Synthesis and evaluation of "AZT-HEPT", "AZT-pyridinone", and "ddC-HEPT" conjugates as inhibitors of HIV reverse transcriptase.

To test the concept that HIV reverse transcriptase could be effectively inhibited by "mixed site inhibitors", a series of seven conjugates containing both a nucleoside analogue component (AZT 1, ddC 2) and a nonnucleoside type inhibitor (HEPT analogue 12, pyridinone 27) were synthesized and evaluated for their ability to block HIV replication. The (N-3 and C-5)AZT-HEPT conjugates 15, 22, and 23 displayed 2-5 microM anti-HIV activity, but they had no effect on the replication of HIV-2 or the HIV-1 strain with the Y181C mutation. The (C-5)AZT-pyridinone conjugates 34-37 were found to be inactive. In marked contrast, the ddC-HEPT molecule 26 displayed the same potency (EC(50) = 0.45 microM) against HIV-1 (wild type and the Y181C nevirapine-resistant strain) and HIV-2 in cell culture. No synergistic effect was observed for these bis-substrate inhibitors, suggesting that the two individual inhibitor components in these molecules do not bind simultaneously in their respective sites. Interestingly, however, the results indicate that the AZT-HEPT conjugates and the ddC-HEPT derivative 26 inhibit reverse transcriptase (RT) in an opposite manner. One explanation for this difference is that the former compounds interact preferentially with the hydrophobic pocket in RT, whereas 26 (after supposed triphosphorylation) inhibits RT through binding in the catalytic site.

"Mixed inhibitors" of HIV-reverse transcriptase: synthesis and antiviral activity.

Some "AZT-HEPT" and "ddC-HEPT" conjugates were designed, synthesized and evaluated for their anti-HIV activity.

Efficient synthesis of N-4 alkyl derivatives of 2',3'-dideoxycytidine (ddC).

The C-4 triisopropylphenylsulfonyl (TPS) group of the 2,3-dideoxyuridine derivative 2 is readily displaced in situ by nitrogen nucleophiles forming N-4 substituted ddC in acceptable yields.

Alteration of flow-induced dilatation in mesenteric resistance arteries of L-NAME treated rats and its partial association with induction of cyclo-oxygenase-2.

1. We investigated the response to pressure (myogenic tone) and flow of rat mesenteric resistance arteries cannulated in an arteriograph which allowed the measurement of intraluminal diameter for controlled pressures and flows. Rats were treated for 3 weeks with NG-nitro-L-arginine methyl ester (L-NAME, 50 mg kg-1 day-1) or L-NAME plus the angiotensin I converting enzyme inhibitor (ACEI) quinapril (10 mg kg-1 day-1). 2. Mean blood pressure increased significantly in chronic L-NAME-treated rats (155 +/- 4 mmHg, n = 8, vs control 121 +/- 6 mmHg, n = 10; P < 0.05). L-NAME-treated rats excreted significantly more dinor-6-keto prostaglandin F1 alpha (dinor-6-keto PGF1 alpha), the stable urinary metabolite of prostacyclin, than control rats. The ACEI prevented the rise in blood pressure and the rise in urinary dinor-6-keto PGF1 alpha due to L-NAME. 3. Isolated mesenteric resistance arteries, developed myogenic tone in response to stepwise increases in pressure (42 +/- 6 to 847 +/- 10 mN mm-1, from 25 to 150 mmHg, n = 9). Myogenic tone was not significantly affected by the chronic treatment with L-NAME or L-NAME + ACEI. 4. Flow (100 microliters min-1) significantly attenuated myogenic tone by 50 +/- 6% at 150 mmHg (n = 10). Flow-induced dilatation was significantly attenuated by chronic L-NAME to 22 +/- 6% at 150 mmHg (n = 10, p = 0.0001) and was not affected in the L-NAME + ACEI group. 5. Acute in vitro NG-nitro-L-arginine (L-NOARG, 10 microM) significantly decreased flow-induced dilation in control but not in L-NAME or L-NAME + ACEI rats. Both acute indomethacin (10 microM) and acute NS 398 (cyclo-oxygenase-2 (COX-2) inhibitor, 1 microM) did not change significantly flow-induced dilatation in controls but they both decreased flow-induced dilatation in the L-NAME and L-NAME + ACEI groups. Acute Hoe 140 (bradykinin receptor inhibitor, 1 microM) induced a significant contraction of the isolated mesenteric arteries which was the same in the 3 groups. 6. Immunofluorescence analysis of COX-2 showed that the enzyme was expressed in resistance mesenteric arteries in L-NAME and L-NAME + ACEI groups but not in control. COX-1 expression was identical in all 3 groups. 7. We conclude that chronic inhibition of nitric oxide synthesis is associated with a decreased flow-induced dilatation in resistance mesenteric arteries which was compensated by an overproduction of vasodilator prostaglandins resulting in part from COX-2 expression. The decrease in flow-induced dilatation was prevented by the ACEI, quinapril.

Growth hormone treatment in patients with neurosecretory dysfunction.

Twenty-four children (14 boys and 10 girls) with neurosecretory dysfunction, defined by a response greater than 10 ng/ml to two pharmacological tests, and 24-hour GH secretion less than 3 ng/ml/min, were treated with biosynthetic hGH. Mean age was 10 years 8 months +/- 3 years 6 months. Growth retardation was -2.8 +/- 0.8 SD. Eighteen children were prepubertal and six pubertal (P2). Mean peaks in two pharmacological stimulation tests were 25.8 +/- 14.8 and 20.8 +/- 11.5 ng/ml. Somatomedin C/insulin-like growth factor I level was 0.8 +/- 0.6 IU/ml in the prepubertal children. Twenty-four-hour integrated concentration of GH was low, at 2.2 +/- 0.5 ng/ml/min. Analysis of secretory profiles showed 3 types: (1) hyperpulsatile profile with numerous peaks of low amplitude, (2) flat profile, (3) profile with an isolated peak greater than 10 ng/ml. Treatment with hGH (0.42 IU/kg/week) resulted in an increase in growth velocity from 4.9 +/- 1.2 to 6.8 +/- 2.2 cm/year. These results are comparable to those of a group with total GH deficiency receiving the same dose. Analysis of the results showed a group of good responders (n = 14, growth velocity: 8.1 cm/year) and a group of poor responders (n = 10, growth velocity: 4.9 cm/year). Thus, treatment of neurosecretory dysfunction with hGH gives results comparable to those obtained in classic GH secretory dysfunction.