Interrelationships among physicochemical properties, absorption and anthelmintic activities of 2-desoxoparaherquamide and selected analogs.

The interrelationships between physicochemical properties, absorption and potency of 2-desoxoparaherquamide and five analogs, representing a new anthelmintic class, were evaluated in in vitro and in vivo assays. At pH 7.5, rates of drug absorption by the gastrointestinal nematode Haemonchus contortus and jird small intestine, parameterized by the permeability coefficient, P(e), ranged from 1.2-2.4 x 10(-4) cm/min (nematode) to 2.5-5.5 x 10(-3) cm/min (jird). In the jird intestine, absorption was pH-dependent, with P(e) at pH 7.5 being twice that at pH 4.5, reflecting the negative influence of protonation on transport of these weakly basic molecules. Each compound rapidly paralyzed H. contortus during in vitro exposure to therapeutically relevant concentrations (1-10 microm). The kinetics of drug action on motility in vivo mirrored their in vitro effects; motility concentrations were reduced in nematodes collected from jird stomach 3 h following oral drug dosing, by which time > or =50% clearance of the parasites had occurred. The nematode/medium partition coefficient K ranged from 10.1 to 16.1, consistent with the lipophilic nature of the compounds. The time required to reduce motility in vitro by 50% (t50*) and P(e) were used to determine C(n)*, the concentration of drug in the nematode at t50*, as an indicator of intrinsic potency. In the jird, the apparent potencies of the compounds were insensitive to route of administration (i.e. oral = i.v. = i.p. = i.m.) for H. contortus and two other gastrointestinal nematodes, Ostertagia ostertagi and Trichostrongylus colubriformis; topical administration, however, required three to 10-fold higher doses for equivalent efficacy.

Synthesis and biological activity of analogues of the antidiabetic/antiobesity agent 3-guanidinopropionic acid: discovery of a novel aminoguanidinoacetic acid antidiabetic agent.

3-Guanidinopropionic acid (1, PNU-10483) has been demonstrated to both improve insulin sensitivity and to promote weight loss selectively from adipose tissue in animal models of non-insulin-dependent diabetes mellitus (NIDDM). However, 1 has also been shown to be a substrate for both the creatine transporter and creatine kinase, leading to marked accumulation in muscle tissue as the corresponding N-phosphate 4. In an effort to identify novel entities that maintain antidiabetic potency without susceptibility to creatine-like metabolism, an analogue program was undertaken to explore the effects of various structural modifications, including homologation, simple substitution, single atom mutations, and bioisosteric replacements for the guanidine and carboxylic acid. Overall, the scope of activity encompassed by the set of new analogues proved to be exceedingly narrow. Notable exceptions demonstrating equivalent or improved antidiabetic activity included the alpha-amino derivative 29, aminopyridine 47, isothiourea 67, and aminoguanidine 69. On the basis of its superior therapeutic ratio, aminoguanidine 69 was selected for preclinical development and became the foundation for a second phase of analogue work. Furthermore, in vitro studies demonstrated that 69 is markedly less susceptible to phosphorylation by creatine kinase than the lead 1, suggesting that it should have less potential for accumulation in muscle tissue than 1.

Synthesis and biological activity of aminoguanidine and diaminoguanidine analogues of the antidiabetic/antiobesity agent 3-guanidinopropionic acid.

3-Guanidinopropionic acid (1) has been demonstrated both to improve insulin sensitivity and to promote weight loss selectively from adipose tissue in animal models of non-insulin-dependent diabetes mellitus (NIDDM). However, 1 has also been shown to be a substrate for both the creatine transporter and creatine kinase, leading to marked accumulation in muscle tissue as the corresponding N-phosphate. The corresponding aminoguanidine analogue 2 was recently discovered to retain the antidiabetic activity of 1 while being markedly less susceptible to creatine-like metabolism, suggesting that it should have less potential to accumulate in muscle. Further structural modification of 2 was undertaken to investigate whether the antidiabetic potency could be augmented while maintaining resistance to creatine-like metabolism. Modifications such as alpha-alkylation, homologation, and bioisosteric replacement of the aminoguanidine all were detrimental to antidiabetic activity. However, the simple regioisomeric aminoguanidinoacetic acid 9 and diaminoguanidinoacetic acid analogue 7 were found to be equipotent to 2, leading eventually to the discovery of the significantly more potent diaminoguanidinoacetic acid regioisomers 52 and 53. Further attempts to modify the more active template represented by 52 led only to reductions in antidiabetic activity. Each of the new active analogues displayed the same resistance to creatine-like metabolism as 2. Further testing of 7, 9, and 53 in obese diabetic ob/ob mice confirmed that weight loss is induced selectively from adipose tissue, similar to the lead 1. Administration of 53 to insulin-resistant rhesus monkeys led to reductions in both fasting and post-prandial plasma glucose levels with concomitant reductions in plasma insulin levels, suggesting that the compound improved the action of endogenous insulin. Compounds 7 and 53 were selected for further preclinical development.

Strategies toward predicting peptide cellular permeability from computed molecular descriptors.

The therapeutic efficacy of an orally administered drug is dictated not only by its pharmacological properties such as potency and selectivity, but also its pharmacokinetic properties such as its access to the site of activity. Thorough evaluation of the physicochemical and biological barriers to drug delivery is essential to the selection and successful development of drug candidates. We have demonstrated previously that cellular permeability, as a primary component of drug delivery, is principally dependent upon the desolvation potential of the polar functionalities in the molecule and, secondarily, upon the solute lipophilicity [Conradi, R.A., Hilgers, A.R., Ho, N.F.H., Burton, P.S. (1992). The influence of peptide structure on transport across Caco-2 cells. II. Peptide bond modification which results in improved permeability. Pharm. Res. 9, 473-479]. Increasingly sophisticated computational methods are becoming available for describing molecular structural features proposed to correlate with such molecular physicochemical determinants of permeability. Herein we examine the relationships of various computationally derived molecular geometric descriptors for a set of peptides and peptidomimetics, in the context of experimentally measured hydrogen-bond potentials and lipophilicities, with their cellular permeabilities. These descriptors include molecular volume, polar and non-polar surface areas and projected molecular cross-sectional areas. Particular attention is paid to the roles of solvation treatments and other computational factors in descriptor generation, deconvolution of cellular transport mechanisms and statistical analyses of the resulting data for the development of valid, structure-based and mechanistically meaningful models of cellular permeability. No significant correlation of cellular permeability with computed descriptors was found. This was primarily because of our inability to identify surrogates for hydrogen-bond desolvation potential for the solutes from among these descriptors.

In vitro metabolism of ceftiofur in bovine tissues.

The metabolism of ceftiofur in bovine kidney, liver, muscle and lung, and the effects of the presence of cystine and glutathione in the media were evaluated using S-9 and microsomal tissue fractions. Conversion of ceftiofur to desfuroylceftiofur (DFC) was catalyzed by an esterase which was most active in kidney, followed by liver. It was not very active in muscle and lung. After DFC was liberated, it rapidly bound primarily to tissue proteins (> 56%), and was also conjugated to cysteine and glutathione. Production of DFC-cysteine by disulfide exchange of DFC with cystine and production of DFC-glutathione by conjugation of DFC to glutathione occurred in buffer if glutathione and cystine were present in the medium. These conjugations were also observed in incubations with tissue fractions, indicating that they were not inhibited by the tissues endogenous molecules. In addition, the metabolism of DFC-glutathione to DFC-cysteine was observed when tissue proteins were present. The metabolism of DFC-glutathione to DFC-cysteine was faster in kidney than in liver. Metabolites devoid of an intact beta-lactam ring were not observed in these in vitro studies.

The oxazolidinone eperezolid binds to the 50S ribosomal subunit and competes with binding of chloramphenicol and lincomycin.

The oxazolidinones are a novel class of antibiotics that act by inhibiting protein synthesis. It as been reported that the drug exerts its primary activity on the initiation phase of translation. In order to study the possibility of direct interaction between the drug and the ribosome, we have developed a binding assay using 14C-labelled eperezolid (PNU-100592; formerly U-100592). Eperezolid binds specifically to the 50S ribosomal subunit of Escherichia coli. The specific binding of eperezolid is dose dependent and is proportional to the ribosome concentrations. Scatchard analysis of the binding data reveals that the dissociation constant (Kd) is about 20 microM. The binding of eperezolid to the ribosome is competitively inhibited by chloramphenicol and lincomycin. However, unlike chloramphenicol and lincomycin, eperezolid does not inhibit the puromycin reaction, indicating that the oxazolidinones have no effect on peptidyl transferase. In addition, whereas lincomycin and, to some extent, chloramphenicol inhibit translation termination, eperezolid has no effect. Therefore, we conclude that the oxazolidinones inhibit protein synthesis by binding to the 50S ribosomal subunit at a site close to the site(s) to which chloramphenicol and lincomycin bind but that the oxazolidinones are mechanistically distinct from these two antibiotics.

Mechanistic roles of neutral surfactants on concurrent polarized and passive membrane transport of a model peptide in Caco-2 cells.

The transport of the model peptide Acf(NMef)2NH2 across Caco-2 cell monolayers was studied in the apical (AP) to basolateral (BL) and the BL to AP direction in the presence of Polysorbate 80 or Cremophore EL in the AP compartment. Increasing surfactant concentrations resulted in increasing AP-->BL peptide permeability and decreasing BL-->AP permeability. In either direction, limiting permeabilities were achieved at concentrations less than the critical micellar concentrations (cmc's) of the surfactants, and remained constant at much higher concentrations. These plateau permeabilities were not equivalent in the two directions. This residual assymetry was abolished by increasing the peptide concentration. Altogether, the observations support the presence of at least two pumps in Caco-2 cells for this peptide, polarized in the BL-->AP direction. These experimental results were analyzed within the context of a quantitative biophysical model incorporating concurrent passive diffusion across the AP and BL membranes accompanied by surfactant-inhibitable active polarized efflux across the AP membrane. The model was also used to locate the additional transport activity at the BL membrane as an uptake pump. Under conditions of complete inhibition, the intrinsic passive diffusional permeability of Acf(NMef)2NH2 was found to be 13 x 10(-6) cm/s, essentially identical with results reported earlier with this peptide utilizing verapamil as an inhibitor. With respect to the mechanism of surfactant inhibition of the apical efflux transport, the monomeric species was found to be responsible with no contribution from micelles. Modeling the mode of inhibition as a noncompetitive Michaelis-Menten process gave identical Kis of 0.5 microM for the two surfactants. Finally, increase of either surfactant beyond 750 microM resulted in a decrease of peptide permeability in the AP-->BL direction. This was attributed to weak association of the peptide with micelles in the AP compartment, which effectively decreased the thermodynamic activity of the peptide at surfactant concentrations greater than 20 times their cmc. Both the experimental approach and accompanying theoretical model demonstrated in this work will allow for further characterization of the inhibitory potencies of surfactants for the nonpassive efflux pathway in vitro and in vivo.

Ceftiofur hydrochloride: plasma and tissue distribution in swine following intramuscular administration at various doses.

Twelve mixed-breed swine (26.5-42.5 kg) received three daily intramuscular (i.m.) doses of 14C-ceftiofur hydrochloride. Three males and three females, received 6.76 +/- 0.83 mg of 14C-ceftiofur free acid equivalents (CFAE)/kg body weight (b.w.)/day, while the other group received 4.41 +/- 0.97 mg.CFAE/kg b.w./day. The swine were slaughtered 12 h following the last dose. Total dose accountability for the 6.76 mg dose was 91.44 +/- 16.11% (72.51% in urine; 12.63% in faeces). For the 4.41 mg dose, accountability was 100.35 +/- 20.45% (82.48% in urine; 12.85% in faeces). Within the tissues used for residue monitoring, the highest concentrations were observed in the kidneys (10.68 and 6.33 micrograms.CFAE/g for the 6.76 and 4.41 mg doses, respectively), followed by the injection sites, lungs, liver and muscle. In a separate study, twelve mix-breed swine (23.1-39.7 kg) received 14C-ceftiofur hydrochloride at 3.08 mg.CFAE/kg b.w. once daily for 3 days. Two males and two females were slaughtered at either 12, 72 or 120 h after the last dose. Total dose accountability for the 3.08 mg dose was > 83% (> 68% in urine; > 13% in faeces). In swine slaughtered 12 h after last dose, residue concentrations closest to the safe concentrations were observed in the kidneys (3.62 micrograms.CFAE/g), followed by the injection sites, lungs, liver and muscle.

Theoretical perspectives on anthelmintic drug discovery: interplay of transport kinetics, physicochemical properties, and in vitro activity of anthelmintic drugs.

This multidisciplinary study demonstrates the utility of the biophysical model approach to assess biological activity of anthelmintics in light of drug-delivery principles. The relationships between drug absorption and efficacy for a set of structurally disparate anthelmintics were determined in cultures of Haemonchus contortus, a nematode that parasitizes the ruminant gastrointestinal tract. Uptake, parameterized by the permeability coefficient, Pe, was shown to occur by absorption across the cuticle. Rates of drug appearance in nematode carcasses paralleled rates of drug disappearance from the medium, and absorption reached an apparent equilibrium within a few hours. The parasite/medium partition coefficient, K, was derived from the ratio of drug concentration in the parasite vs the medium at equilibrium. Pe and K values for each anthelmintic were correlated with lipophilicity (as measured by the partition coefficient (PC) in n-octanol/water) and both parameters plateaued at log PC approximately 2.5, with maximum Pe approximately 8 x 10(-4) cm/min and log K < or = 2.0. Absorption kinetics were related to in vitro potency by monitoring motility of H. contortus. The time required to reduce motility by 50% (t* 50) and Pe were used to calculate Cn*, the drug concentration in the parasite at t* 50, as an indicator of intrinsic potency. The quantitative interplay of apparent biological activity expressed as t* 50, dose, and intrinsic potency highlights the important contribution of drug-uptake kinetics.

Effects of U-75875, a peptidomimetic inhibitor of retroviral proteases, on simian immunodeficiency virus infection in rhesus monkeys.

U-75875 inhibits human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus (SIV) proteases and blocks Gag-Pol protein processing and viral maturation and replication in vitro. Rhesus monkeys were treated with vehicle alone or with formulated U-75875 at doses of 7 or 20 mg/kg of body weight per day for 26 days by continuous intravenous infusion beginning 6 h prior to intravenous inoculation with 10 monkey 50% infectious doses of SIV Delta B670, and the monkeys were monitored until death. The effects of treatment on the level of SIV p26 antigenemia, the infectious virus titer in serum, and the level of proviral DNA in blood mononuclear cells evaluated by PCR were assessed. SIV infection of the controls resulted in an initial viral antigenemia that began 5 to 10 days postinoculation (p.i.), reached peak values on days 10 to 14 p.i., and lasted for more than 15 days. Proviral DNA was detectable in peripheral blood mononuclear cells by 7 to 11 days p.i., reached the mean peak level by 11 days p.i., and remained at high levels through day 24 p.i. Infectious virus was detected in serum from all of the infected controls by 24 days p.i. Treatment with U-75875 for 26 days resulted in a dose-related delay in the day of the peak level of antigenemia (P = 0.034). The level of proviral DNA in peripheral blood mononuclear cells at 11 days p.i. was significantly decreased in a dose-related fashion in the treated monkeys ( P

Long-term effects of Aroclor 1254 (PCBs) on plasma lipid and carnitine concentrations in rhesus monkey.

Sixty-seven female rhesus monkeys (Macaca mulatta) were orally dosed daily for 152 weeks with 0, 5, 20, 40, and 80 micrograms Aroclor 1254 (PCB)/kg body wt. Blood polychlorinated biphenyl (PCB) concentrations were highly positively correlated (r = 0.92, P < 0.001) with doses of PCB administered. A comprehensive analysis of plasma lipids/lipoproteins revealed a PCB-associated increase in plasma triglycerides and decreases in plasma total cholesterol, high-density lipoprotein cholesterol (HDL-chol), very-low plus low-density lipoprotein cholesterol (VLDL+LDL-chol), and total carnitine (which is involved in fatty acid metabolism). All of the lipid/lipoprotein changes were significantly (P < or = 0.05) correlated with blood PCB concentration. These data, obtained after 152 weeks of continuous daily exposure of a primate model to PCB support a causal relationship between plasma lipid changes and PCB intake. Previously, causality has been refuted on the premise that the commonly observed elevation of triglycerides with increasing concentration of blood PCB is a reflection, not of PCB dose, but of the partitioning of PCB between tissues (adipose) and blood in proportion to the blood lipid present. The mechanism of the plasma lipid changes was not investigated in this study but the altered lipid/lipoprotein pattern is discussed with respect to known cardiovascular risk profiles.

Dietary polyunsaturated to saturated fatty acid ratio alters hepatic LDL transport in cynomolgus macaques fed low cholesterol diets.

To determine effect of interaction between dietary cholesterol and triglyceride, i.e., polyunsaturated to saturated (P:S) fatty acid ratio, on LDL metabolism, male cynomolgus macaques were fed purified diets for 83 wk with cholesterol levels of 0.01, 0.06 and 0.50 mg/kJ and P:S ratios of 0.5 and 0.9, oleic acid constant. There were six groups of five animals each (cholesterol, mg/kJ--P:S ratio): Group 1, 0.01--0.5; Group 2, 0.01--0.9; Group 3, 0.06--0.5; Group 4, 0.06--0.9; Group 5, 0.50-0.5; Group 6, 0.50-0.9. LDL (1.019 less than d less than 1.063 kg/L) and glucosylated LDL were iodinated for turnover studies. Hepatic LDL transport was determined using 125I-tyramine-cellobiose-LDL as tracer. Plasma cholesterol increased in proportion to dietary cholesterol, and concentrations (mmol/L) at 77-78 wk were (mean +/- SEM): Group 1, 434 +/- 0.31; Group 2, 3.03 +/- 0.14; Group 3, 8.28 +/- 1.48; Group 4, 7.34 +/- 1.31; Group 5, 15.54 +/- 1.44; Group 6, 15.54 +/- 1.41. LDL cholesterol was 45% higher in Group 1 (2.43 mmol/L) than in Group 2 (1.68 mmol/L). In vivo studies showed that LDL clearance was suppressed by excess dietary cholesterol; receptor-independent LDL clearance was relatively constant. Hepatic LDL protein transport was greater in Group 2 (P:S 0.9) compared with Group 1 (P:S 0.5). The LDL protein synthetic rate was lower in Groups 2, 4 and 6 (P:S 0.9) relative to Groups 1, 3 and 5 (P:S 0.5). We conclude that in this model hepatic LDL receptor activity is altered by degree of saturation in dietary triglycerides when dietary cholesterol is minimal, and that saturated dietary triglycerides enhance LDL protein secretion when dietary cholesterol is ample.

Spontaneous hypercholesterolemia in cynomolgus monkeys: evidence for defective low-density lipoprotein catabolism.

Spontaneously hypercholesterolemic (SH) cynomolgus monkeys were identified that have average plasma cholesterol of 202 mg/dl, while that in normal monkeys is 119 mg/dl. The LDL from these SH monkeys have lower affinity for fibroblast LDL receptors in vitro. The amount of LDL2 (1.030 mean value of d 1.063 g/ml) required to displace 50% of [125I]LDL was 3.8 micrograms/ml for normal LDL2 and 6.6 micrograms/ml for SH-LDL2. The binding affinity of LDL1 (1.019 mean value of d 1.030 g/ml) was the same in normal and SH animals. LDL turnover experiments showed that the SH monkeys were comprised of two populations. Normal LDL2 was cleared much slower in two of the SH monkeys than in normocholesterolemic animals, suggesting that these two animals have an LDL receptor defect. However, LDL2 isolated from these two SH monkeys was cleared normally in normal monkeys. LDL2 isolated from two other SH monkeys is cleared slower than is normal LDL2 in normal animals, suggesting that these animals have an LDL defect. Thus, the hypercholesterolemia of these SH monkeys is associated with defective LDL catabolism; two animals appear to have functionally defective LDL receptors, and two animals appear to have functionally defective LDL.

Effects of epidermal growth factor on apo B mRNA levels and apo B accumulation in the media of primate hepatocytes in culture.

EGF has been shown to augment albumin and apolipoprotein A-I secretion by cynomolgus monkey hepatocytes in primary culture without stimulating cell division. This study was undertaken to determine what effect EGF had on apo B secretion by those hepatocytes. The results indicate that EGF (3 nM final concentration) severely inhibits the rate at which apo B accumulates in the culture medium of primate hepatocytes. That effect was evident within 48 hours of treatment, and by 72 hours the rate that apo B accumulated was less than half that of cells treated with a hormone-free medium. However, the apo B mRNA levels in the EGF-treated cells were more than double those of hepatocytes given the hormone-free medium. These data indicate that EGF has a potent effect on the rate at which apo B accumulates in the culture medium of primate hepatocytes and that the effect is independent of apo B gene expression.

L-carnitine administration and withdrawal affect plasma and hepatic carnitine concentrations, plasma lipid and lipoprotein composition, and in vitro hepatic lipogenesis from labeled mevalonate and oleate in normal rabbits.

Carnitine was administered to normal rabbits to investigate the possible effects of pharmacologic doses on various aspects of normal lipid and lipoprotein metabolism. Carnitine concentrations were measured in the plasma and liver of normal rabbits that received L-carnitine orally [40 mg/(kg.d)] for 21 d and after withdrawal from the carnitine supplement for 21 d. Plasma lipids, plasma lipoprotein composition and in vitro hepatic lipid biosynthesis from [2-14C]mevalonate and [1-14C]oleate were also measured. Threefold elevations in plasma carnitine with carnitine treatment were essentially reversed after 48 h of carnitine withdrawal, but elevated hepatic carnitine accumulation (twofold) persisted for 21 d, suggesting that the accumulated carnitine constituted a pool that is only slowly miscible with plasma. The rabbits withdrawn from L-carnitine for 21 d experienced a 35% decrease in plasma cholesterol, a 50% decrease in VLDL cholesterol, and an increase in the protein content of HDL and of intermediate density lipoprotein + LDL. Additionally, the proportion of [14C]oleate incorporated into hepatic phospholipids increased 35% at the expense of triglyceride and the ratio of hepatic [14C]cholesterol to [14C]squalene derived from [14C]mevalonate increased over twofold following carnitine withdrawal. These studies provide evidence that normal lipid homeostasis can be altered by supplemental carnitine and that the perturbations are reflected by changes in plasma lipids and lipoproteins and in the proportions of the hepatic lipids synthesized.

Cholesterol lowering bile acid binding agents: novel lipophilic polyamines.

A series of novel lipophilic polyamines was synthesized by the sodium cyanoborohydride-mediated reductive amination of various ketones and aldehydes with the polyamine tris(2-aminoethyl)amine. Two of these compounds, N,N-bis[2-(cyclododecylamino)ethyl]-N'-benzyl-1,2-ethanediamine trihydrochloride (36.3HCl) and N,N-bis[2-(cyclododecylmethylamino)ethyl]-N',N'-dimethyl-1,2-ethan ediamine (23), are 29 and 24 times more potent than colestipol hydrochloride, respectively, for lowering animal serum cholesterol levels.

Desolvation energy: a major determinant of absorption, but not clearance, of peptides in rats.

The oral delivery of peptidic drugs is problematic because of their degradation in the gastrointestinal tract and low absorption through the intestinal mucosa. Earlier in vitro studies with two series of digestion-resistant, radiolabeled peptides that varied in physical properties (molecular weight, lipophilicity, and hydrogen bonding sites) had suggested that intestinal transport of these peptides was most influenced by the number of hydrogen bonding sites, the major determinant of desolvation energy. To determine whether this correlation could be confirmed in vivo, intestinal absorption was determined by comparing the biliary and urinary recovery of these radiolabeled peptides in rats given intravenous or intraduodenal doses. Absorption was inversely correlated to the number of calculated hydrogen bonding sites for the model peptides, similar to what had been found in vitro. Clearance by liver and kidneys appeared to be unaffected by desolvation energy but was well correlated with lipophilicity.

Planning the purification process of active cDNA in expression cloning strategies.

In principle, it is possible to clone the gene for any receptor that can be expressed in the Xenopus laevis frog oocyte and assayed electrophysiologically (E. S. Levitan, 1988, TINS 11, 41-43). Repeated fractionation of a lambda vector cDNA library made from mRNA which encodes the receptor protein will eventually lead to a single cDNA clone. This strategy poses the question as to how large should a lambda vector cDNA aliquot be at any fractionation stage in order that one may be relatively certain that the aliquot contains the clone of interest, and how many times should the fractionation be repeated in order that one isolate the single clone of interest? The purification of active cDNA is an iterative process. At each fractionation stage we specify the probability of at least one active cDNA in the total aliquot to be high. The required size of the aliquot taken depends upon this specified probability and the additional probability of selecting at random an individual cDNA which is active. We require an estimate of the latter probability for the initial stage. For subsequent stages Baye's estimators of these probabilities are used in the formula for calculating the aliquot size at each stage. We show how to perform this calculation when there is equal amplification of the active and remaining sequences and when the active sequence has a non-representative (unequal) amplification. When equal amplification holds a relatively simple formula is provided for calculating the expected number of stages needed in the process. When unequal amplification holds there is no simple calculation for this quantity and the entire sequence of calculations leading to termination of the process must be performed. In either case the minimum lambda vector amplification (growth) factor required at each stage to yield an adequate amount of cDNA for analysis is calculable.