fumaryl moiety was incorporated into the spacer in order to determine the effect of conformational restriction of the spacer on the relationship between spacer length and opioid antagonist potency. While it was found that the fumaryl and succinyl series (11) possessed a very similar structure-potency profile with respect to antagonism at mu opioid receptors, the interaction of these two series at kappa receptors differed substantially from one another. This difference was manifested by the longer spacer requirement for peak kappa antagonist potency in the fumaryl relative to the succinyl series. It is concluded that the conformational restriction imposed by the fumaryl group in a short spacer (n = 0) prevents effective interaction of both pharmacophores with vicinal recognition sites of the kappa receptor system; as the spacer is lengthened (n = 2) and becomes more flexible, the simultaneous occupation of vicinal recognition sites occurs with greater facility.

Persistent binding of fatty acyl derivatives of naltrexamine to opioid receptors.

A series of fatty acid derivatives of naltrexamine and naltrexhydrazine were synthesized and evaluated for their persistent binding to opioid receptors. Members of this series were found to require greater than five washes for removal from mouse brain membranes when the fatty acyl chain was saturated. The presence of unsaturation in the fatty acyl groups enhanced the persistent binding. In this regard the persistent binding increased as a function of the number of double bonds, with the unsaturated congeners requiring greater than 10 washes for removal of the ligand from the membranes. The results of this study are consistent with the apparently important role of polyunsaturated fatty acids in the binding of ligands to opioid receptors.

Opioid agonist and antagonist activities of morphindoles related to naltrindole.

A series of naltrindole-related ligands (4-10) with an N-methyl,N-phenethyl,N-cinnamyl, or an unsubstituted basic nitrogen were synthesized and tested for opioid agonist and antagonist activity in smooth muscle preparations and in mice. The nor compounds (4 and 6) and the phenethyl derivatives (5 and 8) displayed full agonist activity (IC50 = 85-179 nM) in the mouse vas deferens preparation (MVD) while the other members of the series exhibited partial agonist or weak antagonist activity. In the guinea pig ileum preparation (GPI), all compounds except 8 were partial agonists. The ligands that were evaluated in mice were found to produce antinociception that was not selectively mediated via delta opioid receptors. However, two of these ligands (4 and 5) appeared to be delta-selective opioid receptor antagonists at subthreshold doses for antinociception. The finding that all of the compounds bind selectively to delta opioid receptors in guinea pig brain membranes together with the in vitro pharmacology and in vivo antagonist studies suggests that the lack of delta agonist selectivity in vivo may be due to a number of factors, including a basic difference between the delta receptor system in the MVD and in the mouse brain. Further, it is suggested that the constellation of message and address components in the morphindole nucleus may tend to stabilize delta receptors in the brain in antagonist state.

7-Spiroindanyl derivatives of naltrexone and oxymorphone as selective ligands for delta opioid receptors.

A series consisting of spiroindanyl (5-7), benzospiroindanyl (8-10), and spiroperinaphthyl (11) derivatives of naltrexone and oxymorphone were synthesized in order to investigate the role of an orthogonal-oriented "address" for delta opioid receptors. All of the ligands exhibited a preference for delta receptors in vitro. The 7-benzospiroindanyl derivative 8 (BSINTX) was the most selective delta opioid receptor antagonist in vitro. In mice BSINTX antagonized the delta 1-selective agonist, [D-Pen2,D-Pen5]enkephalin without significantly affecting the antinociceptive potency of delta 2, mu, and kappa agonists. The results of this study are consistent with an orthogonally-oriented address favoring delta 1 activity.

Synthesis and pharmacologic characterization of an alkylating analogue (chlornaltrexamine) of naltrexone with ultralong-lasting narcotic antagonist properties.

Chlornaltrexamine (CNA) produces ultralong-lasting (3--6 days) narcotic antagonism in mice and persistent stereospecific binding to rat-brain homogenate. Protection studies in mice suggest that CNA mediates its narcotic antagonist effects by interacting with the same receptors that are occupied by naloxone. A single icv dose of CNA also has been found to inhibit the development of physical dependence in mice for at least 3 days. These studies suggest that CNA exerts its sustained effects by selective covalent association with opioid receptors.

Binaltorphimine-related bivalent ligands and their kappa opioid receptor antagonist selectivity.

In an effort to develop selective antagonists for kappa opioid receptors, bivalent ligands that contain opioid antagonist pharmacophores derived from naltrexone or other morphinans were synthesized and tested on the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. The minimum requirements for kappa selectivity are at least one free phenolic OH group and one N-cyclopropyl or N-ally substituent. Several compounds (3, 8, 10) with kappa selectivity as good as or better than norbinaltorphimine (nor-BNI, 2) were discovered. The structure-activity relationship revealed that the pyrrole ring functions strictly as a spacer and does not contribute to kappa selectivity. The pharmacologic data suggest that only one antagonist pharmacophore may be required for kappa selectivity and that the other morphinan portion of the molecule confers selectivity by interacting with a unique subsite proximal to the antagonist pharmacophore recognition locus.

Importance of C-6 chirality in conferring irreversible opioid antagonism to naltrexone-derived affinity labels.

A series of five epimeric pairs of naltrexone derivatives that contain an electrophilic substituent at the 6 alpha- or 6 beta-position was synthesized and tested on the guinea pig ileal longitudinal muscle (GPI) and mouse vas deferens (MVD) preparations in order to determine if the orientation of the electrophile is important for covalent bonding to opioid receptors. In the GPI all compounds were pharmacologically active as reversible agonists, but only the 6 beta-isomers of the fumaramate ester 2b (beta-FNA) and isothiocyanate 6b exhibited covalent reactivity, involving a selective irreversible antagonism of the mu agonist, morphine, without affecting kappa agonists. The 6 alpha-isomer 2a (alpha-FNA) was itself nonalkylating but was able to protect the GPI against alkylation by its epimer, beta-FNA, indicating that the two epimers bind to the same receptor. These results suggest that the proper orientation of the electrophilic substituent is required for covalent bonding with a proximal nucleophile in the case of mu receptor blockade. Moreover, the lack of covalent bonding to kappa receptors by these or other ligands in this series indicates the possible absence of sufficiently reactive nucleophiles on this recognition site. In the MVD, 2b, but not 2a, irreversibly antagonized morphine (as in GPI), whereas neither epimer exhibited irreversible antagonism toward the delta agonist, [D-Ala2,D-Leu5]enkephalin (DADLE). In contrast, both of the isothiocyanate epimers (6a,b) irreversibly blocked mu and delta receptors. Evidence suggesting differences between mu receptors in the MVD and GPI was obtained with the beta-iodoacetamide 5b, which was an irreversible blocker of morphine only in the MVD. When analyzed together with those of previous studies with the nitrogen mustard analogues, alpha- and beta-chlornaltrexamine, the data suggest that the receptor-alkylating ability of each isomer in an epimeric pair differs most when the electrophile possesses a narrow spectrum of reactivity.

Activity of N-methyl-alpha- and -beta-funaltrexamine at opioid receptors.

The N-methyl analogues (2a, 2b) of the nonequilibrium mu opioid receptor antagonist beta-funaltrexamine (1b) were synthesized and evaluated in the guinea pig ileum preparation (GPI). These analogues are highly potent, reversible opioid agonists and possess no nonequilibrium antagonist activity. The ineffectiveness of 2b in protecting against irreversible blockage of mu opioid receptors by 1b and the fivefold lower reactivity of 2b with cysteine suggest that N-methyl substitution adversely affects both the first and second recognition steps that are essential for effective covalent blockage of opioid receptors.