Tumor targeting by covalent conjugation of a natural fatty acid to paclitaxel.

Certain natural fatty acids are taken up avidly by tumors for use as biochemical precursors and energy sources. We tested in mice the hypothesis that the conjugation of docosahexaenoic acid (DHA), a natural fatty acid, and an anticancer drug would create a new chemical entity that would target tumors and reduce toxicity to normal tissues. We synthesized DHA-paclitaxel, a 2'-O-acyl conjugate of the natural fatty acid DHA and paclitaxel. The data show that the conjugate possesses increased antitumor activity in mice when compared with paclitaxel. For example, paclitaxel at its optimum dose (20 mg/kg) caused neither complete nor partial regressions in any of 10 mice in a Madison 109 (M109) s.c. lung tumor model, whereas DHA-paclitaxel caused complete regressions that were sustained for 60 days in 4 of 10 mice at 60 mg/kg, 9 of 10 mice at 90 mg/kg, and 10 of 10 mice at the optimum dose of 120 mg/kg. The drug seems to be inactive as a cytotoxic agent until metabolized by cells to an active form. The conjugate is less toxic than paclitaxel, so that 4.4-fold higher molar doses can be delivered to mice. DHA-paclitaxel in rats has a 74-fold lower volume of distribution and a 94-fold lower clearance rate than paclitaxel, suggesting that the drug is primarily confined to the plasma compartment. DHA-paclitaxel is stable in plasma, and high concentrations are maintained in mouse plasma for long times. Tumor targeting of the conjugate was demonstrated by pharmacokinetic studies in M109 tumor-bearing mice, indicating an area under the drug concentration-time curve of DHA-paclitaxel in tumors that is 8-fold higher than paclitaxel at equimolar doses and 57-fold higher at equitoxic doses. At equimolar doses, the tumor area under the drug concentration-time curve of paclitaxel derived from i.v. DHA-paclitaxel is 6-fold higher than for paclitaxel derived from i.v. paclitaxel. Even at 2 weeks after treatment, 700 nM paclitaxel remains in the tumors after DHA-paclitaxel treatment. Low concentrations of DHA-paclitaxel or paclitaxel derived from DHA-paclitaxel accumulate in gastrocnemius muscle; which may be related to the finding that paclitaxel at 20 mg/kg caused hind limb paralysis in nude mice, whereas DHA-paclitaxel caused none, even at doses of 90 or 120 mg/kg. The dose-limiting toxicity in rats is myelosuppression, and, as in the mouse, little DHA-paclitaxel is converted to paclitaxel in plasma. Because DHA-paclitaxel remains in tumors for long times at high concentrations and is slowly converted to cytotoxic paclitaxel, DHA-paclitaxel may kill those slowly cycling or residual tumor cells that eventually come into cycle.

Tumor targeting by conjugation of DHA to paclitaxel.

Targeting an anti-cancer drug to tumors should increase the Area Under the drug concentration-time Curve (AUC) in tumors while decreasing the AUC in normal cells and should therefore increase the therapeutic index of that drug. Anti-tumor drugs typically have half-lives far shorter than the cell cycle transit times of most tumor cells. Tumor targeting, with concomitant long tumor exposure times, will increase the proportion of cells that move into cycle when the drug concentration is high, which should result in more tumor cell killing. In an effort to test that hypothesis, we conjugated a natural fatty acid, docosahexaenoic acid (DHA), through an ester bond to the paclitaxel 2'-oxygen. The resulting paclitaxel fatty acid conjugate (DHA-paclitaxel) does not assemble microtubules and is non-toxic. In the M109 mouse tumor model, DHA-paclitaxel is less toxic than paclitaxel and cures 10/10 tumored animals, whereas paclitaxel cures 0/10. One explanation for the conjugate's greater therapeutic index is that the fatty acid alters the pharmacokinetics of the drug to increase its AUC in tumors and decrease its AUC in normal cells. To test that possibility, we compared the pharmacokinetics of DHA-paclitaxel with paclitaxel in CD2F1 mice bearing approximately 125 mg sc M109 tumors. The mice were injected at zero time with a bolus of either DHA-paclitaxel or paclitaxel formulated in 10% cremophor/10% ethanol/80% saline. Animals were sacrificed as a function of time out to 14 days. Tumors and plasma were frozen and stored. The concentrations of paclitaxel and DHA-paclitaxel were analyzed by LC/MS/MS. The results show that DHA targets paclitaxel to tumors: tumor AUCs are 61-fold higher for DHA-paclitaxel than for paclitaxel at equitoxic doses and eight-fold higher at equimolar doses. Likewise, at equi-toxic doses, the tumor AUCs of paclitaxel derived from i.v. DHA-paclitaxel are 6.1-fold higher than for paclitaxel derived from i.v. paclitaxel. The tumor concentration of paclitaxel derived from i.v. paclitaxel drops rapidly, so that by 16 h it has fallen to the same concentration (2.8 microM) as after an equi-toxic concentration of DHA-paclitaxel. In plasma, paclitaxel AUC after an MTD dose of DHA-paclitaxel is approximately 0.5% of DHA-paclitaxel AUC. Thus, the increase in tumor AUC and the limited plasma AUC of paclitaxel following DHA-paclitaxel administration are consistent with the increase in therapeutic index of DHA-paclitaxel relative to paclitaxel in the M109 mouse tumor model. A phase I clinical study has been completed at The Johns Hopkins Hospital to evaluate the safety of DHA-paclitaxel in patients with a variety of solid tumors. Twenty-one patients have been treated to date. The recommended phase II dose is 1100 mg/m(2), which is equivalent to 4.6 times the maximum approved paclitaxel dose on a molar basis. No alopecia or significant peripheral neuropathy, nausea, or vomiting have been observed. Asymptomatic, transient neutropenia has been the primary side effect. Eleven of 22 evaluable phase I patients transitioned from progressive to stable disease, as assessed by follow-up CT. Significant quality of life improvements have been observed. Thus, DHA-paclitaxel is well tolerated in patients and cures tumors in mice by targeting drug to tumors.

Fatty acid derivatives of clozapine: prolonged antidopaminergic activity of docosahexaenoylclozapine in the rat.

Stable amides of clozapine derived from fatty acids prominent in cerebral tissue might enhance the central activity of clozapine and reduce its exposure to peripheral tissues. Such derivatives might enhance the safety of this unique drug, which is the only agent with securely established superior antipsychotic effectiveness, but with a risk of potentially lethal systemic toxicity. Amide derivatives of clozapine were prepared from structurally varied fatty acid chlorides and evaluated for ability to inhibit behavioral arousal in rat induced by dopamine agonist apomorphine and to induce catalepsy. Their duration-of-action and potency were compared to free clozapine, and concentrations of clozapine were assayed in brain and blood. Selected agents were also evaluated for affinity at dopamine receptors and other potential drug-target sites. Clozapine-N-amides of linoleic, myristic, oleic, and palmitic acids had moderate initial central depressant activity but by 6 h, failed to inhibit arousal induced by apomorphine. However, the docosahexaenoic acid (DHA) derivative was orally bioavailable, 10-times more potent (ED(50) 5.0 micromol/kg) than clozapine itself, and very long-acting (>/= 24 h) against apomorphine, and did not induce catalepsy. DHA itself was inactive behaviorally. Clozapine showed expected dopamine receptor affinities, but DHA-clozapine was inactive at these and other potential target sites. After systemic administration of DHA-clozapine, serum levels of free clozapine were very low, and brain concentrations somewhat lower than after administering clozapine. DHA-clozapine is a long-acting central depressant with powerful and prolonged antidopaminergic activity after oral administration or injection without inducing catalepsy, and it markedly reduced peripheral exposure to free clozapine. It lacked the receptor-affinities shown by clozapine, suggesting that DHA-clozapine may be a precursor of free, pharmacologically active clozapine. Such agents may represent potential antipsychotic drugs with improved central/peripheral distribution, and possibly enhanced safety.

Conformational studies of paclitaxel analogs modified at the C-2' position in hydrophobic and hydrophilic solvent systems.

The conformations of two paclitaxel analogs modified at the C-2' position, 2'-deoxypaclitaxel and 2'-methoxypaclitaxel, were studied in hydrophobic and hydrophilic solvent systems by a combination of NMR spectroscopy, CD measurements, and molecular modeling. Both analogs have hydrophobic and hydrophilic conformations that resemble those of paclitaxel itself in the same media. Since the two have diminished biological activities in a number of bioactivity assays and the hydrogen-bonding capability of the 2'-hydroxyl group has been eliminated, we postulate that this group is involved in hydrogen bonding with tubulin and plays an important role in molecular recognition. The results of this study are in agreement with our earlier report on paclitaxel 2'-acetate, an analog in which the 2'-hydroxyl group hydrogen-bonding capacity has also been eliminated.

NMR and molecular modeling study of the conformations of taxol 2'-acetate in chloroform and aqueous dimethyl sulfoxide solutions.

Taxol 2'-acetate, an analog of the antitumor drug taxol, displays no significant in vitro microtubule polymerization activity, thus underscoring the importance of a free 2'-OH group to the biological activity of taxol. Previous work had suggested that the inactivity of taxol 2'-acetate is not due to steric interference by the acetyl group. The present study examined the conformations of taxol 2'-acetate in deuteriochloroform and (2)H2O-deuteriodimethyl sulfoxide solutions and found them to be essentially the same as the respective conformations adopted by taxol itself. Thus, neither destabilization of an active taxol conformation by the acetyl group nor the formation of an important taxol conformation determining role for the 2'-OH group appears likely. The implication of these findings is that the taxol 2'-OH group interacts directly with a protein residue in the taxol-microtubule complex, perhaps as a hydrogen bond donor.

Relationship between the structure of taxol and other taxanes on induction of tumor necrosis factor-alpha gene expression and cytotoxicity.

Taxol is an antitumor drug with cytotoxic properties that correlate with its microtubule-stabilizing activities. It has been reported that taxol parallels lipopolysaccharide in its effects on the induction of tumor necrosis factor-alpha (TNF-alpha) gene expression in macrophages (C. Bogdan and A. Ding, J. Leukocyte Biol., 52: 119-121, 1992; C. L. Manthey, M. E. Brandes, P. Y. Perera, and S. Vogel, J. Immunol., 149: 2459-2465, 1992; J. M. Carboni, C. Singh, and M. A. Tepper, Natl. Cancer Inst. Monogr., 15: 95-101, 1993). Structure-activity studies using taxol and related taxanes have been done to determine the relationship between the effects of taxol on TNF-alpha gene expression and its cytotoxic and microtubule-stabilizing activities. Using Northern blot analysis, it was found that changes in the structure of taxol that did not alter cytotoxicity did prevent the induction of TNF-alpha gene expression. The data presented in this paper demonstrate that the effects of taxol on TNF-alpha gene expression are distinct from its known cytotoxic properties.

Characterization of two taxol photoaffinity analogues bearing azide and benzophenone-related photoreactive substituents in the A-ring side chain.

Taxol is a structurally novel and clinically effective antitumor drug, which, unlike other antimitotic agents, induces the assembly of tubulin into microtubules. To characterize the binding site(s) of taxol on the microtubule, taxol-based photoaffinity reagents 1 and 2 bearing photoreactive groups on the A-ring side chain were prepared and evaluated. Taxol analogue 1 exhibits better microtubule assembly activity, greater cytotoxicity toward J774.2 cells, and more specific and efficient photolabeling of the beta-subunit of tubulin than does analogue 2. Therefore, it would appear that 1 is the better candidate for further studies aimed at the characterization of the taxol binding site on the microtubule.

3'-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-tubulin.

Taxol possesses an unusual chemical structure, a unique mechanism of action, and demonstrated activity in human malignancies. It is the only antitumor agent that has a binding site on the microtubule polymer. The interaction of Taxol with the microtubule polymer results in the formation of stable bundles of cellular microtubules that are resistant to depolymerization. Although it has become evident that the microtubule, specifically beta-tubulin, is the target for Taxol, no information is available on the binding site for the drug. In this report, we demonstrate that 3'-(p-azidobenzamido)taxol, an analogue with similar biological activities as Taxol, covalently binds to the N-terminal domain of beta-tubulin after irradiation of the microtubule-drug complex. Taxol competes with [3H]3'-(p-azidobenzamido)-taxol binding, suggesting that the photoaffinity analog and Taxol are binding at the same or overlapping sites. Formic acid cleavage of [3H]3'-(p-azidobenzamido)-taxol-photolabeled beta-tubulin and subsequent protein sequence and mass analyses have identified the N-terminal 31 amino acids as the major site for [3H]3'-(p-azidobenzamido)taxol photoincorporation.

Antifungal properties of taxol and various analogues.

The antimitotic agent taxol was tested for toxicity towards fungi from different taxonomic groups and found to be particularly active against oomycete fungi. In germinating zoospore cysts of the oomycete Phytophthora capsici the mechanism of action of taxol was shown to involve inhibition of mitosis, presumably resulting from an effect on microtubules. Various taxol analogues with deleted A-ring C-13 side chain substituents were tested for toxicity towards P. capsici and Aphanomyces cochlioides to provide insight into structural features required for activity. The importance of the side chain was shown by the much lower activity as compared to taxol of analogues lacking all or part of the side chain. The effect of stereochemistry at the C-2' position on fungitoxicity towards oomycetes was similar to that reported previously on mammalian microtubule assembly.

Poststroke depression: neurologic, physiologic, diagnostic, and treatment implications.

Poststroke depression is a serious disorder that can compromise the overall rehabilitative process, including speech-language treatment. To provide patients with the maximum opportunity for recovery and therapeutic gains, speech-language pathologists need to increase their awareness of depression and understand the neurologic, physiologic, diagnostic, and treatment implications of this disorder. This article defines depression and reviews the theories that explain the nature of the disorder as well as methods for diagnosing and treating stroke patients who are clinically depressed.

Biologically active taxol analogues with deleted A-ring side chain substituents and variable C-2' configurations.

Taxol, a potent inhibitor of cell replication, enhances the assembly of tubulin into stable microtubules and promotes the formation of microtubule bundles in cells. In addition to its unique mechanism of action, taxol exhibits unusual promise as an antitumor agent, but its application in cancer chemotherapy is hampered by its limited availability. In order to better define the structure-activity profile of taxol for the design of more accessible drugs and to provide insight into the chemical features of the taxol-microtubule interaction, taxol analogues 3-8, with deleted A-ring side chain substituents and both R and S C-2' configurations, were synthesized from baccatin III through esterification at the hindered 13-hydroxyl. Employing an improved hydroxyl protection strategy, lactate analogues 3 and 4 were prepared with reasonable efficiency owing to their simple side-chain structures, while N-benzoylisoserine analogues 7 and 8 were synthesized through esterification reactions whose rates were enhanced greatly by the participation of the amide functionality. Although less biologically active than taxol, analogues 5-7 were found to promote the polymerization of tubulin and to be cytotoxic; 5 and 6 were considerably more effective than 7, whereas 3, 4, and 8 were least active. Interestingly, tubulin polymerization was sensitive to the C-2' configuration only when the amide substituent was present in the side chain. This observation suggests that the 3'-amide substituent plays an important role in preorganizing the taxol side chain to bind to microtubules.

Predictors of language restitution following stroke: a multivariate analysis.

A consecutive sample of 50 language-impaired patients was evaluated prospectively during the first 3 to 4 months following unilateral left- or right-hemisphere stroke. A multiple logistic linear regression model was used to assess the relative importance of eight predictor variables on the likelihood of language recovery. Those found to be significantly associated with language recovery included age (favoring younger patients) and length of hospital stay (favoring shorter stays). Gender (favoring males), type of stroke (favoring hemorrhages), and side of lesion (favoring right) were only moderate correlates of recovery. Neither race nor history of previous stroke was a significant predictor of language recovery. Multivariate statistical analysis was useful in illuminating the joint relationship between clinical and demographic predictor variables and language recovery.

Characteristics of recovery of drawing ability in left and right brain-damaged patients.

Drawings of a "person" were obtained from 21 patients with left brain damage (LBD) and 13 patients with right brain damage (RBD) at hospital discharge and 1 month postdischarge, corresponding to 2-4 and 6-8 weeks postonset, respectively. Early LBD drawings were simplistic and often incoherent. Most were placed in the upper left quadrant of the page. RBD drawings were scattered, fragmented, and elaborately detailed. Many showed evidence of left-sided neglect. The resolution of drawing disability also differed between the two groups: LBD subjects recovered drawing abilities more rapidly and more completely than RBD subjects. These differences in drawing characteristics and in the resolution of drawing disability were discernable for experienced neuropsychologists.

The labeling problem in aphasia: an illustrative case.

Twenty-five "experts" on neurogenic motor speech disorders participated in a tutorial exercise. Each was given information on M, a patient who had communication difficulties as the result of stroke, and asked to complete a questionnaire about his problem. The information included a detailed case description, an audiotape of M's speech obtained at 4, 9, 13, and 17 days post-stroke, and test results from the Western Aphasia Battery, the Token Test, and a battery for apraxia of speech. The experts were in excellent agreement on M's primary problem, although it was called by seven different names. The experts were in poor agreement on his secondary problem(s), e.g., the presence and type of aphasia and dysarthria. The results suggest that labeling is difficult, even for "experts." Furthermore, the practicing clinician needs to be sensitive to the likelihood of more than one coexisting problem.

An application of exploratory statistical methods to language pathology: analysis of the Western Aphasia Battery's Cortical Quotient in acute stroke patients. Research note.

Two simple exploratory statistical techniques, stem and leaf displays and box plots, are described and applied to the analysis of Western Aphasia Battery Cortical Quotient scores for 111 acute stroke patients. A stem and leaf display highlights the shape of a distribution of data and is useful for quickly ordering and displaying data in the form of a frequency distribution; box plots focus special attention on the tails of a distribution and are valuable for summarizing, analyzing, and comparing group data. These methods were used to determine an internal Cortical Quotient cutoff score to distinguish between normal and cognitively impaired patients. The utility of this Cortical Quotient cutoff score and the relevance of these statistical techniques to other research in communicative disorders are discussed.

Various consequences of subcortical stroke. Prospective study of 16 consecutive cases.

Sixteen consecutive cases of subcortical stroke were studied prospectively and systematically. Behavioral and neurological manifestations of different sides, sites, and causes of lesion were evaluated by means of daily, standard observations during the patient's hospitalization and formal testing at monthly intervals thereafter. Language and cognitive impairments were observed following hemorrhagic and nonhemorrhagic strokes to both the left and right thalami and basal ganglia. The patterns were unlike those characteristic of traditional aphasia syndromes. One patient was asymptomatic; four demonstrated only dysarthria or dysarthria with abnormal affect; and 11 displayed a combination of speech, language, and cognitive deficits. Language skills recovered more rapidly and completely than did cognitive skills. Recovery was most dramatic within the first six to eight weeks after onset.